Re: 악성종양의 최초 발생과 전이 diriver 만성염증에 대한 2024 네이처 논문 연구 탐구!!!

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오랫동안 궁금했던 

악성종양 발생 환경

전이 환경에 대한 구체적인 탐구들!

만성염증

암세포미세환경의 토양

초기 암발생 --> 성장 --> 전이...

모두 만성염증이 토양이다!!

Tumor Initiation and Early Tumorigenesis: Molecular Mechanisms and Interventional Targets

• 저자: Shangyao Zhang, Tianyi Xiao, Yuanning Du et al.
• 저널: Signal Transduction and Targeted Therapy (Nature 시리즈)
• 출판: 2024년 6월 10일 (Volume 9, Article 149)
• 요약: 종양 발생 초기 단계에서 만성 염증이 돌연변이 세포의 선택과 증식을 촉진한다고 리뷰. Furman et al.을 암의 에티올로지 맥락에서 지지하며, chronic inflammation이 acute에서 unresolved 상태로 전환되어 cytokine(IL-1, IL-6, TGF-β) 경로를 통해 MAPK, PI3K-AKT, NF-κB를 활성화—Furman의 SCI multi-level 기전(분자: inflammasome, 세포: SASP)과 일치.
• 위험인자: 만성 감염, 비만, 흡연이 염증 microenvironment를 형성해 CHIPs(clonal hematopoiesis) 등 종양 위험 증가.
• 함의: anti-inflammatory 약물(NSAIDs, IL-1 억제제)이 초기 종양 예방에 유용, Furman의 common soil hypothesis를 암 특화.
•  

"The 'seed and soil' hypothesis revisited" (Paget의 종양 전이 가설 재고찰)에 관한
2008년 Lancet Oncology 논문(또는 관련 서신/코멘터리) 일부이고,



1. "The 'seed and soil' hypothesis revisited" (Paget의 'seed and soil' 가설 재고찰) 원문 요약 및 해석

이 텍스트는 1889년 Stephen Paget가 제안한 "seed and soil" 가설
(종양 세포(seed)가 특정 장기(soil)에서만 잘 자라는 이유를 설명하는 고전 이론)을
현대적으로 재평가한 내용입니다.

원래 Paget는 유방암 환자의 부검 기록(735명)을 분석해
전이가 무작위가 아니라 특정 장기(간, 폐, 뇌, 뼈 등)에 집중된다는 점을 지적하며,
"종양 세포(seed)는 모든 장기에 떨어지지만,
적합한 토양(soil)이 있어야만 자란다" 고 주장했습니다.

• 주요 역사적 내용:
  • 1889년 Paget 논문: 유방암 전이가 간·폐·뇌·뼈에 빈번하고, 비장·신장 등에는 드물다는 관찰 → 전이가 수동적·무차별적이지 않음.
  • 1929년 James Ewing의 도전: 혈관 구조·순환 역학으로 설명 가능하다고 반박 → "seed and soil" 가설이 수십 년간 주류에서 밀려남.
  • 20세기 후반~현재: 선택적 전이(selective metastasis) 증거 축적 → Paget 가설이 다시 인정받음 (e.g., 장기 특이적 microenvironment, stroma, immune niche가 "soil" 역할).
  • 현대 재해석: "seed" (종양 세포) + "soil" (장기 미세환경: stroma, 혈관, 면역세포, cytokine 등)의 상호작용이 전이 성공 결정. 순수 기계적 요인(혈류)만으로는 설명 불가.
• 키 포인트:
  • Paget의 1889년 발견: 전이가 무작위가 아님 → 뼈 전이는 유방·전립선암에서 높고, 비장·신장에서는 낮음 → "토양"의 역할 강조.
  • Ewing의 반박에도 불구하고, 오늘날 대부분의 종양학자들은 seed and soil 를 받아들임 (terminology는 바뀌었을 뿐: "metastatic niche", "pre-metastatic niche" 등).
  • 결론: 전이 병인(pathogenesis)은 종양 세포와 숙주 장기 간 cross-talk 에 달려 있음.

이 내용은
Furman et al. (2019)의 SCI(전신 만성 염증)가 암·전이의 "soil" 을 악화시킨다는 관점과
연결될 수 있습니다
(만성 염증이 pro-tumorigenic microenvironment 형성).

https://www.nature.com/articles/s41392-024-01848-7

이 리뷰는

종양 발생 초기 단계 (tumor initiation & early tumorigenesis) 를 다루며,

단순 유전자 돌연변이가 아닌 유전자·후성유전·외부 환경 요인 의 복합 상호작용이

정상 세포를 암으로 전환시키는 과정을 통합적으로 설명합니다.

Furman et al. (2019)의 SCI (systemic chronic inflammation) 개념을 직접 인용하며 (ref. 130),

만성 염증이 암의 주요 위험인자로 작용한다는 점을

강하게 지지·확장합니다.

1. 배경 및 핵심 주장 (Abstract & Introduction)

종양 발생은 multistep 과정으로,

정상 세포의 oncogenic mutation (e.g., TP53, KRAS)이 clonal advantage를 주지만,

정상 조직에도 이러한 돌연변이가 흔함에도 암으로 진행되는 경우는 드물다.

→ 추가 driver events (genetic + epigenetic + environmental)가 필요

→ irreversible heterogeneous lesion으로 전환.

• 핵심 paradox: 정상 조직 somatic mutation burden 높음 (e.g., sun-exposed skin TP53 clones) → 하지만 cancer incidence 낮음 → extrinsic selective pressure와 tissue context가 결정적.
• 주장: 초기 clonal expansion과 malignant evolution은 염증·노화·대사·미생물 등 extrinsic factors가 oncogenic potential을 "release"하며, microenvironment가 tumor-suppressive → supportive로 전환.
• 함의: premalignant stage interception (예방·조기 개입)이 가능 → precision prevention.

• 클론 수준 진화 (무성생식): 재조합·독립 분리 없음 → 전체 유전체가 그대로 복제 → 유해 돌연변이 축적(Muller's ratchet) → 클론 쇠퇴.
• 유전자 수준 진화 (성생식): 독립 분리 + crossing-over(교차) → allele이 섞여 독립 선택 → 유해 돌연변이 제거 쉬움, 유익 조합 생성 → 장기 안정성·적응력 ↑.

재조합의 역할 차이:

클론(무성)은 재조합 부재로 "전체 유전체가 하나의 운명"을 공유 → 돌연변이 축적(Muller's ratchet) → 쇠퇴 및 단기 수명.

유전자(성생식)는 재조합으로 "개별 유전자가 독립 운명" → 돌연변이 제거 + 다양성 생성 → 장기 안정성.

2. 주요 기전 (Key Mechanisms — Figure 중심)

리뷰는 genetic/epigenetic/external drivers의 co-evolution을 강조.

• Genetic drivers: SNVs, CNAs (aneuploidy, ecDNA), structural variants (chromothripsis). 초기 단계에서 TP53 biallelic loss가 CNAs 허용 (esophageal cancer).
• Epigenetic rewiring: DNA hyper/hypomethylation (tumor suppressor inactivation, plasticity 유도), histone mods (Polycomb repression). Aging-related methylation이 Wnt pathway 활성화 → BRAF sensitivity ↑.
• Environmental/external drivers (Furman SCI 직접 연결):
  • Chronic inflammation: unresolved damage → tumor risk factor. Cytokine (IL-1, IL-6, TNF-α) → NF-κB/STAT3 활성 → clonal expansion 촉진. Pancreas: KRAS + inflammation → acinar-to-ductal metaplasia (ADM) → PanIN → PDAC.
  • Metabolic dysregulation (metaflammation): 고당·고지방 식이 → cGAS-STING 활성 → 염증·glycolysis ↑.
  • Microbiome dysbiosis: pks+ E. coli colibactin → genotoxicity + epigenetic modulation (CRC).
  • Aging/inflammaging: SASP → EMT 촉진, immune surveillance ↓ (Furman의 immunosenescence 연계).

→ Figure 1: Multistage tumorigenesis — 정상 → premalignant → malignant progression, microenvironment evolution (suppressive → supportive: TAMs, CAFs, ECM stiffening).

Multistage tumorigenesis 설명 
다단계 종양 형성 (Multistage tumorigenesis)

정상 조직에서는 체세포 돌연변이(somatic mutation)가 산발적으로 발생하며,
이는 종양 억제 기전(tumor-suppressive mechanisms)에 의해 제거되거나,
증식 우위를 얻어 클론(clone)을 형성합니다.

이러한 돌연변이 클론은
추가 자극에 노출되기 전까지는
여전히 항상성(homeostasis)을 유지할 수 있습니다.

그러나
추가 자극에 노출되면 증식이 통제 불능 상태가 되고,
악성 전환(malignant transformation)이 시작됩니다.

이 과정에서
전암성 병변(premalignant lesion)에서
진행성 종양(advanced tumor)으로 점진적으로 발전합니다.

이 과정 동안 변형된 세포는
점차 추가적인 유전자 돌연변이와 후성유전적 변화(epigenetic alterations)를 축적하며,
면역 회피(immune evasion), 구조적 파괴, 침윤(invasion) 등
점점 더 악성인 특성을 나타냅니다.

동시에
세포 주변 미세환경(microenvironment)도
종양 억제적(tumor-suppressive) 상태에서
악성 종양을 지지하는(supportive of malignancy) 상태로 진화합니다.

이는 다음과 같은 변화를 포함합니다:

• 기능 장애를 일으키는 면역 감시(dysfunctional immunosurveillance)
• 종양 촉진성 염증(tumor-promotive inflammation)의 출현
• 섬유아세포(fibroblasts)가 점차 암 관련 섬유아세포(CAFs: cancer-associated fibroblasts)로 전환
• 세포외기질(ECM: extracellular matrix)의 경직화(stiffening)

주요 용어

• CAF: cancer-associated fibroblast (암 관련 섬유아세포)
• TAM: tumor-associated macrophage (종양 관련 대식세포)
• MDSC: myeloid-derived suppressor cell (골수 유래 억제 세포)
• ECM: extracellular matrix (세포외기질)

→ Figure 3: Driver events convergence — inflammation, metabolism, microbiome, aging이 NF-κB/STAT3/ROS/cGAS-STING 등 공통 경로로 모임 

종양 발생 초기 단계에서의 oncogenic driver events 간 상호작용 (Interactions between oncogenic driver events)

a 유전독성(genotoxicity) 외에도 화학적·라디칼 손상은 세포 손상(cell injury), 분화(differentiation), 세포사(apoptosis)를 유도할 수 있습니다. 이러한 손상에 대한 저항성을 부여하는 oncogenic mutation은 증식 우위(proliferative advantages)를 제공합니다. 반대로 이러한 손상은 전사적·후성유전적 조절(transcriptional and epigenetic regulation)을 통해 증식(proliferative) 및 자가재생(self-renewal) 경로를 자극합니다. 면역세포 또한 활성화되어 변형된 세포의 운명(transformed cell fate)을 조절하고 종양 형성(tumorigenesis)을 촉진할 수 있습니다.

b 불건강한 식이 패턴(unhealthy diet patterns)은 고혈당(hyperglycemia)과 고인슐린혈증(hyperinsulinemia)을 유발하며, 이는 인슐린 신호에 대한 차별적 반응(differential response)을 초래합니다. 이로 인해 Src 또는 Ras 돌연변이를 가진 세포가 경쟁 우위를 얻어 종양 형성을 촉진할 수 있습니다. 높은 수준의 지방산(fatty acids) 또한 대사 재구성(metabolism remodeling)과 미토콘드리아 막 전위(mitochondrial membrane potential) 회복을 통해 세포 경쟁(cell competition)에서 Ras 돌연변이 세포의 유지(retention)를 촉진합니다. 또한 지방산과 포도당은 면역 반응과 염증을 조절하는 신호 분자(signaling molecules)로서 종양 형성에 참여합니다.

c 미생물군집(microbiota)은 변형된 세포와 상호작용하여 숙주의 DNA 메틸화(DNA methylation), 전사(transcription), 대사(metabolism), 면역 미세환경(immune microenvironment)에 영향을 미쳐 악성 전환(malignant transformation)에 관여합니다.

d 노화(aging)는 노화된 기질 세포(senescent stromal cells)가 SASP(senescence-associated secretory phenotype)를 분비하게 하여 세포 경쟁의 결과를 역전시키고 돌연변이 세포의 상피-중간엽 전이(EMT: epithelial-to-mesenchymal transition)를 촉진합니다. 노화는 또한 자발적 메틸화(spontaneous methylation)를 유발하여 돌연변이 주도 종양 형성(mutation-driven tumorigenesis)을 더욱 촉진합니다.

e
위에서 언급된 병리 과정들은
모두 염증(inflammation)으로 수렴(converge)하며,
이는 확장 클론(expansive clones)의 종양 형성 잠재력(tumorigenic potential)을 방출합니다.

염증은
oncogenic pathway를 활성화하고
후성유전적 가소성(epigenetic plasticity)을 증가시킵니다.

예를 들어,
췌장 염증(pancreatic inflammation)으로 유도된 가소성 상태(plastic state)에서는
ADM(acinar-to-ductal metaplasia) 과정에서
Kras 돌연변이 세포가 악성 상태로 전환될 가능성이 높아지지만,
염증이 없는 경우 Kras는 PanIN(pancreatic intraepithelial neoplasia)만 유도하고
PDAC(pancreatic ductal adenocarcinoma)로 진행되지 않습니다.

약어 목록

• EMT: epithelial-to-mesenchymal transition (상피-중간엽 전이)
• ROS: reactive oxygen species (활성산소종)
• nAChR: nicotinic acetylcholine receptor (니코틴성 아세틸콜린 수용체)
• MDSC: myeloid-derived suppressor cell (골수 유래 억제 세포)
• PDK: pyruvate dehydrogenase kinase (피루브산 탈수소효소 키나아제)
• TCF4: T cell factor 4 (T 세포 인자 4)
• HGF: hepatocyte growth factor (간세포 성장인자)
• TET2: tet methylcytosine dioxygenase 2 (테트 메틸시토신 디옥시게나아제 2)
• EGFR: Epidermal growth factor receptor (상피세포 성장인자 수용체)
• UV: ultraviolet (자외선)
• Gata6: GATA Binding Protein 6 (GATA 결합 단백질 6)
• EZH2: enhancer of zeste homolog 2 (zeste homolog 2의 enhancer)
• PPAR-δ: peroxisome proliferator-activated receptor-delta (퍼옥시좀 증식체 활성화 수용체-델타)
• FGF21: fibroblast growth factor 21 (섬유아세포 성장인자 21)
• CCL2 PDK4: pyruvate dehydrogenase kinase 4 (피루브산 탈수소효소 키나아제 4)
• ADM: acinar-to-ductal metaplasia (선포-관상 전이)
• PanIN: pancreatic intraepithelial neoplasms (췌장 상피내 종양)
• PDAC: pancreatic ductal adenocarcinoma (췌관 선암)
• ETBF: enterotoxigenic Bacteroides fragilis (장독성 Bacteroides fragilis)

→ Figure 4: Cell-autonomous processes — stem cell origin, dedifferentiation, trans-differentiation hijacking.

종양 형성에서의 세포 자율적 과정 (Cell-autonomous processes in tumorigenesis)

유전적·후성유전적 돌연변이를 획득한 후, 변형된 세포(transformed cells)는
악성 연속체(malignant continuum) 에 진입합니다.

이 과정에서 세포는
발달 경로(developmental pathways)를 재프로그래밍(reprogram)하여
점차 통제 불가능한 자가재생(self-renewal) 능력과
비정상적인 분화 잠재력(aberrant differentiation potential)을 획득합니다.

이는 주로 다음 세 가지 메커니즘을 통해 이루어집니다:

1. 줄기세포 기원 (originating from stem cells)
2. 계통 결정 세포로부터의 탈분화 (dedifferentiating from lineage-committed cells)
3. 전이분화(trans-differentiation) 과정 중 중간 상태의 탈취 (hijacking intermediate states during trans-differentiation)

→
이 설명은
종양 세포가 외부 요인 없이도 스스로 악성 특성을 강화하는
cell-autonomous 과정(세포 자율적 과정)을 강조합니다.

돌연변이가 축적되면서
줄기세포 특성(stemness) 회복, 분화 경로 탈선, 또는 중간 분화 상태를 이용해
더 악성인 표현형으로 진화한다는 내용입니다.

이는 Zhang et al. (2024)의 Signal Transduction and Targeted Therapy 리뷰에서
Figure 4와 연계되는 핵심 개념으로,
SCI(만성 염증)가 이러한 과정의 "촉매" 역할을 한다는
Furman et al. (2019)과 연결됩니다.

Table 3 Evolution of transformed cells and microenvironment in tumorigenesis

From: Tumor initiation and early tumorigenesis: molecular mechanisms and interventional targets

Components Tissue NEarly     evolution                                                                                  Refs

Transformed cells	colon	128a	Conventional adenomas originate from WNT-driven expansion of stem cells, while SSLs develop from lineage-committed cells. Downregulation of CDX2 in serrated specific cells supports a loss of regional identity and emergence of a fetal gene expression‎ signature.	262
	colon	81a	Stem-like cells form a malignancy continuum from early and late polyps to CRC, along which the WNT signal increases and the glutathione peroxidase increases to reduce the oxidative stress.	261
	colon	72a	Proliferation and DNA damage repair increase, while mitochondrial function and lipid metabolism decrease from normal tissue to CRC.	279
	skin	52	Sequential dedifferentiation that recapitulates the ordered cascade of differentiation in reverse is predominant in melanoma progression	493
	esophagus	43	Differentiated BE cells decrease during the transition from BE to EAC while the undifferentiated BE phenotype is maintained; transcription factor HNF4A is activated in differentiated BE cells and MYC is activated in undifferentiated BE cells.	494
	esophagus	29	Quiescent progenitor, cycling, mucosal defense, and terminal differentiation programs decrease expression‎, while hypoxia-related stress, reactive oxygen species-related stress, deoxidation, and antigen presenting programs increase from normal to LGIN-HGIN-ESCC.	307
	blood	31	Differentiation defects, distinct stemness, self-renewal and quiescence signatures of TP53 wild-type pre-LSCs HSCs in sAML are distinct from HSCs in healthy and myelofibrosis samples. Chronic inflammation enhances the fitness of TP53-mutant cells for clonal expansion.	135
	lung	25	Energy metabolism and ribosome synthesis programs are upregulated in AT2-like cells that emerge during AAH, which is diverged from normal AT2 cells and gain stemness as LUAD progresses.	62
	lung	9	Differentiation decreases from normal to cancer cells.	495
	stomach	25	mTOR signaling, RAS pathway, and VEGF signaling, functioning in IGC, are highly enriched in tumor cells.	496
	oral cavity	9	EMT, mTORC1, and FRA pathways increase during the OSCC initiation.	414
Immune cells	colon	128a	The cytotoxic immune response is more significant in serrated polyps compared to conventional adenomas, and the distinction persists in advanced tumors.	262
	colon	72a	The relative abundance of plasma cells, B cells, CD8 + T cells, CD4 + T cells, Treg cells, γδ T cells and NK cells decrease, and that of macrophages increase from normal tissue to carcinoma.	279
	colon	81a	The number of Tregs increases from polyps to carcinoma; Exhausted T cells only appear in CRC.	261
	stomach	43	The number of IgA+ plasma cells increases in chronic atrophic gastritis and intestinal metaplasia, and declines in GAC; Myeloid cells transition from immune-activating to immune-suppressive properties as they progress from intestinal metaplasia to GAC.	497
	lung	62	AIS lesions that can regress to normal have more infiltrating immune cells, while progressive lesions have developed immune escape mechanisms in precancerous stages.	281
	lung	53	Immunosuppression initiates at the preneoplastic stage, characterized by a reduction in T cell infiltration and clonality, and an increase in Tregs.	280
	lung	41	T cell infiltration is associated with mutation-induced neoantigens, alongside PD-L1 upregulation in T cells from AAH to ADC.	415
	breast	35a	DCISs have active immune responses while transforming to suppressive one in IDCs.	498
	bone marrow	65	Mature B cells, NK cells, and CD14+ monocytes decrease and CD16+ monocytes, tumor-associated macrophages increase from normal to smoldering MM.	452
	bone marrow	32	The number of CD8 + T cells decreases in smoldering MM and MM compared with healthy and MGUS.	499
	bone marrow	14	The proportion of T cells decreases from smoldering MM to MM, while monocytes contribute to the largest proportion in primary MM.	500
	pancreas	30	Myeloid cells and CD4 + T cells are more enriched in ADM and PanIN compared to normal pancreas.	501
	esophagus	29	Changes in immune cells are late events in ESCC tumorigenesis, and the proportion of CD8 + T cells is similar from normal to LGIN and HGIN while increasing in ESCC.	307
	esophagus	19	Macrophages increase from esophageal squamous precancerous lesions to ESCC but M2 macrophage cells and Treg cells decrease in ESCC compared to the precancerous lesions stages.	502
Fibroblast /CAFs	colon	81a	Pre-CAFs are enriched in polyps and express RUNX1 to epigenetically regulate CAF programs.	261
	colon	72a	The relative abundance of fibroblasts increases from normal tissue to carcinoma.	279
	breast	79	Normal fibroblasts transit to CAFs from primary DCIS to later invasive breast cancer.	366
	stomach	43	myCAFs are dominant in intestinal metaplasia and enriched in SDC2 expression‎, which is associated with aggressive progression and poor prognosis.	497
	stomach	5	The PDGFRα+ fibroblasts expand in metaplasia and cancer compared with normal.	503
	stomach	24	CAFs occur from the precancerous state, and the number increases in the malignancy; The iCAFs exhibits pro-stemness property and may promote diffuse type GAC tumorigenesis.	496
	pancreas	30	PanINs are surrounded by fibroblasts and the diversity of fibroblasts gradually increase from normal to tumors.	501
	pancreas	6	Fibroblasts activation and myCAF subtype transformation occur from LGD-IPMNs to HGD-IPMNs.	504
	esophagus	29	CAFs are activated in the HGIN and ESCC, rather than in normal and LGIN tissues.	307
	skin	14	Fibroblasts have not yet been fully activated in actinic keratosis; myCAFs increased more than iCAFs in tumorigenesis	505
	oral cavity	9	CAFs increase from normal epithelium to dysplasia and tumors, among which myCAFs are the dominant subtype.	414
Micro-organisms	colon	616	Fusobacterium nucleatum spp. elevates continually from intramucosal carcinoma to advanced tumors. Atopobium parvulum and Actinomyces odontolyticus only increase from LGD to intramucosal carcinoma. Fecal metabolites, including BCAAs and phenylalanine, increase from intramucosal carcinoma, while bile acids increase from LGD.	201
	colon	431	A panel of gut microbiome-associated serum metabolites alters from normal, through adenoma to CRC.	202
	colon	386	Integration of fecal metabolites and microbiome analysis can distinguish CRCs with adenomas and normal tissues.	203

1. SSL sessile serrated lesion, CRC colorectal cancer, BE barrett’s esophagus, EAC esophageal adenocarcinoma, LGIN low grade intraepithelial neoplasia, HGIN high grade intraepithelial neoplasia, ESCC esophageal squamous cell carcinoma, Pre-LSCs pre-leukemic stem cells, HSCs hematopoietic stem cells, LGD low-grade dysplasia, HGD high-grade dysplasia, AT2 alveolar type 2 cell, sAML secondary acute myeloid leukemia, IPMNs intraductal papillary mucinous neoplasms, OSCC oral squamous cell carcinoma, OLK oral leukoplakia, PanIN pancreatic intraepithelial neoplasia, CAFs cancer-associated fibroblasts, MDSCs myeloid-derived suppressor cells, AAH atypical adenomatous hyperplasia, AIS adenocarcinoma in situ, ADC adenocarcinoma, IPNs indeterminate pulmonary nodules, DCIS ductal carcinoma in situ, IDCs invasive ductal carcinomas, SDC2 Syndecan 2, GAC gastric adenocarcinoma, MM multiple myeloma, MGUS monoclonal gammopathy of undetermined significance, NK natural killer, ADM acinar-to-ductal metaplasia, BCAAs branched-chain amino acids, ECM extracellular matrix, Treg regulatory T cells, myCAF myofibroblastic CAF, LUAD lung adenocarcinoma, IGC intestinal-type gastric cancer, EMT epithelial-mesenchymal transition, iCAF inflammatory CAF
2. aNumbers of samples, the others are number of individuals

→ Figure 5: Cell competition — mutant clones가 wild-type 제거 (e.g., Apc-mutant Notum secretion).

조직 간 세포 경쟁 (Cell competition across tissues)

a 단순 장 상피(simple intestinal epithelium)에서는 살아있는 세포가 세포 간 신호 전달(intercellular communications)과 세포골격 재배열(cytoskeleton rearrangement)을 통해 상피 밖으로 밀려나게 됩니다(extruded).

b 장 줄기세포(intestinal stem cells)는 장와(crypt) 바닥에 위치한 줄기세포 틈새(stem cell niche) 내에서 우위를 놓고 경쟁합니다. 돌연변이를 가진 슈퍼경쟁자(supercompetitors) 는 줄기세포성(stemness)을 유지하고, 정상형(wild-type) 세포를 대체하며, ISC 틈새를 점령한 뒤 전체 장와(crypt)를 장악할 가능성이 더 높습니다. 밀려난 정상형 세포(“losers”)는 분화(differentiate)되어 장와를 따라 위쪽으로 이동한 뒤 결국 상단에서 탈락(shed)됩니다.
줄기세포의 운명은 슈퍼경쟁자로부터 직접 분비되는 신호(secretory signals)와, 슈퍼경쟁자에 의해 자극받은 주변 기질 세포(stromal cells)로부터 간접적으로 오는 신호에 의해 조절됩니다. 줄기세포성을 억제하는 신호(stemness inhibitory signals) — 예: BMP 활성제(BMP activators)와 NOTUM — 는 정상형 세포와 슈퍼경쟁자에 차별적으로 작용합니다. 정상형 세포의 줄기세포성 유지 능력을 방해하지만, 슈퍼경쟁자에는 상대적으로 영향이 적습니다.

c 층상 상피(stratified epithelium)에서도 줄기세포 경쟁의 결과는 세포 운명 결정(cell fate decisions)에 의해 조절됩니다. 그러나 장와와 같은 특정 미세구조에 국한되지 않고, 승리 클론(winner clone)은 더 넓은 영역으로 확장될 잠재력을 가집니다.

약어

• WT: wild-type cells (정상형 세포)
• BMP: bone morphogenic protein (골형성단백질)
• ISCs: intestinal stem cells (장 줄기세포)

→ Figure 6: Micro-environment interactions — immune (TAMs IL-1β feedback), fibroblasts → CAFs, ECM → YAP/TAZ.

변형된 세포와 미세환경 구성 요소 간 상호작용
(Interactions of transformed cell and microenvironmental components)

a 변형된 세포(transformed cells)에서 발생하는 비정상적인 유전적·후성유전적·전사적 신호(abnormal genetic, epigenetic and transcriptional signals)는 역설적으로 면역 활성화(immune activation)를 유도하면서 동시에 면역 회피(immune evasion) 전략을 개발합니다. 이들 간의 crosstalk(교차 작용)은 주로 직접 세포-세포 상호작용 신호(direct cell-cell interaction signals) 와 파라크린 신호(paracrine signals) — 예: 케모카인(chemokines), 사이토카인(cytokines), 성장인자(growth factors) — 를 통해 매개됩니다.
긍정적 피드백(positive feedback)으로서, 종양 지지 면역세포(tumor supportive immune cells) — 예: TAMs(tumor-associated macrophages) — 는 IL-1β 신호 등을 생산하여 악성 진화(malignant evolution)를 더욱 촉진합니다.

b 변형된 세포는 환경 스트레스(environmental stress)와 유전자 변화(genetic alterations)와 함께 분비 신호(secretory signals) 및 접촉 신호(contact signals)를 통해 섬유아세포(fibroblasts)를 활성화시켜, 다양한 종양 촉진 특성(tumor-promoting properties)을 가진 암 관련 섬유아세포(CAFs: cancer-associated fibroblasts) 로 전환시킵니다.
반대로 CAFs는 줄기세포성 신호(stemness signals)를 분비하여 세포 경쟁(cell competition) 과정에서 돌연변이 세포와 정상형 세포(wild-type cells)를 차별적으로 조절합니다.

c 환경 신호(environmental signals)는 세포외기질(ECM: extracellular matrix)의 재구성(remodeling)을 유도하며, ECM 접착(adhesion) 상실을 보이는 단일 변형 세포는 자신의 생존을 지원하기 위해 ECM을 생산할 수도 있습니다.
반대로 병리적 조건(염증, 노화, 상처 치유, 제2형 당뇨병(T2DM) 등)에서 발생하는 비정상적인 기계적 신호(abnormal mechanical signals) — 예: 경직도(stiffness), 점탄성(viscoelasticity) — 는 YAP/TAZ 경로 활성화를 통해 돌연변이 세포의 악성 진행(malignant progression)을 촉진합니다. 이 pro-tumorigenic 효과는 RTK-Ras 경로 돌연변이에 의해 더욱 악화됩니다.
또한 경직된 ECM은 filamin이 핵주변(perinuclear areas)에서 정상형 세포와 돌연변이 세포의 경계면(interface)으로 이동하는 것을 억제하여, 돌연변이 세포의 밀려남(extrusion)을 더욱 억제합니다.

약어 목록

• TAMs: tumor-associated macrophages (종양 관련 대식세포)
• MDSC: myeloid-derived suppressor cell (골수 유래 억제 세포)
• cGAS: cyclic GMP-AMP synthase (고리형 GMP-AMP 합성효소)
• STING: stimulator of interferon genes (인터페론 유전자 자극인자)
• IRF3: IFN regulatory factor 3 (인터페론 조절 인자 3)
• T2DM: type 2 diabetes mellitus (제2형 당뇨병)
• YAP: yes-associated protein (예-연관 단백질)
• TAZ: transcriptional co-activator with PDZ-binding motif (PDZ 결합 모티프를 가진 전사 공활성제)
• LPAR4: lysophosphatidic acid receptor 4 (리소포스파티딕산 수용체 4)

→
이 설명은
변형된 세포(transformed cell)가
미세환경(microenvironment)과 어떻게 양방향 상호작용을 통해
악성 진화를 가속화하는지를 보여줍니다.

특히
염증, ECM 경직, 면역세포(TAMs), 섬유아세포(CAFs)가
핵심 mediator로 작용하며,
Furman et al. (2019)의 SCI(전신 만성 염증)가
이러한 상호작용의 "촉매" 역할을 한다는 점과 강하게 연결됩니다.

3. Furman et al. (2019)과의 직접적 연계 & SCI 지지

• 직접 인용 (Furman D. et al., Nat Med 2019): "chronic inflammation in the etiology of disease across the life span"을 ref. 130으로 언급.
• SCI가 암 etiology의 핵심으로 작용: unresolved persistent damage → pro-tumorigenic microenvironment remodeling.
• Inflammaging (age-related SCI) → immunosenescence + SASP → clonal fitness ↑ + surveillance ↓ → cancer risk ↑.
• Metaflammation (metabolic inflammation in obesity/diabetes) → hyperglycemia → cGAS-STING → tumorigenesis 촉진 (Furman의 대사염증 연장).
• modifiable factors (식이, microbiome, aging-related exposures)가 SCI를 통해 암 initiation driver로 작용 — Furman의 common soil hypothesis를 암 특화.

4. 임상·연구 함의 & Interventional Targets

• Risk prediction: Normal tissue somatic burden (TP53 clones), epigenetic signatures (methylation panels), multi-omics (scRNA-seq + spatial)으로 high-risk identification.
• Interception strategies:
  • Chemoprevention: Aspirin (CRC ↓), metformin (metabolism modulation), statins (microbiota/immunity), canakinumab (IL-1β blockade → lung cancer ↓).
  • Immunoprevention: HPV/HBV vaccine, tumor antigen vaccine (HER2, MUC1).
  • Lifestyle: Mediterranean diet, fiber (microbiota modulation → inflammation ↓), NAD+ precursors.
• 미래 방향: Premalignant longitudinal sampling, animal models (organoids), single-cell/spatial omics로 co-evolution mapping → targeted interception.

발표 팁 (대학원 수준)

• 강조 포인트: “Furman 2019의 SCI가 암 initiation의 'enabling hallmark'로 작용 — 만성 염증이 oncogenic mutation의 잠재력을 release하고 microenvironment를 pro-tumorigenic으로 재구성한다.”
• Figure 활용: Fig. 1 (multistage overview), Fig. 3 (driver convergence — inflammation 중심), Fig. 6 (microenvironment remodeling) 순서로 설명.
• 비판적 관점: 주로 observational·animal data 기반, 인간 premalignant intervention RCT 부족. 그러나 aspirin/metformin 등 기존 약물 repurposing으로 translational potential 높음. Furman 인용으로 SCI-cancer link의 최신 증거 강화.

이 논문은 Furman 2019의 SCI 프레임워크를 암 발생 초기에 특화·확장한 2024년 최신 Nature 리뷰로, inflammaging·metaflammation이 cancer의 unifying driver임을 강력히 뒷받침

Table 1 Dynamics of genome evolution in tumorigenesis

From: Tumor initiation and early tumorigenesis: molecular mechanisms and interventional targets

TissueNormalPrecancerousTumorEvolutionRefs

Blood	DNMT3A, TET2, TP53, CUX1, ASXL1, SRSF2, EZH2, SF3B1, JAK2, BCORL1	MDS: SF3B1, TET2, SRSF2, ASXL1, DNMT3A, RUNX1 MDS: ASXL1, DNMT3A, BCOR, SRSF2, U2AF1, TET2 MF: TP53	CMML: TET2, SRSF2, ASXL1, KRAS, ZRSR2, CBL, RUNX1 AML: SFRS2, TET2, DNMT3A, AXSL1, TP53, BCORL, RUNX1, BCORL1	DNMT3A and PPM1D mutations are more frequent in CHIP than in myeloid malignancies; TP53 mutation increases from MF to AML.	104,135,348,355,457,458
Bladder	KMT2D, KDM6A, ARID1A, RBM10, EP300, STAG2, NOTCH2, CDKN2A	Not reported	TP53, MLL2, ARID1A, KDM6A, CDKN2A, PIK3CA, YWHAZ, NCOR1	TP53 mutations are preval‎ent in cancer but are rare in normal urothelium.	39,459
Bronchus	TP53, NOTCH1, FAT1, ARID1A, ARID2, CHEK2, PTEN, IDH1, CREBBP, EP300	ADH: KRAS, BRAF, ERBB2, PDGFRA, HERC2, PDGFRA, ARID1A	ADC: EGFR, RBM10, TP53, KRAS, LRP1B, STK11, BRAF, HERC2	Not reported	162,460
Colon	NFKBIZ, ARID1A, PIGR, ERBB3, ERBB2, AXIN2, FBXW7, PIK3CA, STAG2	UC dysplasia: TP53, RNF43, BRAF, APC, KRTAP4, CTNNB1, KRAS SSL: BRAF, KRTAP4, MK167 AD: APC, MK167, APOB, KRAS, LRP1B, FAT2	TP53, KRAS, APC, PIK3CA, SMAD4, FBXW4, SOX9, RNF43, ARID1B	The frequency of TP53 mutations increases during CRC tumorigenesis. APC mutations occur from the precancerous state and are not detected in normal colons	117,137,334,347
Esophagus	NOTCH1, TP53, FAT1, NOTCH2, PPM1D, ZFP36L2, NOTCH3, CHEK2	Squamous dysplasia: TP53, NOTCH1, ZFHX4, CDKN2A, FAT1, ZNF750 BE:TP53, CDKN2A, KDM6A, ARID1A, ARID1B, APC, ERBB2	ESCC: TP53, NOTCH1, KDM6A, KMT2D, LRP1B, ZNF750, CDKN2A, SMAD4, CCSER1, SOX2, BCL6, CCDN1 EAC: TP53, CDKN2A, ARID1A, EYS, SYNE1, ABCB1	ESCC: The Frequency of TP53 mutations increases and NOTCH1 mutations decreases during ESCC tumorigenesis. EAC: The frequencies of TP53 and ERBB2 mutations increase from BE to EAC.	54,59,461,462,463,464,465
Endometrium	ERBB2, ERBB3, PIK3CA, ARHGAP35, PIK3R1, KRAS, FBXW7, PPP2R1A, ZFHX3, FOXA2	AEH: PTEN, ARID1A, PIK3CA, CHD4, CTNNB1	PTEN, TP53, PIK3CA, CTNNB1, KRAS, CTCF, ARID1A, PIK3R1, FBXW7, ARHGAP35	ERBB2 and ERBB3 mutations are positively selected in normal epithelium, but not in cancer.	466,467,468,469,470
Liver	ALB, ACVR2A, HMCN1, ARID2, APC, ESRRG, ARID1A	ARLD: JACK1, ARID2, RP1L1, TERT, CDH9 Cirrhosis: PKD1, PPARGC1B, KMT2D, ARID1A, STARD9, APOB, ALMS1, ALB, TP53	TERTp, TP53, CTNNB1, ARID1A, ARID2, RPS6KA3, NFE2L2, KRAS, PIK3CA, AXIN1, CDKN2A	Mutations of TERTp mutation occur early in dysplastic nodules and are not detected in healthy or cirrhotic livers.	136,332,471,472,473
Skin (squamous cell)	NOTCH1, FAT1, NOTCH2, TP53, NOTCH3, RBM10, KMT2D	AKs: TP53, NOTCH1, FAT1, KIF24, HMCN1, KMT2C, PIK3CA	TP53, CDKN2A, KMT2D, FAT1, NOTCH1, NOTCH2	NOTCH1 and FAT1 mutations are more common in normal tissues than in tumors.	52,474,475,476
Skin (melanocyte)	BRAF, CBL, MAP2K1, NF1, RASA2, ARID2	TERTp, BRAF, NRAS, GNA11, HRAS, CDKN2A, RB1, PPP6C, MAP2K1	BRAF, TERTp, NRAS, CDKN2A, PTEN, ARID1B, ARID1A	BRAFV600E occurs from nevi to tumors. The frequency of CDKN2A mutation increases from intermediate lesions to melanoma.	57,477
Breast	PIK3CA, PIK3R1, TP53, PTEN	BBL with atypia: PIK3CA, GATA3, PTEN, RUNX1 MAP3K1, CBFB BBL without atypia: PIK3CA DCIS: CDH1	Ductal carcinoma: PIK3CA, GATA3, PTEN, AKT1, CBFB, ATRX, NOTCH2	PIK3CA mutations are found in both normal and proliferative lesions, although they are less common in normal lobules.	454

Table 4 Molecular markers for cancer risk prediction

From: Tumor initiation and early tumorigenesis: molecular mechanisms and interventional targets

ClassPredictive markersUsageRefs

Colon
Protein	Hemoglobin, fecal calprotectin	Diagnose high-risk adenoma and polyp	506
Genetic mutations	20 genes that differentiate conventional adenoma and non-hypermutated CRC	Distinguish adenoma and CRC	347
Bacteria	Fusobacterium nucleatum, hemoglobin	Detect advanced adenoma	507
Metabolites	Benzoic acid	Discriminate healthy controls and patients with adenoma	508
Metabolite	8 gut microbiome-associated serum metabolites altering in both CRC and adenoma	Discriminate CRC and adenoma from normal samples	202
Circulating immune cells	Relative FOXP3+ regulatory T cell counts	Predict risk of CRC	509
Esophagus
Copy number variations	Gains of 1q, 8q, 9q, 12p, 20q, losses on 9p and 17p, aneuploidy and tetraploidy	Predict neoplastic progression in BE	351,352,353,354
Methylated DNA	CDKN2A, RUNX3, HPP1, NELL1, TAC1, SST, AKAP12, CDH13 methylation	Predict neoplastic progression in BE	362
Methylated DNA	UP10, UP35-1, CG6522, YPEL3, POU3F1 methylation	Discriminate NDBE from HGD and EAC	363
Methylated DNA	TFAP2E, OTX1, OPLAH, CHAD, MARCH11, GALR1, HOXA9 methylation	Predict ESCC risk	510
Blood
Genetic mutations	DNMT3A, TET2 mutations, clinical characteristics (e.g. age, sex)	Predict risk of AML	355
Genetic mutations	CHIP gene mutations, variant allele frequency, clinical characteristics (e.g. age, sex)	Predict risk of AML, MDS and MPN	350
Breast
TME	Coordinated spatial and functional changes in the TME of ductal carcinoma in situ	Predict risk of invasive progression from ductal carcinoma in situ	366
Genetic mutations	Loss-of-function variants in predisposition genes of breast cancer, polygenic risk score, clinical characteristics (e.g. family history)	Predict 2-year breast cancer risk	511
Metabolites	Histidine, N-acetyl glycoproteins, glycerol, ethanol	Predict breast cancer risk	512
Circulating immune cells	Relative CD8+ T cell counts	Predict breast cancer risk	509
Lung
Protein	Pro-SFTPB, clinical characteristics (e.g. age, sex)	Predict lung cancer risk	513
Protein	CA125, CEA, CYFRA 21-1, pro-SFTPB, clinical characteristics (age, smoking)	Predict lone year lung cancer risk	514
Metabolite	Cystine, valine, asparagine, 3-chlorotyrosine, 12:0-carnitine, glutamate, phosphocholine	Distinguish invasive adenocarcinoma and its precursors with benign diseases	173
Gene expression‎	Genes in the airway field associated with premalignant lesions, such as TOMM22 and COX4I1	Predict risk of lung premalignant lesion	515
Circulating immune cells	Relative counts of FOXP3+ regulatory T cells	Predict lung cancer risk	509
Stomach
Serum	H. pylori IgG, pepsinogen I, pepsinogen I/II ratio, gastrin-17	Diagnose gastric precancerous lesions	516,517
Protein	APOA1BP, PGC, HPX, DDT, H. pylori infection, clinical characteristics (e.g. age, sex)	Predict gastric lesion progression	518
Methylated DNA	TFAP2E, OTX1, OPLAH, CHAD, MARCH11, GALR1, HOXA9 methylation	Predict gastric cancer	510
Cervix
Methylated DNA	MAL and miR-124-2 gene methylation	Detect cervical intraepithelial neoplasia	519
Methylated DNA	Number of hypervariable CpGs and hypermethylated CpGs associated with age	Predict risk of cervical neoplasia, detect pre-invasive neoplasia and cervical cancer	520
Serum	HPV DNA/mRNA, E6 oncoprotein, HPV genotyping, p16/Ki-67, clinical characteristics (e.g. age, cytology)	Predict risk of CIN2+	521
Pancreas
Serum	IP-10, IL-6, PDGF, CA19-9	Discriminate pancreatic cancer from benign pancreatic disease	522

Table 5 Potential agents for cancer prevention

From: Tumor initiation and early tumorigenesis: molecular mechanisms and interventional targets

AgentTargetMechanismsIndicationClinical evidence

Hormonotherapy
Tamoxifen	Estrogen receptor inhibitor	Counteract estrogen effects and inhibit cell proliferation	Breast cancer	FDA approved523
Raloxifene	Estrogen receptor inhibitor	Counteract estrogen effects and inhibit cell proliferation	Breast cancer	FDA approved523
Anastrozole	Aromatase inhibitor	Inhibit the enzyme aromatase to reduce estrogen levels	Breast cancer	USPSTF recommend368,369
Exemestane	Aromatase inhibitor	Inhibit the enzyme aromatase to reduce estrogen levels	Breast cancer	USPSTF recommend368,369
Dutasteride	5α-reductase	Inhibit the conversion of testosterone to dihydrotestosterone	Prostate cancer	RCT372
Finasteride	5α-reductase	Inhibit the conversion of testosterone to dihydrotestosterone	Prostate cancer	RCT371
Anti-inflammation
Aspirin	COX1/COX2	Inhibit prostaglandin synthesis, platelet activation, Wnt-β-catenin signaling, and inflammation	Colorectal cancer	USPSTF recommend524
			Barrett’s esophagus	RCT525
Sulindac	COX1/COX2		Colorectal polyps	RCT526
Celecoxib	COX2		Colorectal adenomas	RCT527
Small molecular inhibitor
Ruxolitinib	JAK1/JAK2	Decrease STAT3 phosphorylation and induce cell apoptosis	Premalignant breast disease	Ongoing RCT (NCT02928978)
Erlotinib	EGFR	Induce growth inhibition, apoptosis and cell cycle arrest	Liver cancer	RCT (NCT02273362)
Metabolic agents
Metformin	Mitochondrial complex I, MAPK and mTOR to modulate energy metabolism	Activate AMPK, which inhibits the mTOR pathway and reduces cyclin D1 expression‎ and RB phosphorylation	Colorectal adenomas	RCT383
			Oral cancer	Ongoing RCT (NCT02581137, NCT05536037, NCT05237960)
			Lung cancer	Ongoing RCT (NCT04931017)
			Multiple myeloma	Ongoing RCT (NCT04850846)
Atorvastatin	HMG-CoA reductase	Disrupt the meval‎onate pathway with downstream effects on membrane integrity, cell signaling, protein synthesis, and cell cycle progression	Colon cancer	Ongoing RCT (NCT04767984)
Immunotherapy
HPV vaccine	HPV	Elicit both T-cell and antibody responses to HPV infected cells	Cervical intraepithelial neoplasia	FDA approved523
HBV vaccine	HBV	Inhibit the replication of HBV and promote its clearance	Primary liver cancer	RCT528
MUC1 vaccine	MUC1	Target aberrantly post-translationally modified antigen MUC1 in various cancer cells	Colorectal adenomas	RCT407
			Lung cancer	Ongoing RCT (NCT03300817)
			Ductal carcinoma in situ	Ongoing RCT (NCT06218303)
KRAS vaccine	KRAS	Target driver mutation	Pancreatic cancer	Ongoing RCT (NCT05013216)
EGFR vaccine	EGFR	Target driver mutation	Lung cancer	Ongoing RCT (NCT04298606)
HER-2/neu vaccine	HER-2/neu	Target driver mutation	Ductal carcinoma in situ	Clinical Trial405
Nivolumab	PD-1	Block the binding of PD-1 to PD-L1 and enhance the role of T cells in recognizing and killing tumor cells	Oral leukoplakia	RCT416
			Squamous dysplasia	Ongoing RCT (NCT03347838)
			Melanoma	Ongoing RCT (NCT04099251)
Pembrolizumab	PD-1		Oral Leukoplakia	Ongoing RCT (NCT03603223)
			Cervical intraepithelial neoplasia	Ongoing RCT (NCT04712851)
Calcipotriol+ 5-fluorouracil	TSLP	Induce TSLP expression‎ and CD4+ T cell immune response	Skin cancer	RCT411
Micronutrient supplement
Vitamin A	NA	Combine with retinol-binding protein to inhibit cell growth, induce differentiation, regulate cell-cycle-mediated stem cell plasticity	Skin cancer	RCDCIST529
Nicotinamide	NA	Boost cellular energy, enhance DNA repair and reduce level of immunosuppression	Nonmelanoma skin cancers, AK	RCT445
			Nonmelanoma skin cancer	RCT530
			Keratinocyte Carcinoma	Ongoing RCT (NCT05955924)
Vitamin D	NA	Bind to vitamin D receptors located in cell nuclei to inhibit proliferation and angiogenesis and induce differentiation and apoptosis	Colorectal adenomas	RCT531
			Hepatocellular carcinoma	Ongoing RCT (NCT02779465)
Calcium	NA	Bile acid-binding capacity, direct effect on calcium-sensing receptors on colonocytes	Colorectal adenomas	RCT532
Vitamin D+ calcium	NA	Reduce cell proliferation, induce differentiation and apoptosis, downregulate inflammatory mechanisms and regulate immune response	Breast cancer	RCT448
Folic acid	NA	Affect DNA replication, repair, and methylation through the one-carbon metabolic pathway	Colorectal adenomas	RCT449
			Cervical cancer	RCT (NCT00703196)
Long-chain omega-3 (PUFA)	Components of phospholipids that form cell membranes	Have potent anti-inflammatory effects, reduce cell proliferation and increase apoptosis	Colorectal adenomas; serrated polyps	RCT441
			DCIS; ADH	RCT (NCT00627276)
Selenium	NA	Depress carcinogen bioactivation, cell proliferation and cell cycling, increase apoptosis	Colorectal adenomas	RCT533
Squaric acid dibutylester	NA	Trigger innate immunity	Melanoma	Ongoing RCT (NCT04999631)
Berberine	NA	Change the structure of the microbiota, modulate the TME and block the activation of tumorigenesis-related pathways	Colorectal adenomas	RCT442

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Tumor initiation and early tumorigenesis: molecular mechanisms and interventional targets

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• Review Article
• Open access
• Published: 19 June 2024

Tumor initiation and early tumorigenesis: molecular mechanisms and interventional targets

• Shaosen Zhang, 
• Xinyi Xiao, 
• Yonglin Yi, 
• Xinyu Wang, 
• Lingxuan Zhu, 
• Yanrong Shen, 
• Dongxin Lin & 
• Chen Wu 

Signal Transduction and Targeted Therapy volume 9, Article number: 149 (2024) Cite this article

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Abstract

Tumorigenesis is a multistep process, with oncogenic mutations in a normal cell conferring clonal advantage as the initial event. However, despite pervasive somatic mutations and clonal expansion in normal tissues, their transformation into cancer remains a rare event, indicating the presence of additional driver events for progression to an irreversible, highly heterogeneous, and invasive lesion. Recently, researchers are emphasizing the mechanisms of environmental tumor risk factors and epigenetic alterations that are profoundly influencing early clonal expansion and malignant evolution, independently of inducing mutations. Additionally, clonal evolution in tumorigenesis reflects a multifaceted interplay between cell-intrinsic identities and various cell-extrinsic factors that exert selective pressures to either restrain uncontrolled proliferation or allow specific clones to progress into tumors. However, the mechanisms by which driver events induce both intrinsic cellular competency and remodel environmental stress to facilitate malignant transformation are not fully understood. In this review, we summarize the genetic, epigenetic, and external driver events, and their effects on the co-evolution of the transformed cells and their ecosystem during tumor initiation and early malignant evolution. A deeper understanding of the earliest molecular events holds promise for translational applications, predicting individuals at high-risk of tumor and developing strategies to intercept malignant transformation.

초록

종양 형성은 다단계 과정으로,

정상 세포에서 발생한 종양유발 돌연변이가

클론 우위를 부여하는 것이 초기 사건이다.

그러나

정상 조직에서 광범위한 체세포 돌연변이와 클론 확장이 흔히 관찰됨에도 불구하고,

이들이 암으로 전환되는 것은 매우 드문 사건으로 남아 있어,

돌이킬 수 없고 고도로 이질적이며 침윤성 병변으로 진행하기 위한

추가적인 driver 사건의 존재를 시사한다.

최근 연구자들은

환경성 종양 위험 인자와 후성유전학적 변화가 돌연변이 유발과 독립적으로

초기 클론 확장과 악성 진화를 강력하게 영향을 미친다는 점을 강조하고 있다.

또한, 종양 형성 과정에서의 클론 진화는

세포 내적 정체성(cell-intrinsic identity)과

다양한 세포 외적 요인(cell-extrinsic factor) 간의 복합적인 상호작용을 반영하며,

이러한 요인들은 선택 압력을 가하여 비제어적 증식을 억제하거나

특정 클론이 종양으로 진행될 수 있도록 허용한다.

그러나 이러한 driver 사건들이

세포 내적 역량(intrinsic cellular competency)을 유도하는 동시에

환경 스트레스를 재구성하여 악성 전환을 촉진하는 메커니즘은 아직 완전히 밝혀지지 않았다.

본 리뷰에서는

종양 발생과 초기 악성 진화 동안 변형된 세포와

그 미세환경 생태계의 공진화(co-evolution)에 영향을 미치는

유전적·후성유전적·외부적 driver 사건들을 정리하고,

이들의 역할을 요약한다.

종양 형성 초기의 분자적 사건들을 더 깊이 이해하는 것은

고위험군 개인을 예측하고 악성 전환을 차단하는 전략을 개발하는 등

번역적 응용(translational application)에 큰 가능성을 제시한다.

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Introduction

It is generally believed that tumorigenesis is a multi-stage process, wherein the initial step is the occurrence of an oncogenic mutation in a single somatic cell. The mutation endows cells with clonal advantages, allowing the mutant clone to expand and accumulate additional genetic and epigenetic alterations, ultimately resulting in an irreversible, highly heterogeneous, and invasive lesion1 (Fig. 1). Mutations that confer growth competitiveness and promote cancer evolution are referred to as cancer driver mutations. Identifying driver mutations and revealing their roles in tumors represent key areas of focus in cancer genome research. Recent advancements in sampling and sequencing technologies facilitate the detection of somatic mutations and clonal expansion in normal tissues. It is surprising that even though driver mutations harbored by positively selected clones overlap to a great extent with cancer driver mutations and are pervasive in morphologically normal tissues, only a low annual incidence rate of cancer is diagnosed in populations. It is suggested that mutations alone are insufficient for tumor formation, and other prerequisite molecular events need to be identified. Additionally, humans have evolved various strategies to maintain homeostasis and defend oncogenic transformation. However, environmental insults and aging often disrupt the balance and increase the risk of cancer formation.2,3 Although the mechanisms of these risk factors contributing to cancer progression have been widely explored, how they are involved in early tumorigenesis and interact with specific oncogenic mutations are still not completely understood. The non-genetic effects of external signaling may explain the paradox of genetic mutation and tumorigenesis. Epigenetic rewiring can serve as another impetus to release uncontrollable growth and survival potential.

서론

종양 형성은 일반적으로 다단계 과정으로 여겨지며,

그 초기 단계는 단일 체세포에서

종양유발 돌연변이(oncogenic mutation)가 발생하는 것이다.

이 돌연변이는

세포에 클론 우위(clonal advantage)를 부여하여

돌연변이 클론이 확장되고 추가적인 유전적·후성유전적 변화를 축적하게 하며,

궁극적으로 돌이킬 수 없고 고도로 이질적이며 침윤성 병변으로 이어진다¹ (Fig. 1).

돌연변이를 가진 클론을 유전자 수준에서 설명하면 다음과 같습니다.
1. 기본 개념: 클론(clone)이란?

• 한 세포에서 시작해서 모든 자손 세포들이 동일한 유전적 배경을 공유하는 세포 집단을 클론이라고 합니다.
• 정상 조직에서도 세포 분열 시 미세한 체세포 돌연변이(somatic mutation)가 생길 수 있지만, 대부분은 중립적이거나 해롭지 않습니다.
• 그러나 특정 driver mutation(암 driver 돌연변이)이 생기면 그 세포와 그 자손들은 유전자 수준에서 동일한 변이를 가지게 되고, 이 집단 전체를 mutant clone(돌연변이 클론)이라고 부릅니다.

2. 유전자 수준에서 돌연변이 클론의 구성

• Trunk mutation (줄기 돌연변이 / clonal mutation): 클론의 공통 조상 세포에서 처음 생긴 driver mutation. → 이 클론에 속하는 모든 세포가 100% 이 돌연변이를 가지고 있습니다. (heterozygous 또는 homozygous 상태로 고정) 예: KRAS G12D, TP53 R175H, EGFR exon 19 deletion 등. 이 변이는 클론의 유전적 지문(fingerprint) 역할을 합니다.
• Subclonal mutation (하위 클론 돌연변이): 나중에 trunk mutation을 가진 세포 중 일부가 추가로 얻은 새로운 돌연변이. → 클론 전체가 아니라 일부 하위 집단(subclone)만 가지고 있습니다. → 종양이 커지면서 branched(가지치기) 진화가 일어나 여러 subclonal population이 생깁니다.
• Passenger mutation: driver와 무관하게 우연히 쌓인 중립적/무해한 변이들. 그러나 clonal evolution 과정에서 함께 확장되므로, 클론 식별의 marker로도 쓰입니다.

3. 유전자 수준에서 클론이 확장되는 과정 (clonal expansion)

1. 정상 세포 → driver mutation 1개 발생 (예: APC loss in 대장암 초기) → 이 세포와 자손들이 동일한 변이를 공유 → founding clone (창시 클론) 형성
2. 이 클론이 주변 정상 세포보다 생존·증식 우위 → clonal sweep (선택적 확장) → 대부분의 세포가 이 동일한 driver mutation을 가진 상태로 대체됨
3. 추가 driver mutation 발생 → 새로운 subclone 탄생 예: founding clone (KRAS mutant) → 일부 세포에서 TP53 mutation 추가 → TP53+KRAS double mutant subclone → 이 subclone이 다시 확장되면서 종양이 더 악성으로 진화

4. 시각적·유전자 수준 비유

• 트리 구조로 보면:
  • 뿌리(trunk): founding driver mutation (모든 세포 공유)
  • 가지(branches): subclonal driver mutations (각 가지마다 다른 추가 변이)
  • 잎사귀: 개별 세포 수준의 ultra-rare or passenger mutations
• DNA 시퀀싱으로 보면:
  • Variant allele frequency (VAF)가 높은 변이 → clonal (트렁크)
  • VAF가 낮은 변이 → subclonal (가지)

요약 한 줄

돌연변이를 가진 클론이란,
특정 driver mutation(또는 여러 개의 공통 변이)을
유전자 수준에서 모든 세포가 동일하게 공유하는 세포 집단으로,
이 변이 덕분에 정상 세포를 밀어내고 점점 커지는(expand) 세포 무리입니다.

이 과정이 반복되면서
종양은 유전적으로 점점 더 복잡하고 이질적인 구조(heterogeneous clonal architecture)를
가지게 됩니다.

성장 경쟁력을 부여하고 암 진화를 촉진하는 돌연변이를

암 driver 돌연변이(cancer driver mutations)라고 부른다.

driver 돌연변이를 식별하고

종양 내에서의 역할을 밝히는 것은 암 게놈 연구의 핵심 분야이다.

최근 샘플링 및 시퀀싱 기술의 발전으로

정상 조직에서 체세포 돌연변이와 클론 확장을 검출할 수 있게 되었다.

긍정적으로 선택된 클론이 보유한 driver 돌연변이가

암 driver 돌연변이와 상당 부분 겹치며,

형태적으로 정상적인 조직에서도 광범위하게 존재함에도 불구하고,

인구에서 진단되는 암의 연간 발생률은 매우 낮다는 점이 놀랍다.

이는 돌연변이만으로는 종양 형성이 충분하지 않으며,

다른 필수적인 분자 사건(prerequisite molecular events)을

식별해야 한다는 것을 시사한다.

또한,

인간은 항상성을 유지하고

종양유발 전환(oncogenic transformation)을 방어하기 위한 다양한 전략을 진화시켜 왔다.

그러나

환경적 손상(environmental insults)과 노화는

종종 이 균형을 깨뜨려 암 형성 위험을 증가시킨다²,³.

이러한 위험 인자들이

암 진행에 기여하는 메커니즘은 광범위하게 연구되어 왔으나,

초기 종양 형성 과정에서 어떻게 관여하며

특정 종양유발 돌연변이와 어떻게 상호작용하는지는 여전히 완전히 이해되지 않았다.

외부 신호의 비유전적 효과(non-genetic effects)는

유전적 돌연변이와 종양 형성 사이의 역설을 설명할 수 있다.

후성유전적 재배선(epigenetic rewiring)은

제어 불가능한 성장과 생존 잠재력을 해방시키는 또 다른 원동력이 될 수 있다.

Fig. 1

Multistage tumorigenesis. In normal tissue, somatic mutations sporadically arise and either are eliminated by tumor-suppressive mechanisms or gain proliferative advantages to form clones. The mutant clones can still maintain homeostasis until they are exposed to additional stimulus. Their proliferation becomes uncontrolled, and malignant transformation initiates, progressing from premalignant lesions to advanced tumors. During this process, the transformed cells gradually accumulate additional genetic mutations and epigenetic alterations, exhibiting increasingly malignant traits such as immune evasion, structural disruption, and invasion. Simultaneously, the microenvironment of these cells evolves from being tumor-suppressive to supportive of malignancy. This includes dysfunctional immunosurveillance, the emergence of tumor-promotive inflammation, gradual transformation of fibroblasts to CAFs, as well as stiffening of the ECM. CAF cancer associated fibroblast, TAM tumor associated macrophages, MDSC myeloid-derived suppressor cell, ECM extracellular matrix. Created with BioRender.com

Full size image

다단계 종양 형성(Multistage tumorigenesis)

정상 조직에서는

체세포 돌연변이(somatic mutations)가 산발적으로 발생하며,

이러한 돌연변이는 종양 억제 기전에 의해 제거되거나,

증식 우위를 얻어 클론을 형성합니다.

돌연변이 클론은

추가적인 자극에 노출될 때까지 항상성을 유지할 수 있습니다.

그러나 추가 자극에 노출되면

증식이 비제어적으로 변하고,

악성 전환(malignant transformation)이 시작되어

전암성 병변(premalignant lesions)에서

진행성 종양(advanced tumors)으로 진행됩니다.

이 과정에서 변형된 세포(transformed cells)는

점차 추가적인 유전 돌연변이와 후성유전학적 변화를 축적하며,

면역 회피(immune evasion),

구조적 파괴(structural disruption), 침윤(invasion) 등 점점 더 악성적인 특성을 나타냅니다.

동시에 세포의 미세환경(microenvironment)도

종양 억제적 상태에서 악성을 지지하는 상태로 진화합니다.

이는 기능 장애를 일으키는

면역 감시(dysfunctional immunosurveillance),

종양 촉진성 염증(tumor-promotive inflammation)의 출현,

섬유아세포가 암 관련 섬유아세포(CAFs)로 점진적 전환,

그리고 세포외기질(ECM)의 경직화(stiffening) 등을 포함합니다.

(CAF: cancer-associated fibroblast, 암 관련 섬유아세포 TAM: tumor-associated macrophage, 종양 관련 대식세포 MDSC: myeloid-derived suppressor cell, 골수유래 억제세포 ECM: extracellular matrix, 세포외기질)

Cells capable of forming a neoplastic phenotype after acquiring genetic and epigenetic alterations will henceforth be referred to as “transformed cells”. Their clonal evolution is the result of a balance between intrinsic competency and extrinsic selective pressures, which is influenced by neighboring competitors, the microenvironment, and the cooperative tissue architecture. It used to be difficult to detect the rare precursors of tumors, while being armed with innovative technology, the identities of transformed cells and their interactions with the environment are being elucidated. In this review, we explore the driver events that enhance the transforming competency of a cell into full-fledged tumors, and examine the key transitions underlying tumor initiation and early tumorigenesis driven by these events. In addition, given that numerous interventional strategies for advanced tumors are limited by their heterogeneity, premalignant stage is regarded as a promising timing for intervention.4 Therefore, we also summarize how the molecular processes can be utilized to predict patients who are at high-risk of developing consequential cancer, and to develop preventive strategies that intercept malignant transformation.

유전적·후성유전적 변화를 획득한 후 종양성 표현형(neoplastic phenotype)을 형성할 수 있는 세포를 이하 “변형된 세포(transformed cells)”라고 지칭합니다. 이들의 클론 진화(clonal evolution)는 세포 내적 역량(intrinsic competency)과 세포 외적 선택 압력(extrinsic selective pressures)의 균형 결과이며, 이는 인접 경쟁 세포(neighboring competitors), 미세환경, 그리고 협력적 조직 구조(cooperative tissue architecture)에 의해 영향을 받습니다. 과거에는 종양의 희귀한 전구체(pre-cursor)를 검출하기 어려웠으나, 혁신적인 기술의 도움으로 변형된 세포의 정체성과 미세환경과의 상호작용이 점차 밝혀지고 있습니다. 본 리뷰에서는 세포를 완전한 종양으로 전환시키는 역량(transforming competency)을 강화하는 driver 사건들을 탐구하고, 이러한 사건들에 의해 주도되는 종양 발생(tumor initiation)과 초기 종양 형성(early tumorigenesis)의 핵심 전환 과정을 검토합니다. 또한, 진행성 종양의 높은 이질성(heterogeneity)으로 인해 수많은 중재 전략이 제한되는 점을 고려할 때, 전암 단계(premalignant stage)는 중재 개입의 유망한 시점으로 여겨집니다⁴. 따라서 본 리뷰에서는 이러한 분자 과정들을 활용하여 중대한 암 발병 위험이 높은 환자를 예측하고, 악성 전환을 차단하는 예방 전략을 개발하는 방법도 요약합니다.

The research history of tumor initiation and early tumorigenesis

The earliest explanation for the origin of cancer can be dated back to the early 1900s, cell-free extracts of a diseased animal were able to transmit tumors to healthy animal, suggesting that tumors originate from a unit smaller than a cell5 (Fig. 2). In 1914, Theodor Boveri proposed the somatic mutation theory after observing chromosomal abnormalities in tumor cells.6 Subsequent studies validated DNA as the genetic material and revealed that tumorigenesis requires the accumulation of approximately six or seven mutations.7,8 The term “oncogene” was introduced in 1960s when genetic material of certain viruses was verified to contribute to malignant transformation.9 The first specific tumor gene was identified in 1976 by Michael Bishop and Harold Varmus, that part of the DNA of avian sarcoma virus hybridized in the genomes of birds transforming normal cells to tumor cells, and named it as SRC.10 This indicated that the genetic material in our genome is capable of transforming normal cells. Subsequently, the first proto-oncogene, RAS, and tumor suppressor gene, RB1, were cloned in the early 1980s.11,12,13 Following this, a significant number of these two classes of cancer genes were identified, accompanied by discovery of other forms of variations, including copy number alterations, translocations and promoter hypermethylation.14 In the middle of 2000s, benefiting from next-generation sequencing, cancer genomics flourished and promoted the launch of large-scale tumor sequencing initiatives, such as The Cancer Genome Atlas (TCGA) in 2006 and the International Cancer Genome Consortium (ICGC) in 2007.15 The TCGA consortium published its Pan-Cancer Analysis of Whole Genomes (PCAWG) data in 2020, which contained the whole genomic sequencing data of 38 tumor types from more than 2800 patients, largely expanding our understanding of cancer genomics.16 According to the influence in cancer development, mutations can be categorized as driver mutations and passenger mutations. The driver mutations confer fitness advantage for clone expansion while other preexisting mutations, lacking positive selection properties, are referred to as passenger mutations,17 and over 3,000 cancer driver genes have been identified experimentally or computationally to date.18 Notably, in the last decade, deep sequencing from low-input samples has helped to identify somatic mutations in normal tissues, which are highly concordant with the tumor driver mutations.19 It reveals a limitation of the somatic mutation theory, that is the mere presence of mutations is insufficient for tumorigenesis, suggesting that there are other driver events.

종양 발생과 초기 종양 형성의 연구 역사

암의 기원에 대한 가장 초기의 설명은 1900년대 초로 거슬러 올라갑니다. 당시 병든 동물의 세포가 없는 추출물(cell-free extracts)이 건강한 동물에게 종양을 전파할 수 있다는 사실이 밝혀져, 종양이 세포보다 작은 단위에서 기원한다는 가설이 제기되었습니다⁵ (Fig. 2). 1914년 테오도르 보베리(Theodor Boveri)는 종양 세포에서 관찰된 염색체 이상을 바탕으로 체세포 돌연변이 이론(somatic mutation theory)을 제안했습니다⁶. 이후 연구들은 DNA가 유전 물질임을 입증하였고, 종양 형성에는 약 6~7개의 돌연변이 축적이 필요하다는 사실을 밝혔습니다⁷,⁸. 1960년대에는 특정 바이러스의 유전 물질이 악성 전환에 기여한다는 사실이 확인되면서 “종양유전자(oncogene)”라는 용어가 도입되었습니다⁹. 1976년 마이클 비숍(Michael Bishop)과 해롤드 바르무스(Harold Varmus)는 조류 육종 바이러스(avian sarcoma virus)의 DNA 일부가 조류 게놈에서 정상 세포를 종양 세포로 전환시키는 것을 확인하고, 이를 SRC라고 명명하였습니다¹⁰. 이는 우리 게놈 내의 유전 물질이 정상 세포를 변형시킬 수 있음을 시사했습니다. 이어 1980년대 초에 최초의 원종양유전자(proto-oncogene)인 RAS와 종양 억제 유전자(tumor suppressor gene)인 RB1이 클로닝되었습니다¹¹,¹²,¹³. 이후 이 두 부류의 암 유전자가 대량으로 식별되었으며, 복제수 변이(copy number alterations), 전좌(translocations), 프로모터 과메틸화(promoter hypermethylation) 등 다른 형태의 변이도 발견되었습니다¹⁴.

2000년대 중반 차세대 시퀀싱(next-generation sequencing)의 혜택으로 암 게놈학(cancer genomics)이 급성장하였고, 2006년 The Cancer Genome Atlas (TCGA), 2007년 International Cancer Genome Consortium (ICGC)와 같은 대규모 종양 시퀀싱 프로젝트가 시작되었습니다¹⁵. TCGA 컨소시엄은 2020년 Pan-Cancer Analysis of Whole Genomes (PCAWG) 데이터를 발표하였으며, 이는 38개 종양 유형의 2,800명 이상 환자에서 얻은 전ゲ놈 시퀀싱 데이터를 포함하여 암 게놈학에 대한 이해를 크게 확장하였습니다¹⁶. 암 발달에 미치는 영향에 따라 돌연변이는 driver mutation과 passenger mutation으로 분류됩니다. Driver mutation은 클론 확장에 적합성 우위(fitness advantage)를 부여하는 반면, 긍정적 선택 압력이 없는 기존 돌연변이는 passenger mutation으로 불립니다¹⁷. 현재까지 실험적·계산적으로 3,000개 이상의 암 driver 유전자가 식별되었습니다¹⁸.

특히 지난 10년간 저입력 샘플(low-input sample)로부터의 심층 시퀀싱(deep sequencing)이 정상 조직 내 체세포 돌연변이를 식별하는 데 도움을 주었으며, 이들 돌연변이는 종양 driver 돌연변이와 높은 일치도를 보였습니다¹⁹. 이는 체세포 돌연변이 이론의 한계를 드러내는 것으로, 돌연변이의 존재 자체만으로는 종양 형성이 충분하지 않으며, 다른 driver 사건들이 존재한다는 것을 시사합니다.

Fig. 2

Research history of tumor initiation and early tumorigenesis. The upper section emphasizes the role of somatic mutations in tumorigenesis, while the lower section demonstrates the evidence of the driver events beyond genetic events. ICGC the International Cancer Genome Consortium, TCGA the Cancer Genome Atlas, PCAWG the Pan-Cancer Analysis of Whole Genomes, HTAN the Human Tumor Atlas Network

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On the other hand, Victor A. Triolo first proposed that cancer is a tissue-based disease in 1965.20 Following studies have verified that the capability of mutated malignant cells to induce tumors is context-dependent. Injecting tumor cells into normal mouse blastocysts can result in the development of normal embryos, indicating that malignant cells alone do not necessarily lead to tumors.21 The role of tissue injury in Rous sarcoma virus-mediated tumorigenesis,22,23 and tumors induced by carcinogen-treated extracellular matrices24,25 both further confirmed that extrinsic factors influence the outcome of tumorigenesis. Accordingly, tissue organization field theory was proposed in 2011.26 The theory posits that aberrant tissue organization and cell-cell interactions contribute to tumorigenesis, with carcinogens targeting the entire tissue. In 2018, the Human Tumor Atlas Network (HTAN) was launched,27 aiming at setting three dimensional atlases at crucial transitions of multiple tumors, including tumor initiation and local expansion, based on single-cell and spatial methods, and elucidating complex interactions between cells and their dynamic tumor ecosystem. It is expected to help us better understand how microenvironmental factors and transformed cells cooperatively promote the early transformation. Furthermore, the pan-cancer analysis of epigenome, transcriptome, proteome, and post-translational modification were recently published,28,29,30,31,32,33,34 providing multidimensional information of the tumor biology and possibly giving insights for the research of tumorigenesis.

Molecular drivers of tumorigenesis

Genetic alterationsSingle nucleotide variants

Single nucleotide variants continuously accumulate through lifespan, originating from errors during DNA replication and repair processes, resulting from both endogenous factors (e.g., cellular metabolites, reactive oxygen species, nitrogen species, and transposable elements) and exogenous factors (e.g., radiation, tobacco, alcohol, and other chemical mutagens). Spontaneous chemical modifications can also serve as mutagens.35,36 Somatic mutations in morphologically normal tissues can establish a baseline for studying cancer genome evolution and for identifying key drivers of malignant transformation. In recent years, a series of studies have analyzed the mutational landscape across nonmalignant tissues, shedding light on tissue-specific mutational burdens, mutational signatures, and the spectrum and frequency of driver mutations and their clonal expansions (Table 1), which can be influenced by stem cell dynamics, tissue turnover patterns, and environmental exposures.3,19 Mutational signatures, developed to depict various DNA damage and repair processes, offer insights into mutagenic mechanisms.35 It shows that age-related signatures, such as single base substitution signature 1 (SBS1) and SBS5, are preval‎ent across phenotypically normal tissues, although their contributions vary.37,38 These signatures are the primary mutagenic factors in most types of tissues, especially those with high rates of cellular proliferation, such as the intestines.37,38 In contrast, exogenous mutational signatures often play a relatively minor role. However, there are some exceptions, such as the SBS22 mutational signature associated with aristolochic acid, which is common in the liver samples37 and is also significantly enriched in the urothelial samples from Chinese donors.39

Table 1 Dynamics of genome evolution in tumorigenesis

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To explore intra-individual heterogeneity, our laboratory analyzed 9 normal organs from the same donors, and found that the liver exhibited the highest mutational burden, significantly surpassing that of other epithelial tissues, whereas the pancreas had the lowest level of mutation burden.37 In addition, we compared the mutational signatures across organs and found that aging induced mutagenesis was the most preval‎ent, although it varied significantly among different tissues. Certain organs, such as livers, were largely influenced by exogenous mutagens. We also spatially reconstructed clonal architecture at sub millimeter resolution, and revealed how clone expansions associate with tissue microstructures, harbored mutations, and environmental factors.37 Similar phenomena have also been observed in other studies.38,40

Similar to driver mutations in cancer, mutations conferring fitness are positively selected and promote clonal expansion in nonmalignant tissues. Intriguingly, although most driver mutations are classical cancer mutations, they can maintain homeostasis in normal tissues, and exert opposite effects on tumorigenesis.19 Furthermore, some mutations are less common in tumors than in normal tissues and have been validated to play a tumor-suppressive role through outcompeting oncogenic clones, exemplified by NOTCH1 loss of function (LOF) in the esophagus.41 In contrast, the frequency of some mutations increases in tumors, like TP53 in skin, esophageal and endometrial cancers and PTEN in endometrial cancer, indicating their contribution to tumor development.3 Given that these mutations are generally tolerable in normal tissues, there should exist other factors to further promote their proliferative potential and initiate malignant evolution. To accurately identify additional driver events and the timing they emerge, multiple sampling is required. We recently revealed more detailed genomic changes throughout the entire process of esophageal squamous cell carcinoma (ESCC) formation, using multistep tumorigenesis samples ranging from normal tissue, through low-grade and high-grade intraepithelial neoplasia, to tumors from the same individuals.42 We also reconstructed their temporospatial evolutionary dynamics and confirmed that biallelic loss of TP53 in low-grade intraepithelial neoplasia is one of the earliest steps in initiating malignant transformation, serving as a prerequisite for copy number alterations (CNAs) in oncogenic genes involved in the cell cycle, DNA repair, and apoptosis pathways.42 It was also verified in mouse models of esophageal and pancreatic tumorigenesis that Trp53 loss of heterozygosity (LOH) is a critical step for genomic instability and malignant transformation. Meanwhile, heterozygous Trp53 mutation can maintain clonality only to a limited extent in normal tissues.43,44

Copy number alterations and structural variations

Large-scale chromosomal alterations are another widespread form of genetic mutations, encompassing numerical and structural variations and constituting 80–90% of cancer genomes.45,46 CNAs comprise aneuploidy, whole-genome duplications (WGDs), and extrachromosomal DNA (ecDNA), while structural variations include genomic catastrophes such as chromothripsis, chromoplexy, and breakage-fusion-bridge cycles. The complex genomic rearrangements have a reciprocal causation with chromosomal instability (CIN), an ongoing state in which cells accelerate the production of aneuploidy, and both of which can converge onto initial chromosome segregation errors.47,48 It is speculated that chromosomal alterations occur very early in the evolution of specific cancer types, suggesting their potentially pivotal roles in tumor initiation.16,49,50,51 Indeed, while CNAs and aneuploidy are rarely observed in normal tissues,19,52,53,54,55 they can be detected in precancerous lesions, albeit at much lower levels than that in fully formed tumors.56,57,58,59,60,61 Furthermore, the levels of CNAs and CIN in precancerous lesions they indicate can serve as indicators of malignant progression.60,62,63 ecDNA, a unique form of CNAs, consists of double-stranded circular chromatids, and may serve as a robust driver of tumor genome evolution due to the absence of centromeric sequences and uneven distribution in daughter cells during mitosis.64 Notably, ecDNA has been detected early in the progression from high-grade dysplasia in Barrett’s esophagus to esophageal adenocarcinoma (EAC).64,65 Their copy number and structural complexity increased along the tumor evolutionary trajectory. Patients who progressed to EAC exhibited higher levels of ecDNA compared to those who did not.65

Benefiting from multi-region sampling and single-cell sequencing, ongoing CIN and complex evolutionary processes of CNAs and structural variations can be depicted accurately.66,67,68,69 Through multi-region sampling of Barrett’s esophagus concurrently containing different states of dysplasia and microscopic EAC foci, it has been reported that the evolution of CNAs during EAC tumorigenesis can be launched ahead of the development of dysplasia. Multigenerational CIN was initiated by mitotic errors and subsequent genomic catastrophes, including WGD, and inactivation of TP53 played an enabling role in the propagation of CIN, aggravating the accumulation of CNAs.69 Recently, signatures of CNAs and CIN have been summarized from pan-cancer studies, encompassing numerous structural and copy number-related biological phenomena, such as WGD, aneuploidy, LOH, homologous recombination deficiency, chromothripsis, and haploidization.46,70 It is expected to facilitate integrated analysis of CNAs and structural variations, so as to better elucidate mutational processes and genomic complexity.

Chromosomal abnormalities promote tumorigenesis through their effects on abnormal gene expression‎, including disruption or loss of tumor suppressors, oncogene amplification, and formation of oncogenic fusion genes.47,64 Loss of the 3p arm, harboring tumor suppressor genes such as VHL, PBRM1, BAP1, and SETD2, can be an initiating event in clear-cell renal cell carcinoma. An increased frequency of LOH at 9p has been observed from precancerous lesions to cutaneous squamous cell carcinoma (CSCC), possibly driven by loss of tumor suppressive gene CDKN2A in this region.71,72 Driver fusion genes such as EML4-ALK in non-smoker lung adenocarcinoma (LUAD) are speculated to be generated from complex chromosomal rearrangements, including chromothripsis and chromoplexy, and to arise in early years of life.73 Specifically, ecDNA can both promote gene amplification and function as mobile enhancers regulating the expression‎ of oncogenes.74,75 Nevertheless, it is worth noting that the role of CNAs and structural variations in tumorigenesis are context-dependent.76 Complex chromosomal aberrations are likely to exert deleterious cellular effects, inducing senescence, DNA damage, proteotoxicity, essential and toxic gene changes.77 However, under specific conditions, aneuploid cells can be preserved, for instance, when WGD occurs ahead, providing extra copies of essential genes to alleviate deleterious alterations.78 Furthermore, TP53 inactivation often occurs earlier to support the occurrence of WGD and clonal expansion.79,80 There are also paradoxical immune activation and evasion induced by CIN. Chromosomal mis-segregation generates micronuclei, from which DNA leakage into the cytoplasm can activate the immune system, leading to the clearance of genomic unstable cells via cGAS-STING and type I interferon (IFN) pathway.81 At some points, tumor cells develop strategies to overcome the IFN signaling. Simultaneously, the secretome induced by CIN stimulate chronic inflammation and pro-tumorigenic effects.77,82

Epigenetic alterations

The epigenome is another layer of information to encode cell identity and could be passed onto daughter cells. Upon development, natural aging and environmental exposure, there are dynamic changes in DNA and histone covalent modifications that remodel chromatin states and structures, and the heritable epigenetic marks, such as DNA methylation, being referred to as “epimutations”, serve as another important impetus of malignant evolution independent to genetic mutations.83,84,85 Accumulating evidence suggests that clones with aberrantly rewired epigenetic programs show increased tumor susceptibility in morphologically normal tissues.58,86 Particularly, age-induced DNA methylation changes are parallel to those seen in malignant states, including increased CpG island methylation and global hypomethylation.84,87 During precancerous evolution, epigenomes undergo a stepwise progression, culminating in a high level of intra-tumor heterogeneity in invasive lesions. For instance, a gradual increase of methylation aberrations was observed transitioning from precursors to invasive LUAD.88 Actinic keratosis, a precancerous lesion of CSCC, displayed classic cancerous methylome features, with two distinct methylation patterns suggesting different progression pathways to malignancy.89 Precancerous colorectal adenomas have also already undergone genome-wide methylation changes and showed preliminary heterogeneity at the adenoma stage.90 In specific tumors, such as ependymomas, it seems that epigenetic alterations play a decisive role, with only minimal genetic alterations detected.91

Tumor driver events induced by epigenetic reprogramming are presented as overly either restriction or permission states for gene expression‎, which can induce all hallmarks of cancer.88 Highly repressive states induced by DNA hypermethylation lead to gene inactivation, often occurring in tumor suppressor gene related pathways, including DNA repair, cell cycle regulation, and p53 signaling.92 Additionally, hypermethylation of promoter CpG islands is frequently observed in lineage-specific transcription factor (TF) sequences that carry bivalent H3K4me3 and H3K27me3 modifications, transforming these previously poised sequences into inactive states that promote dedifferentiation and tumorigenesis.93,94 We have confirmed this process in early esophageal tumorigenesis.95 Overly permissive states, also known as epigenetic plasticity, can stochastically induce expression‎ of pro-carcinogenic programs. For example, hypomethylation in enhancers and lineage-committed TF regions serves as an important mechanism in leukogenesis,96,97 which has already been leveraged by DNMT3AR822 clone in nonmalignant hematopoiesis, leading to chaotic transcriptional phenotypes and increased tumor risks.96,97,98 Another way to induce permissive states and enhance cellular plasticity involves the suppression of Polycomb repressors, such as through the inactivation of histone methyltransferases, as exemplified by early lung tumorigenesis induced by KMT2D inactivation.99,100 Although DNA hypermethylation mainly induces suppressive states, they can also promote gene expression‎ through dysfunctional chromosomal topology.101 Abnormal hypermethylation at cohesin and CCCTC-binding factor (CTCF)-binding sites reduces the binding of insulator protein and formation of insulators, thereby promoting aberrant regulatory interactions like the activation of a constitutive enhancer for the tyrosine kinase gene PDGFRA to upregulate its expression‎.101 An integrative multi-omics atlas of 11 major cancer types indicated that tumor-specific and concurrent epigenetic driver events are associated with cancer transition, with enhancer accessibility playing a more specific role in transition from normal to different types of tumors.28 The evidence above suggests that roles of distal regulatory regions and chromatin topology in tumorigenesis warrant further exploration.

Epigenetic alterations and genetic mutations have complex interactions in promoting tumor initiation, with genetic mutations possibly serving as primers to induce epigenetic changes, or epigenetic reprogramming potentiating oncogenic competence of genetic mutations.102,103 Genes that encode epigenetic modifiers are common driver mutations in specific cancers and can occur in precancerous stage, such as TET2, DNMT3A and ASXL1 in hematologic malignancies,104,105 and SWI/SNF chromatin remodeling complexes in solid tumors.106,107 Recurrent tumor driver mutations also have capabilities to mediate epigenetic remodeling. For instance, one of the tumor-suppressive roles of p53 is to safeguard epigenetically regulated lineage commitment, and its role in limiting cell fate reprogramming has been proven in several cell types.108,109,110,111 The oncogenic effects of Kras mutations mediated by chromatin remodeling have also been documented.112 Conversely, epigenetic priming might precede genetic mutations, rendering cells more susceptible to oncogenic signals, exemplified by aging-related DNA methylation which can activate the Wnt pathway to be more sensitive to Braf mutation induced colon transformation.113,114 Furthermore, epigenetic abnormalities play a role in accumulating mutations, such as through spontaneous deamination of DNA methylation,115 and DNA hypomethylation induced CIN.63 In addition, methylated promoters of DNA repair genes underlie a field wherein the colorectal cancer (CRC) with higher rate of mutations arise.116 Multi-region single-gland genome, epigenome, and transcriptome profiling of concomitant colorectal adenomas and tumors demonstrated that genetic and epigenetic mutations mutually promoted accumulation of each other. Mutational signature showed that the epigenome alterations induced DNA mutation, while driver mutations were also found in chromatin modifier genes.117 However, the functions of chromatin accessible driver genes and genetic driver mutations were independent. Some accessible drivers were devoid of mutations.117 Parallel evolution of methylome and genome was also observed in lung tumorigenesis, where global hypomethylation was associated with high mutation burden, CNAs and allelic imbalance, as well as immune infiltration.88 Beyond genetic and epigenetic interactions, it is recently reported that chromatin accessibility could also be modified by RNA modification, another regulatory layer for gene expression‎, being known as epitranscriptome. N6-methyladenosine (m6A) modifications of RNA are the most common form of mRNA modification, and their roles in regulating transcript stability, translation and localization have been proven to be intricately involved in tumorigenesis.118 Recently, specific crosstalk between RNA m6A and epigenetic marks, such as histone modifications and DNA methylations, is being unveiled.119,120,121,122,123 Our work indicated that m6A in super-enhancer RNA is capable of activating YTHDC2 and recruiting H3K4 methyltransferase MLL1 for co-transcriptionally directing H3K4me3 demethylation as well as being accessible to oncogene transcription.124 In addition, we also found that m6A in RNA could be the cause of DNA demethylation in nearby genomic loci in both normal and cancer cells, which is mediated by RNA m6A modification reader FXR1 to recruit DNA dioxygenase TET1.125 Altogether, different aspects of chromatin regulation are integrated to regulate cell fate and function. A deeper understanding is warranted to explore their roles and causal relationships.

Environmental factors

There are diverse environmental and systemic factors that have been epidemiologically confirmed as tumor risk factors, encompassing chemical and radical insults, unhealthy metabolic behaviors, specific pathogen infections, as well as aging. They induce versatile alternations in whole or at local positions, including both induction of genetic and epigenetic alterations in transformed cells and profound impacts on microenvironmental components that predispose to tumor initiation (Table 2). Since inflammation is a convergent response to various environmental alterations, we discuss its role in this part at first, which is followed by context-specific mechanisms of other risk factors.

Table 2 Gene-environment interactions in tumorigenesis

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Inflammation

Inflammation is a conserved response to potential insults, being involved in tissue repair, regeneration, and homeostasis regulation by stimulating cytokine production and mobilizing innate and adaptive immune systems to remove insults and protect the integrity of the tissue.126,127 While acute inflammation aims to solve damage and has tumor-suppressive effects, chronic inflammation caused by unresolved and persistent damage is a well-known tumor risk factor and is considered an enabling hallmark of cancer.128 It can be triggered by numerous external stimuli associated with tumors, including chemical carcinogens, radiation, and infections.129 Additionally, aberrant autoimmune reactions, such as reflux esophagitis, inflammatory bowel disease, and atrophic gastritis, as well as systemic and subclinical inflammation related to ageing and obesity, can trigger similar pro-tumorigenic effects.129,130 The mechanisms by which inflammation is involved in early tumorigenesis include not only oxidative stress and DNA damage, but also priming or releasing the expansion and transformative potential of cells harboring oncogenic mutations.131,132 This process can be exemplified by inflammation-stimulated TP53 mutation clone expansion in colonic and leukemic transformations.133,134,135 Notably, expanding mutant clones in inflammation can play roles independent of tumorigenesis, such as the regeneration role of ARID1A, KMT2D and PKD1 in liver injury136 and tumor-suppressive NFKBIZ mutation in colitis.137

It has been widely confirmed that cytokines and growth factors in chronic inflammation play pro-tumorigenic roles, such as interleukin 1 (IL-1), IL-6, transforming growth factor beta (TGF-β), IL-17A, and IL-22, and their functions, which regulate cell survival, proliferation and cell fate determination can be hijacked by cells harboring mutations, activating mitogen-activated protein kinases (MAPK), phosphatidylinositol-3-kinase (PI3K) -AKT, Janus kinase (JAK) -STAT and NF-κB pathways to increase the risk of tumors.131,132 Specifically, inflammatory mediators can play a decisive role in early malignant evolution. For example, the cooperation between Sox2 overexpression‎ and inflammation activated STAT3 is capable of inducing ESCC, while in the absence of environmental stimuli, mutations alone may only enhance proliferation without progressing towards tumors.138 Liver injury induced dedifferentiation is also a promoter of tumorigenesis, where both mature hepatocytes and cholangiocytes have the potential to give rise to different type of primary liver cancers, comprised of hepatocellular carcinoma and intrahepatic cholangiocarcinoma.139 The lineage commitment is dependent on both mutation backgrounds and epigenetic regulations of the injury signaling.140,141 Hepatocytes harboring oncogenic mutations induced intrahepatic cholangiocarcinoma upon stimulation of damage-associated molecular patterns (DAMP)-associated cytokines induced by liver cell necroptosis.141 By contrast, apoptotic microenvironment promotes transformation of hepatocytes with the same mutation background to hepatocellular carcinoma.141 In hematological system, since chronic inflammation leads to stem cell differentiation and exhaustion, mutations conferring resistance to inflammation stress, such as TET2 and DNMT3A, can be positively selected and form clonal hematopoiesis of indeterminate potentials (CHIPs). Tet2 LOF hematopoietic stem/progenitor cells (HSPCs) upregulated TLR-TRAF6 in response to inflammation, resulting in a shift from the canonical NF-κB pathway to the noncanonical NF-κB pathway, thereby avoiding inflammatory damage to mutated stem cells, and facilitating the Tet2 mutation-induced progression of myelodysplastic syndrome.142 Dnmt3A LOF CHIP could also prevent hematopoietic stem cells from terminal differentiation through increasing methylation of IFNγ signaling pathways.143

The epigenetic plasticity conferred by inflammation lowers the barriers for malignant transformation. A typical example is pancreatic tumorigenesis initiated from Kras mutant acinar cells and promoted by injury and pancreatitis. Kras mutation is insufficient for transformation, and injury-induced inflammation is indispensable in the development of pancreatic intraepithelial neoplasms (PanIN) and pancreatic ductal adenocarcinoma (PDAC).144,145,146 Inflammation induces transdifferentiation of acinar cells to ductal cells, which is a reversible process termed as acinar-to-ductal metaplasia (ADM), and can be resolved as tissue regenerates.145,146 However, the reprogramming can be co-opted by Kras mutations to irreversibly transform the ADM program to PanIN and PDAC programs.147,148,149 Distinct chromatin states between normal regeneration and Kras induced tumorigenesis could be mediated by a chromatin reader, bromodomain and extra-terminal family member reader, BRD4. The divergence was initiated as early as 48 hours after pancreatic injury induced by caerulein in mouse models.148 Besides, another study identified that a precancerous cell subset with ductal identities and oncogenic potential had emerged in ADM, and Kras mutation maintained the pro-oncogenic programs, ultimately resulting in PDAC.149 It is because inflammation activated AP-1 to dominate the pro-oncogenic transcriptional program, and its key components Junb and Fosl1 could be stabilized by Kras mutation.149 Similar cooperation between gene and environment was depicted in oncogenic epidermal wound repair, where stress-induced TFs, such as AP-1, ETS2 and STAT3, induced transient lineage infidelity between epidermal stem cells and hair follicle stem cells. In tumorigenesis, stress-TFs were enhanced, resulting in a permanent lineage infidelity and newly activated oncogenic enhancers for malignant transformation, which were divergent from normal regeneration.150

An emerging field of study of inflammation-induced epigenetic rewiring is tissue memory, which is an adaptation to recurrent stress and has been identified in various tissues, including skin, lung, intestine and pancreas.151,152,153,154,155,156,157 In parallel to the immune memory, epithelial cells set long-term memory based on epigenetic modifications they have adopted during injury, which can be partially maintained after the resolution, enabling a more rapid response to a next similar damage.158 However, there is a trade-off between tissue long-term adaptation and tumorigenesis that the persistent abnormal epigenetic program primes a field permissive for tumorigenesis. For instance, pancreatic epithelium develops tissue memory of ADM to rapidly instigate a protective program for a secondary pancreatic injury and reduce tissue damage,157 which can be enhanced by Kras mutations via MAPK constitutive signaling to increase fitness. Nevertheless, Kras mutations induces an irreversible ADM reprogramming and increase tumor risk simutaneously.157 Similarly, in wound-priming epidermis, there are memory stem cells located in distal intact areas, which are prepared both to respond to another damage adaptively, and to give rise to tumors detrimentally. This is achieved through epigenetic and transcriptional reprogramming and mediated by a long-lasting loss of histone repressive mark H2AK119ub.159

Chemical and radical insults

Environmental carcinogens are preval‎ent in nature, derived from air pollution, cigarettes, alcohol, ultraviolet (UV) radiation, etc. These carcinogens promote tumor progression through various mechanisms, including genotoxicity, epigenetic modification, chronic inflammation, immune suppression, oxidative stress, and activation of receptor-mediated signaling pathways.160,161

In the tumor initiating stage, chemical and radical carcinogens not only induce mutations and contribute to specific mutational signatures,54,162 but also promote clonal expansion of specific mutations. For example, driver mutations of CSCCs, such as NOTCH, TP53, FAT1 and FGFR3, are more preval‎ent in chronically UV exposed skin than in unexposed healthy skin.52,163 Similarly, smoking promotes clonal expansion in the blood, including ASXL1, DNMT3A, and TET2 CHIPs.105,164 Intriguingly, the landscape of clone expansion is likely to be reversible. The high mutational burden and driver mutation frequency in the bronchial epithelium decrease after smoking cessation, likely due to the rescue effect of quiescent cell expansion, which was previously protected from tobacco mutagenic insults.162

The positively selected mutant clones are expected to exhibit resistance to stress. In sun-exposed skin, plasmacytoid dendritic cells with Tet2 LOF are protected from UV-induced cell death, providing a reservoir for the accumulation of more oncogenic mutations and subsequent malignant transformation.165 Mouse esophageal stem cells harboring Trp53 mutations are less vulnerable to radiation-induced oxidative stress and replace differentiated wild-type cells for clone expansion.166 HSPCs with Trp53 mutation were also insensitive to radiation-induced differentiation. Mutant p53 bound to enhancer of Zeste homolog 2 (EZH2), a catalytic subunit of Polycomb repressive complex 2 that is responsible for trimethylation of Lys-27 in histone 3 (H3K27me3), thereby promoting the expression‎ of self-renewal program in Trp53-mutant CHIP.167

In addition to providing a hostile environment, multiple insults can directly activate epithelial cells to induce epigenetic and transcriptional changes, or they can act on immune cells to trigger inflammatory responses, indirectly promoting tumor development. Nicotine activates the AKT-extracellular-regulated kinase (ERK)-MYC pathway via the nicotinic acetylcholine receptor and inhibits the Gata6 promoter, a key regulator of acinar cell differentiation.168 This leads to the dedifferentiation of acinar cells and further promotes the activation of Kras mutation, thereby facilitating the transformation of Kras-mutant ADM and PanIN.168 Chronic exposure to cigarette smoke has also proven to induce time-dependent epigenetic changes, which makes bronchial epithelial cells more susceptible to single Kras mutation induced tumorigenesis.169 Alterations in transformed cells, such as epithelial-to-mesenchymal transition (EMT), anchorage-independent growth, and RAS/MAPK signaling upregulation, are closely associated with gene silencing induced by hypermethylation.169 The Epidermal growth factor receptor (EGFR) gene mutation is identified as a common driver mutation in healthy lung tissue exposed to environmental particulate matter measuring ≤2.5 μm (PM2.5), and is associated with a higher incidence of LUAD.170 Hill et al. showed that PM2.5 induced lung macrophage infiltration and secretion of IL-1β, which mediated the reprogramming of alveolar type (AT) II cells into a progenitor-like state.170

Metabolic factors

Cellular metabolism is regulated by both intrinsic metabolic properties of the cell and the intake of external nutrients. Tumors modify their metabolic patterns to evade nutrient restraints and fulfill their heightened demands for aberrant growth and proliferation. Alternatively, tumors produce oncogenic metabolites that regulate gene and protein expression‎ to promote tumor progression.171,172 Recent findings suggest that metabolic remodeling begins earlier at precancerous stages. In early precancerous lesions of lung squamous cell carcinoma, activities such as fatty acid metabolism, oxidative phosphorylation, and the citric acid cycle are enhanced.173,174 These early metabolic changes in tumorigenesis might play a role in driving tumor initiation by interacting with predisposed mutations.175 There are two primary mechanisms. One is that mutations drive early metabolic alterations and adaptations. The other is that abnormal metabolic environment facilitates transformation of mutated cells. Classical oncogenic mutations, such as PIK3CA, TP53, RAS, and MYC, are all implicated in metabolic regulation by influencing the activity and localization of metabolic enzymes at transcriptional and post-transcriptional levels.176 Specifically, they have the potential to recapitulate epigenetic modifications through upregulating expression‎ of metabolic effectors. In the early stage of pancreatic tumorigenesis, mutant Kras and loss of Trp53 enhance acetyl coenzyme A and α-ketoglutarate synthesis, respectively. The metabolites, in turn, epigenetically promote dedifferentiation and PanIN formation.177,178 Additionally, Kras mutations promote metabolic remodeling via post-translational modification of metabolic enzymes. They suppress ubiquitylation and degradation of branched-chain amino acid transaminase 2, an enzyme essential for the catabolism of branched-chain amino acids and mitochondrial respiration, thereby contributing to the progression of PanINs.179 Apart from recurrent cancer mutations, mutations in genes encoding metabolic enzymes, including succinate dehydrogenase, fumarate hydratase, and isocitrate dehydrogenase 1 or 2, have the capability to accumulate oncometabolites, disrupting dioxygenases and their epigenetic regulatory functions.171 The isocitrate dehydrogenase mutation induced oncometabolite, (R)-2-hydroxyglutarate, was confirmed to promote the early tumorigenesis of acute myeloid leukemia (AML) and gliomas through the inhibition of histone lysine demethylases 5.180

In addition to mutation-driven metabolic remodeling, unhealthy systemic metabolic status, including high-fat and high-carbohydrate diets, and metabolic diseases they induce, such as obesity and type 2 diabetes mellitus, can increase the risk of tumors.181,182 Obesity triggers several pathological processes associated with tumor development, including hyperglycemia-related insulin resistance, abnormal hormone secretion, inflammation and dysregulation of lipid metabolism. Under physical conditions, insulin signaling systematically senses blood glucose levels and promotes proliferation and anabolic metabolism. In the presence of obesity, insulin resistance in metabolic tissues leads to hyperglycemia and hyperinsulinemia, while tumor cells develop strategies to maintain their sensitivity to insulin-induced proliferative signaling.181 Transformed mutant cells can adopt similar strategies, utilizing the proliferative signaling and gaining competitive advantages.183,184 Furthermore, hyperglycemia induced by both glucose and fructose consumption enhances tumorigenesis by accelerating glycolysis and de novo lipogenesis.185 Recently, glucose was reported to act as a signaling molecule, directly binding to and activating NSUN2, thereby activating NSUN2-TREX2 signaling. This led to inhibition of dsDNA accumulation, subsequent cGAS/STING pathway activation, and immune activation.186 In terms of the high-fat diet (HFD), Sasak et al. found that it disrupted cell competition outcomes by enhancing lipid metabolism.187 In normal epithelium, the apical extrusion of RasV12 transformed cells could be mediated by Warburg-like effects and damage to mitochondrial membrane potential. However, HFD increased the levels of free fatty acids and promoted their metabolic transformation to acetyl coenzyme A, which played a role in restoring the mitochondrial membrane potential and inhibited the clearance of RasV12 cells.187,188 Furthermore, inflammation and immune responses play crucial roles in linking the pathological processes of obesity to tumorigenesis.189 Pancreatic Kras mutation can downregulate peroxisome proliferator-activated receptor (PPAR)-γ, exacerbate inflammation and further promote the formation of PanIN, mediated by fibroblast growth factor 21, which is an endocrine regulator for metabolic homeostasis.189 HFD also promotes the activation of PPAR-δ and the secretion of CCL2 in Kras-mutant pancreatic cells. Consequently, immunosuppressive cells are recruited, promoting the transformation from PanIN to PDAC.190 Additionally, the mechanisms by which HFD promotes tumorigenesis are also related to microbial dysbiosis. Alterations in gut microbiota and metabolites are crucial for HFD-associated colorectal tumorigenesis, inducing cell proliferation, impairing gut barriers, and promoting oncogenic gene expression‎.191

Microbiome

The human body harbors diverse microbiome communities that interact with the host in complex ways.192 Dysbiosis has been implicated in the development of numerous diseases, including cancer.128 The tumorigenic effects of specific microorganisms have been well-established across several types of tumors. The World Health Organization has classified several microorganisms as Group 1 carcinogens, including Helicobacter pylori (H. pylori), Epstein-Barr virus (EBV), human papillomavirus, hepatitis B virus (HBV), and hepatitis C virus (HCV).193 Excepted for the classic oncogenic pathogens, many other microorganisms have also been discovered to be associated with tumors,194 and several large-scale pan-cancer studies have revealed the presence of microbes in almost all types of tumors.195,196,197 For example, Fusobacterium nucleatum, polyketide synthase-positive(pks+) Escherichia coli (E. coli), and enterotoxigenic Bacteroides fragilis (ETBF) are associated with the occurrence of CRC.198,199,200 Furthermore, it has recently been discovered that microbiome alterations emerge in early precancerous stages of CRC, indicating their promoting role in early tumorigenesis.201,202,203 In other tumor types, there are also some cues that microbiota is involved in tumor formation, such as Streptococcus anginosus (S. anginosus) in gastric cancer,204 Acidovorax species in lung squamous cell carcinoma,205 and Bacteroides fragilis in breast cancer.206

The microbiome plays a crucial role in tumorigenesis through various mechanisms, including physical binding or secretion of metabolites and toxins, which lead to genotoxicity and epigenomic abnormalities, activation of signaling pathways, and modulation of the immune system and inflammatory responses.207,208 One of the most well-known examples of genotoxicity is the integration of the HBV genome into the host liver cell genome, which results in genetic mutations and chromosomal abnormalities that promote liver cancer.209,210 HBV DNA most commonly integrates into the telomerase reverse transcriptase (TERT) promoter region, disrupting the tight suppression of TERT transcription and leading to abnormal liver cell proliferation.211,212,213 Furthermore, when HBV inserts into the human cyclin A gene, it generates novel tumor-specific chimeric proteins with oncogenic functions.214,215,216 Pathogenic E. coli also promotes tumorigenesis through genomic alterations. The toxin colibactin, secreted by pks+ E. coli, causes interchain crosslinking and double-strand DNA breaks, leading to gene mutations and tumorigenesis.217,218

In addition to genetic mutations, it has been reported that bacteria significantly contribute to epigenetic alterations.219,220 The human commensal bacterium ETBF could promote distal colonic tumorigenesis in the ApcMinΔ716/+ mouse model. When another BrafV600E was induced, new tumors emerged in the midproximal colon, which exhibited similar phenotypes to human BRAF-mutant serrated-like tumors. The colonization of ETBF and Braf mutation synergistically increased the levels of CpG islands DNA methylation and induced characteristic immunophenotypic alterations, including IFN pathway activation, and myeloid-derived suppressor cells and CD8+ T cell infiltration.219 Furthermore, the microbiota can exert an epigenetic modulation role by influencing the oncogenic effects of mutant proteins. Trp53 mutation plays context-specific roles in intestinal tumorigenesis, promoting tumorigenesis in the distal gut while suppressing tumors in the proximal gut.220 The tumor suppressive effect was achieved through disrupting the binding of T cell factor 4 to chromatin and repression of the WNT signaling. A high density of microorganisms in the distal gut, along with their metabolite gallic acid, has the potential to reverse the protective role of mutant p53 and activate the oncogenic WNT pathway. The administration of antibiotics effectively reduced WNT activation and cell proliferation.220 Furthermore, Fu et al. recently discovered that S. anginosus promoted the tumorigenesis of H. pylori-negative gastric cancer through direct interactions.221 The surface protein of S. anginosus, TMPC, could activate gastric epithelial cell receptor ANXA2, enabling colonization of S. anginosus in gastric mucosa and activation of MAPK pathway.221 As a result, S. anginosus damaged the gastric barrier function, promoted cell proliferation, and inhibited apoptosis of epithelial cells, and ultimately induced gastric cancers.221

Microbes can also play a pro-tumoral role by regulating the immune microenvironment. The microbiome in pancreatic cancer selectively activates Toll-like receptors in monocytes, which in turn drives immune suppression by inducing T-cell anergy, ultimately fostering tumorigenesis.222 Fungi migrating from the intestine to the pancreas also experience fungal dysbiosis. They activate the mannose-binding lectin-complement cascade reaction to accelerate PDAC formation.223 On the contrary, some microorganisms play roles in inhibiting immunosuppression and tumor formation.224,225 Ruminococcus gnavus and Blautia producta, belonging to Lachnospiraceae family, could inhibit the growth of colon tumors by degrading dissolved glycerophospholipids, suppressing their immunosuppressive function, and maintaining the immune surveillance function of CD8 T cells.224 Similarly, during the occurrence of CRC, the urea cycle is activated because of the absence of beneficial bacteria with ureolytic capacity. The accumulation of urea could induce macrophages to polarize towards a pro-tumorigenic phenotype, characterized by polyamine accumulation, thereby promoting the tumorigenesis of CRC.225 Altogether, the complex crosstalk between the microbiome and their hosts in tumorigenesis involves both tumor cells and their microenvironmental cells, inducing changes at genetic, epigenetic, transcriptional, and metabolic levels, which warrants further exploration.

Aging

Aging is considered the primary risk factor for tumorigenesis.226 There are systemic and local changes that overlap with that in tumors, including genomic instability, epigenetic alterations, inflammatory responses, and dysbiosis,227 which may have already played a role as early as in tumor initiating stages. Abnormal epigenetic alterations associated with aging underlie mutation-induced tumorigenesis. In mouse-derived organoids, aging-like spontaneous methylation of DNA promoter CpG-island induced colon more susceptible to the BrafV600E-driven proximal colon tumorigenesis by activating Wnt signaling.114 However, there are some aging hallmarks, including telomere attrition, decreased stem cell plasticity, and cellular senescence-associated cell cycle arrest, possessing tumor-suppressive properties.227 Tumor-initiating cells always evade these tumor-suppressive mechanisms through mutations, such as inactivating mutations in TP53, CDKN2A, and CIP1.228 Moreover, mutations in the promoter of TERT, which allow for the maintenance of telomeres, are one of the most common driver mutations in a variety of tumors, and can be detected even in cirrhotic regenerative nodules, preventing cellular senescence and cell-cycle arrest, and thereby enhancing the proliferative potential of the transformed cells.213,229

In addition to transformed cells, various microenvironmental cells, including fibroblasts, immune cells, and endothelial cells, generally exhibit an increased rate of senescence.230,231 This is accompanied by the secretion of a large quantity of senescence-associated secretory phenotype, including various cytokines, growth factors, enzymes, and extracellular matrix (ECM). Although senescence-associated secretory phenotypes promote the clearance of senescent cells by activating the immune system in youth, it exerts immunosuppressive, pro-inflammatory, and pro-fibrotic effects in aging and chronic inflammation, contributing to tumorigenesis by directly targeting tumor cells or indirectly remodeling the microenvironment.232 In cell competition, hepatocyte growth factor, a component of the senescence-associated secretory phenotype secreted by fibroblasts, was confirmed to inhibit RasV12 cell elimination by inducing their EMT and transformation from apical to basal extrusion.233 Furthermore, the senescence and dysfunction of immune cells can lead to immunosuppression, possibly further increasing the risk of cancer.234 Clearance of senescent macrophages was shown to reduce tumor burden and intercept non-small cell lung cancer at early and intermediate tumor stages by promoting immune surveillance in a Kras-driven lung cancer model.235

Key processes required for early tumorigenesis

The identities of transformed cells are the result of the combined influence of intrinsic genetic and epigenetic profiles and external signaling. These factors collectively activate oncogenic pathways and remodel the microenvironment (Fig. 3). Consequently, there are not only cell-autonomous alterations that override cellular quality control mechanisms, enabling the gradual acquisition of hallmarks of cancer, but also adaptations to the extrinsic stress from their surrounding healthy counterparts, microenvironmental components, and tissue architecture. In addition, transformed cells actively reshape the external factors to be tailored for their oncogenic identities.

Fig. 3

Interactions between oncogenic driver events. a In addition to genotoxicity, chemical and radical insults can induce cell injury, differentiation, and apoptosis. Oncogenic mutations that can confer resistance to such injuries provide proliferative advantages. On the other hand, the insults stimulate proliferative and self-renewal pathways by transcriptional and epigenetic regulation. Immune cells can also be activated to regulate transformed cell fate and promote tumorigenesis. b Unhealthy diet patterns induce hyperglycemia and hyperinsulinemia, and further cause differential response to insulin signals, which can facilitate cells harboring Src or Ras mutation in gaining competitive advantages and promote tumorigenesis. High levels of fatty acids also promote retention of Ras-mutant cells in cell competition by metabolism remodeling and mitochondrial membrane potential restoration. In addition, fatty acid and glucose participate in tumorigenesis as signaling molecules by modulating immune response and inflammation. c Microbiota interacts with transformed cells to affect host DNA methylation, transcription, metabolism and immune microenvironment to have an influence on malignant transformation. d Aging induces senescent stromal cells to secrete SASPs, which can reverse the outcome of cell competition and promote EMT of the mutant cell. Aging also cause spontaneous methylation, further promoting mutation-driven tumorigenesis. e The pathological processes mentioned above can converge at inflammation, which releases tumorigenic potential of expansive clones by activating oncogenic pathways and increases epigenetic plasticity. For instance, in pancreatic inflammation induced plastic state, ADM, Kras-mutant cells are more likely to transform to malignant status, while in the absence of inflammation, Kras can only induce PanIN without progression to PDAC. EMT epithelial-to-mesenchyma transition, ROS reactive oxygen species, nAChR nicotinic acetylcholine receptor, MDSC myeloid-derived suppressor cell, PDK pyruvate dehydrogenase kinase, TCF4 T cell factor 4, HGF hepatocyte growth factor, TET2 tet methylcytosine dioxygenase 2, EGFR Epidermal growth factor receptor, UV ultraviolet, Gata6 GATA Binding Protein 6, EZH2 enhancer of zeste homolog 2, PPAR-δ peroxisome proliferator-activated receptor-delta, FGF21 fibroblast growth factor 21, CCL2 PDK4, pyruvate dehydrogenase kinase 4, ADM acinar-to-ductal metaplasia, PanIN pancreatic intraepithelial neoplasms, PDAC pancreatic ductal adenocarcinoma, ETBF enterotoxigenic Bacteroides fragilis, Created with BioRender.com

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Cell-autonomous processes

Cells in normal tissues are hierarchically organized to restrain tumorigenesis. The initiating transformed cells must reprogram their cell fates, so as to gain uncontrollable self-renewal abilities and aberrant differentiation.2 There are mainly three ways, encompassing activation of unlimited proliferative potential in stem cells, dedifferentiation of lineage-committed and differentiating cells, as well as leveraging intermediate states during trans-differentiation as the precursor of cancer (Fig. 4).

Fig. 4

Cell-autonomous processes in tumorigenesis. After acquiring genetic and epigenetic mutations, transformed cells enter a malignant continuum where they reprogram their developmental pathways, allowing gradually gains of uncontrollable self-renewal capabilities and aberrant differentiation potential, primarily through three mechanisms: originating from stem cells, dedifferentiating from lineage-committed cells, and hijacking intermediate states during trans-differentiation. Created with BioRender.com

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It is believed that stem or early progenitor cells are more likely to achieve malignant transformation, based on their inherent self-renewal capacity and longevity.236,237,238 On one hand, stem cells can accumulate more genetic mutations and epigenetic alterations necessary for tumor formation.239 On the other hand, stem cells and early progenitor cells exhibit high levels of cellular plasticity and are highly susceptible to fate transition.240 In the developmental hierarchy of melanocytes, progenitor stages, including neural crest and melanoblasts, are susceptible to transformation by BRAFV600E and additional mutations, while differentiated melanocytes resist these cancerous signals.241 The difference is induced by ATPase family AAA domain-containing 2 (ATAD2) in neural crest and melanoblasts, which regulates chromatin accessibility.241 This enables TFs including SOX10 and MYC to form complexes with ATAD2, initiating the expression‎ of downstream neural crest genes and oncogenic MAPK pathway genes, respectively.240

In addition to stem cells, there is accumulating evidence suggesting that committed cells are also able to give rise to cancer, specifically after undergoing dedifferentiation into stem-like cells upon oncogenic mutations or environmental stimulation.242,243,244 For instance, melanoma induced by BrafV600E and Pten loss can be originated from mature, pigment-producing melanocytes located in the interfollicular regions of mouse tails, which experienced transcriptional reprogramming and dedifferentiation prior to invasion.245 Consistently, Kaufman et al. identified the fate change during melanoma initiation in a BrafV600E and Tp53 loss zebrafish model. Re-expression‎ of neural crest progenitor program in melanoma, characterized by embryonically expressed gene Crestin, was driven by neural crest progenitor transcriptional factors, such as SOX10.246 Another example where dedifferentiation is implicated in tumor initiation is observed in mammary epithelium. Pik3ca mutation in lineage-restricted mammary basal and luminal cells can both induce multipotent stem-like cells, which is followed by development of tumor heterogeneity and multilineage mammary tumors.247 Luminal progenitor cells derived from BRCA1 basal-like breast cancers have also been confirmed to undergo dedifferentiation.248,249 Mechanically, MYC plays a central role in the reprogramming of the lineage-specific cells. It inhibits mammary luminal-specific TFs, leading to the decommissioning of enhancers that disrupts their original transcriptional program. Additionally, MYC activates de novo enhancers and activates oncogenic pathways, such as the WNT pathway, which supports stem cell features and predisposes luminal epithelial cells to tumor initiation.250

Trans-differentiation is a common physiological response to injury, converting cells that are initially committed to one differentiation fate into an entirely different direction, either directly or through a stem or progenitor cell intermediate. The process can be implicated in tumor initiation, as exemplified in lung tumorigenesis that hijacks repair and regeneration programs. Many types of lung epithelial cells are highly plastic, and are capable of abandoning their cell fidelity to differentiate into each other upon injury, such as the transformation of club cells into AT2 cells, and AT2 cells into AT1 cells, which can be leveraged to promote tumorigenesis.111,251,252,253,254 Specifically, the intermediary state during these transformations is likely to be the key progenitor giving rise to tumors. For instance, KRT8 intermediate cells, which transition between AT2 and AT1 cells, have been identified in normal lung tissues adjacent to LUAD lesions.253 The KRT8 cells expand in precancerous and cancerous stages and are implicated in tobacco-associated KRAS-mutant LUAD,253 marked as reduced differentiation, enhanced plasticity and harboring KRAS driver mutations.253 The high-plasticity cells can also play a role in later progression and development of tumor heterogeneity. In a mouse model of LUAD tumorigenesis originating from KrasG12D mutation and Trp53 loss in AT2 cells, a subset of transitional and high-plasticity cells emerging from adenomas was computationally predicted to drive cellular heterogeneity.254Although they are distinct from stem cells, they exhibit high growth and differentiation potential and play a transitional role in giving rise to the most heterogeneous cancer cell identities, which are indispensable for LUAD progression254 Other classic cases include pancreatic and epidemic injury, where lineage infidelity and epigenetic reprogramming at intermediate stages can be exploited by oncogenic mutations to activate malignant programs.147,150,255,256 Specifically, it is suggested that EMT is a drastic state of plasticity, and its intermediate state also exists, which endows cells with the highest capacity of invasion and metastasis.257,258 Recent evidence indicates that the EMT can occur at a very early stage of tumorigenesis.259,260 In squamous cell carcinomas induced by FAT1 LOF, the mutation triggers both a mesenchymal state mediated by YAP1-ZEB1 and a sustained epithelium state through EZH2 inactivation and SOX2 expression‎, illustrating a hybrid EMT phenotype with enhanced stemness and increased metastatic potential.260

Recently, a series of studies analyzing different stages of precancerous samples across various tumor types at single-cell resolution have demonstrated dynamic evolutionary trajectories preceding tumor formation, revealing a continuum of changes that lead to acquisition of hallmarks of cancers, including cell cycle, cell fate regulation, and metabolic reprogramming128 (Table 3). For example, through single-cell multi-omics analysis of HSPCs from patients with myeloproliferative neoplasm, a convergent genomic evolutionary pattern of a double-hit TP53 mutation in hematopoietic stem cells was identified, and based on this trajectory, pre-leukemia stem cells ultimately progressing to secondary AML were found to undergo differentiation arrest prior to TP53 mutation occurrence, and the subsequent P53 mutant clones could be selected by inflammation, leading to clonal expansion.135 Conventional colon adenomas can be traced back to originating from colonic stem cell (CSC). Throughout the progression from normal stem cells to adenomas and then to colon cancer, there is a gradual change in gene expression‎ and chromatin accessibility, including upregulation of stem-like programs and increased antioxidative stress capability.261,262 On the other hand, premalignant phenotypes induced by intrinsic and environmental drivers have been explicitly depicted in preclinical models. In mouse models and organoids, gastric premalignancies resulting from Trp53 mutations and exposures relevant to the disease have demonstrated the acquisition of renewal properties, activation of the WNT pathway independent of exogenous WNT ligands, and the abilities to overcome cell cycle distress and DNA damage stress.263 Similarly, progenitors of pancreatic tumorigenesis, induced by Kras mutations and inflammation, are characterized as gaining proliferative potential, with activation of cell cycle genes and other pathways.150 Furthermore, in colorectal cancer originating from CSCs, CSCs are fixed predominantly on a highly proliferative phenoscape, whereas there is a continuous differentiation phenoscape that spans revival CSCs to proliferative CSCs under normal condition.264 YAP signaling regulates polarization of revival stem cells, which can be activated by fibroblast derived TGF-β, while APC loss and KRASG12D mutation collaboratively activate MAPK-PI3K signaling, trapping CSCs in the cancerous proliferative fate.264 Compared to ECM signaling, the intrinsic mutations exert a more dominant effect in regulating the stem cell fates.264 The evidence above also suggests that the regulation of malignant transformation involves the interplay between intrinsic cellular factors and microenvironmental factors, which needs to be eval‎uated in a tissue-specific context.

Table 3 Evolution of transformed cells and microenvironment in tumorigenesis

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Clonal expansion by cell competition

Multicellular organisms develop surveillance mechanisms that compare cellular fitness with neighboring cells to preserve the most robust populations in environments with limited space and nutrients, a process termed ‘cell competition’. In epithelial tissues, mutant cells that alter fitness often become the losers and are eliminated by neighboring wild-type cells. Therefore, the process is an important tumor-suppressive mechanism, referred to as ‘epithelial defense against cancer’.265 However, in some cases, mutations can endow cells with ‘winner’ properties, allowing them to eliminate surrounding normal cells and gaining space for clonal expansion and tumor development, which is called ‘supercompetitor’.266

The molecular mechanisms to elicit cell competition include mechanical force, cell-cell contact, and secretory signaling, and losers can be eliminated through various forms, including extrusion, apoptosis, differentiation, necroptosis, and entosis, which are quite different from one tissue to another.267 For instance, in mouse pancreas and intestinal epithelium, apical extrusion of living cells was employed to eliminate Ras-mutant cells, through intercellular communications and alterations in cytoskeleton188,268 (Fig. 5a). On the other hand, in self-renewing tissues, stem cell fate is a decisive factor for cell competition. Stem cells compete to occupy stem cell niche, and the winners have persistent self-renewal properties, while the differentiated cells would be removed from the stem cell niche. The structure of stem cell niches varies across tissue, which may be the cause for various clone sizes and structures in different tissues.7 In the intestinal glandular epithelium, the stem cell niche is located at the bottom of the crypt. Accordingly, competitions are confined to a single crypt and clones rarely expand to other crypts. Under normal conditions, intestinal stem cells (ISCs) stochastically differentiate and migrate upward along the crypt, shedding at the top. Otherwise, they maintain self-renewal and occupy the entire niche to form a monoclonal crypt, a phenomenon referred to as ‘crypt fixation’269 (Fig. 5b). Oncogenic mutations, such as KRAS, APC, and PIK3CA, have the potential to disrupt the neutral drift and tend to achieve crypt fixation.270,271,272 The scenario is different in stratified epithelium, where stem/progenitor cells are distributed throughout the entire basal layer without interference from microstructures. Therefore, fitter stem cells have the potential to expand across the entire structure theoretically, until they encounter cells with the same fitness and end the competition (Fig. 5c).

Fig. 5

Cell competition across tissues. a Live cells can be extruded from simple intestinal epithelium by intercellular communications and cytoskeleton rearrangement. b Intestinal stem cells compete for dominance within the stem cell niche located at the bottom of the intestinal crypt. Mutant supercompetitors are more likely to maintain stemness, replace wild-type counterparts, occupy the ISC niche, and subsequently take over the entire crypt. The displaced wild-type cells, referred to as “losers,” differentiate, migrate upward along the crypt, and are ultimately shed at the top. The fate of stem cells can be regulated by secretory signals that come directly from supercompetitors and indirectly from stromal cells surrounding the crypts, stimulated by the supercompetitors. Stemness inhibitory signals, including BMP activators and NOTUM, differentially affect wide-type cells and supercompetitors by preventing wild-type cells from maintaining stemness, while having less effects on supercompetitors. c In stratified epithelium, the outcome of stem cell competition is also regulated by cell fate decisions. However, it is not limited to specific microstructure as the crypt, the winner clone has the potential to expanding to a large area. WT wild-type cells, BMP bone morphogenic protein, ISCs intestinal stem cells. Created with BioRender.com

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Recent studies indicated that oncogene-mutation supercompetitors have the ability to outcompete their wild-type counterparts by both rising their own fitness and decreasing their competitors fitness.270,271,272 Apc–/– ISCs secrete notum palmitoleoyl-protein carboxylesterase, an antagonist of WNT signaling to inhibit wild-type ISC proliferation and to facilitate Apc-mutant clones towards crypt fixation, ultimately contributing to adenoma formation.270,271 Analogously, ISCs carrying Pik3ca or Kras mutation enhanced secretion of BMP ligand, mediating the differentiation of wild-type ISCs.272 Super-competition has also been observed in Asxl1 CHIP, where mutant HSPCs generate mature offspring with elevated expression‎ of pro-inflammatory genes.273 The inflammatory environment induced differentiation of wild-type cells, while the mutant HSPCs upregulated genes that suppress inflammation to protect themselves from differentiation.273

Interactions with microenvironmental components

The microenvironment is composed of diverse immune cells, fibroblasts, and ECM,274 which have sophisticated interactions with transformed cells. On one hand, the healthy microenvironment plays a tumor-suppressive role and exerts the selective pressure to sculp clonal landscape. On the other hand, the transformed cells can remodel the surrounding niche to support their fitness, and accumulating work has identified early transformation of the microenvironment during tumorigenesis (Table 2). In this part, we aim to illustrate the interplays and co-evolutionary dynamics between mutant clones and their microenvironment during tumorigenesis.

Immune cells

The immune system possesses the capacity to suppress and shape tumors. Immune surveillance can be stimulated by mutation-induced neoantigens. Accordingly, immunogenic pressure selects for transformed cells that can evade immune recognition and killing, as well as those with the capability to sculp an immunosuppressive landscape.

A convergent immune identity is present in almost all established tumors, including varying extents of suppression of cytotoxic T lymphocytes, natural killer cells, and dendritic cells, increases in regulatory T (Treg) cells and other suppressive cells, activation of pro-inflammatory cells, as well as transformation of myeloid cells into pro-oncogenic phenotypes275,276,277,278 (Fig. 6a). There is a continuum of immune evolution accompanying the transformation of cells from pre-cancerous stages (Table 3). For example, a stepwise process of CRC tumorigenesis was shown to be accompanied by a shift from pro-inflammatory to immune-suppressive macrophage populations, along with upregulation of ‘don’t eat me’ CD47-SIRPα signaling.279 Moreover, during the progression of preneoplasia to invasive LUAD, the immune system evolves with downregulation of immune-activation pathways, such as dendritic cell maturation and the acute phase reaction pathway, and upregulation of immunosuppressive pathways including T cell exhaustion signaling and poly adenosine diphosphate-ribose polymerase (PARP) signaling pathways.280 More importantly, the immune transformation may play a decisive role in the transition from precancerous lesions to tumors. Lung carcinoma in situ only progresses to cancer if immune evasion occurs while lesions with an active immune response and higher infiltration of CD8+ T cells would regress.281

Fig. 6

Interactions of transformed cell and microenvironmental components. a Abnormal genetic, epigenetic and transcriptional signals in transformed cells can paradoxically induce immune activation, while simultaneously developing strategies to achieve immune evasion. Their crosstalk is primarily mediated by direct cell-cell interaction signals and paracrine signals, such as chemokines, cytokines and growth factors and direct cell-cell interaction signals. As a positive feedback, tumor supportive immune cells, such as TAMs, which can produce IL-1β signals to further promote malignant evolution. b Transformed cells, along with environmental stress and genetic alterations, can activate fibroblasts through both secretory and contact signals, transforming them into CAFs with diverse tumor-promoting properties. In turn, fibroblasts secrete stemness signals to differentially regulate mutant and wild-type cells during cell competition. c Environmental signals can induce ECM remodeling, and a single transformed cell with ECM adhesion loss can also produce ECM to support its survival. In turn, abnormal mechanical signals in the ECM, including stiffness and viscoelasticity, under pathological conditions such as inflammation, aging, wound repair, and T2DM, predispose mutant cells to malignant progression through the activation of the YAP/TAZ pathway. The pro-tumorigenic effects can be aggravated by mutations in the RTK-Ras pathway. Additionally, a stiff ECM inhibits filamin from translocating from perinuclear areas to the interface of wild-type and mutant cells, further inhibiting the extrusion of mutant cells. TAMs tumor associated macrophages, MDSC myeloid-derived suppressor cell, cGAS cyclic GMP-AMP synthase, STING stimulator of interferon genes, IRF3 IFN regulatory factor 3, T2DM type 2 diabetes mellitus, Yap yes-associated protein, TAZ transcriptional co-activator with PDZ-binding motif, LPAR4 lysophosphatidic Acid Receptor 4. Created with BioRender.com

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As mentioned beforehand, many environmental factors change the immune landscape, stimulating chronic inflammation and increasing tumor susceptibility. In addition, the transformed cells can be a key driver of immune remodeling. Mechanically, tumor cells are able to regulate immune cell activation, chemotaxis, and polarization through paracrine secretion of cytokines, chemokines, and growth factors, or through direct cell-cell interaction signals, such as tumor antigens presented by major histocompatibility complex class l (MHC-I), programmed death ligand 1 (PD-L1), and CD47.282 In turn, a remodeled immune ecosystem supports further malignant progression. The crosstalk between transforming cells and the immune microenvironment is complicated and synergistically promotes the co-evolution. Caronni et al. found that transformed cells secreted high-level prostaglandin E2 and tumor necrosis factor (TNF) and thus promoted infiltration of IL-1β expressing tumor-associated macrophages (TAMs), which drove inflammatory reprogramming of neighboring transformed cells, resulting in a positive feedback loop to aggravate inflammation and tumor progression.283 Another case at this point is in Hras-mutant benign cutaneous papilloma. Upregulation of TGF-β pathway induced transcriptional reprogramming of cancer stem cells, resulting in upregulated expression‎ of leptin receptors in cancer stem cells and angiogenesis.284 As a result, benign tumor cells enhanced sensing and responding to circulatory leptin levels, and activated downstream PI3K-AKT-mammalian target of rapamycin (mTOR) pathway, leading to malignant transformation.284

The immunomodulatory roles of transformed cells can be induced by genetic and epigenetic mutations and aberrate signaling. The driver mutations may serve as a major source of heterogeneity in the immune landscape of early tumors. Early transformation of host immunity in lung tumorigenesis was verified to be strongly associated with the type of driver mutations.280 Mutant Kras induced stronger immune activation compared with that of EGFR mutations from normal and premalignant to cancerous states, including CD8+ T cell infiltration, a low ratio of CD4+/CD8+ T cells and Treg/CD8+ T cells, and higher T cell clonality.280 Indeed, the immunomodulatory roles of the two classic tumor driver mutations have been widely explored. Cells harboring Kras mutation acquire capability to activate STAT3, secrete IL-6 and other proinflammatory cytokines. They also activate NLRP3 inflammasome and release chemokines, such as CCL5 and CXCL3, mediating tumor-promoting inflammation and immune modulation, and further promoting tumor progression.285 Similarly, EGFR mutations have been reported to promote Treg infiltration by upregulating CCL22 through activation of JUN amino-terminal kinase (JNK)/cJUN, and impede CD8+ T cell recruitment through downregulation of IRF1 and CXCL10 pathway.286 Pten deletion promoted PI3Kβ-mediated immune evasion through activation of the AKT and BMX-STAT3 pathways with reduced GM-CSF production, inactivation of dendritic cells, downregulation of antigen presentation pathways, and attenuation of IFNγ-mediated anti-tumor responses. In addition, mutations in TP53, another classical tumor suppressor gene, can not only maintain chronic inflammation by secreting IL-8 through the NF-κB pathway,287 but also inhibit innate immune response by disturbing the cytosolic DNA activated STING-TBK1-IRF3 pathway.288

In addition to genetic mutations, epigenetic and transcriptional factors are also involved in shaping the immune microenvironment. Repression of CXCL9 and CXCL10 expression‎, as well as impairment of CD8+ T cell infiltration in tumors, can be induced by mutations in isocitrate dehydrogenase and global hypermethylation.289 Meanwhile, oncogenic pathways, such as WNT-β-catenin, TGF-β, NF-κB and HIF, have the capability to alter the immune landscape by affecting the communication network between immune cells and cancer cells.290 A genome-wide CRISPR screening for genes modulating immune evasion from cytotoxic T lymphocytes in mouse cancer cells identified those involved in regulating IFN-response and TNF-induced cytotoxicity.291 Similarly, Martin et al. performed CRISPR screening in immunodeficient and normal mice, identifying multiple tumor suppressor genes that were positively selected by the adaptive immune system. These tumor suppressor genes are involved in various crucial cellular processes, such as chromatin interaction, antigen presentation, protein stability regulation, TGF-β signaling, and IFNα signaling.292 Although the evidence above is primarily based on research in established tumors, the effects of immune evasion are now being highlighted at the earliest stages of tumorigenesis. SOX17 deregulated IFNγ receptor expression‎ and further lowered the expression‎ of MHC-I and CXCL10, as well as CD8+ T cell infiltration. These changes played indispensable roles in the in vivo adaptation of genetically engineered naïve colon cancer organoids.293 In ESCC tumorigenesis, pathological overexpression‎ of SOX2 activated endogenous retroviral elements and promoted double-stranded RNA formation, which should have activated immune surveillance.294 However, parallel upregulation of ADAR1 in turn attenuated the IFN signaling and contributed to immune escape.294 Interestingly, metabolic identities of tumor cells and immune components can also play a role in their interactions, forming competitive or dependent relationships with each other. On one hand, metabolites of tumor cells promote immunosuppressive effects,295,296 and in turn, phagocytosis of TAMs facilitates nutrient accumulation to meet energy requirement of tumor cells.297 On the other hand, there is nutritional competition between immune cells and tumor cells.298 mTORC1 signaling in TAMs plays a role in regulating the competition.299 Under normal protein diet conditions, the mTORC1 pathway is weakened in TAMs and thereby be enhanced in Myc-overexpressing tumor cells, resulting in a competitive advantage of tumor cells. Conversely, under low-protein diet conditions, activation of the GTPase-activating proteins GATOR in TAMs leads to TFEB/TFE3 nuclear translocation and mTORC1 activation in TAMs. As a consequence, TAMs gain an advantage over tumor cells in metabolic competition, exerting tumor-suppressive effects.299 Whether the mechanism is involved in early tumorigenesis warrants further exploration.

There are some arguments for the timing of immune activation and evasion. It is believed that there is an immune ignorance at the earliest cancerous stage where only a few transformed cells are present, and low levels of neoantigens they produced are deficient to activate immune clearance.275,300 The immune surveillance may not be a decisive factor for the initial clonal expansion.41 A mathematical model was developed to separate the fitness of driver mutations based on positive oncogenicity and negative immunogenicity. It revealed that TP53 mutations in non-cancerous tissues were primarily selected for their pro-oncogenic proliferative advantage rather than negatively selected by immunogenicity.301 When progressing to advanced tumors, the pro-tumoral evolutionary force shifted into powering immune evasion. The shift could also explain the reason for different TP53 hotspot mutations between cancer and normal tissues.301 The timing of switch from immune ignorance to activation and subsequent evasion need further exploration. High-resolution multiregional spatial and single-cell multi-omics sequencing are well suited to assess this issue. For instance, Cody et al. constructed a pseudo-temporal trajectory of colorectal tumorigenesis based on CIN and hypermutated pathways in their spatial multi-omic atlas, and mapped immune state changes along progression pseudotime, thereby facilitating prediction of immune exclusion.302

Fibroblasts

Fibroblasts constitute the primary stromal cellular components and serve major roles in ECM production, tissue structure maintenance, regulation of stem cells, interactions with immune cells, and participation in wound repair. Their role in regulating cell fate through paracrine orchestration can be hijacked by transformed cells to promote tumorigenesis272,303 (Fig. 6b). In the ISC niche, prostaglandin E2 secreted by a rare population of PTGS2-expressing fibroblasts can act on Sca-1+ ISCs and activate Cox2-Yap signaling for ApcMin/+ stem cell expansion and colon tumorigenesis.303 The stem cell niche signals produced by stromal cells also participate in the competition between oncogenic-mutant and wild-type cells. Pik3ca mutant ISCs showed an expansion advantage, partially by inhibiting stromal WNT signaling and creating a detrimental condition for the survival of wild-type ISCs.272

Alternatively, it is well-documented that cancer-associated fibroblasts (CAFs) are an important component in the TME. They can be activated by various stimuli in cancerous tissues, including TGF-β, inflammatory factors such as IL-1, IL-6, and TNF-α, physiological and genomic stress, ECM changes, and contact signals.304,305,306 In addition, CAFs promote tumor growth by remodeling the ECM, inducing immune evasion, and directly interacting with tumor cells.304 It has been confirmed that they emerge and contribute to the earliest stage of tumorigenesis (Table 3). The transformed cells are the main driver for CAF transformation.307,308,309 We identified a reciprocal mechanism between fibroblasts and epithelial cells, evolving synchronously in the multistep ESCC tumorigenesis.307 In the early stage of tumorigenesis, epithelial cells gradually downregulated ANXA1 expression‎ due to the suppression of transcription factor KLF4. Subsequently, the formyl peptide receptor type 2, an ANXA1 receptor on fibroblasts responsible for fibroblast homeostasis, was dysregulated and drove the transformation of CAFs. This process was accompanied by TGF-β secretion from transformed cells, further accelerating CAF transformation.307 Similarly, the epithelial-stromal interactions mediated by JAG1 on ductal carcinoma in situ cells and NOTCH2 on fibroblasts play a role in CAF transformation and mammary tumorigenesis.

Apart from transformed cells, other abnormal signals can prime pro-tumorigenic identity of fibroblasts before transformation. Mutations in fibroblasts, such as BRCA1 and NOTCH1 can also be regarded as the prerequisite of tumorigenesis. In addition, external stimuli, including aging, dense microenvironment and exposure to oncogenic insults can all promote transformation.233,310,311,312 Dermal fibroblasts under UV exposure induce suppression of NOTCH and its effector CSL, and promote the production of inflammatory cytokines, growth factors and matrix metalloproteinases, contributing to precancerous actinic keratosis lesions and CSCC formation.313

Extracellular matrix

The ECM is mainly composed of fibrous proteins and glycosaminoglycans, providing mechanical support, cellular anchoring, and storage for water and various bioactive molecules.314 Additionally, the ECM communicates with cells through local adhesions, converting chemical and mechanical signals into biological signals, regulating key cellular processes such as proliferation, apoptosis, fate decision, and migration. This process is known as mechanosensing and mechanotransduction.315 During tumorigenesis, the ECM experiences remodeling mainly driven by CAFs, tumor cells, and macrophages, leading to increased deposition, cross-linking and stiffness. As a result, the changes promote malignant progression by transducing abnormal biomechanical signals to transformed cells, as well as regulating immune recruitment and activation.314,316

The abnormal ECM has been profoundly investigated in established tumors, however, their earlier roles in regulating clone evolution before cancer formation and the accurate timing for oncogenic disorganization are unclear. Recently, Wu et al. reported the role of ECM remodeling in tumor initiation, where a solitary transformed cell at the very beginning of tumorigenesis met much more stress in a normal microenvironment than that in established tumors, including loss of cell-cell contact between tumor cells and pro-tumor ECM317 (Fig. 6c). The individual pancreatic cancer cell enhanced production of ECM and adapted to isolated stress by increasing expression‎ of the stress-responsive gene lysophosphatidic acid receptor 4 (LPAR4) and promoted the production of fibronectin-rich ECM, which could compensate for the absence of stromal-derived factors and help tumor initiation.318 Furthermore, the ECM could also support neighboring cells without upregulated expression‎ of LPAR4 through integrins α5β1 or αVβ3.318

Ahead of the transformed cell driven ECM remodeling, many pathological conditions, such as chronic inflammation, aging, and tissue injury, can increase stiffness of ECM and prime a tumor-vulnerable state.319 At the initial stage, stiffness influences the epithelial defense of oncogenic mutation. Filamin, an actin filament cross-linking protein located at the interface of wild-type and mutant cells, facilitates the extrusion of mutant cells under normal physiological conditions. However, when ECM is stiff, filamin relocates to the perinuclei and leads to the failure of epithelial defense against cancer and causes tumorigenesis.320 Furthermore, stiff signaling plays a role in cell fate regulation, and further regulates the susceptibility to oncogenic transformation.321,322 In the condition of SmoM2 induced basal cell carcinoma, the back skin, which has a denser collagen I network compared with the skin of the ear, was not susceptible to the mutation-induced progenitor state reprogramming and tumor initiation.321 Chronic UV exposure and aging can decrease the expression‎ of collagen, overcoming the natural resistance and increasing the risk of tumorigenesis.321 Orthogonally, oncogenic mutations render tumor-initiating cells to be more sensitive to signals of ECM stiffness. Even slight changes in ECM rigidity can trigger abnormal responses in cells harboring mutated oncogenes in the RTK-Ras pathway, such as human epidermal growth factor receptor 2 (Her2) and Kras.322 Stiffness and the mutations synergistically activated the YAP/transcriptional co-activator with PDZ-binding motif (TAZ) pathway, subsequently promoting the transformation of precancerous states.322 In addition to stiffness, viscoelasticity is another pro-tumorigenic mechanical property of ECM, which can be induced by advanced glycation end-products (AGEs) accumulation by type 2 diabetes mellitus. It is characterized by decreased interconnectivity of collagen matrix, shorter fiber length and greater heterogeneity, activating integrin-β1-tensin-1-YAP pathway and promoting cancer progression.323 Notably, YAP/TAZ serves as a molecular hub for mechanosensing and mechanotransduction, which is activated by mechanical signals transmitted by cytoskeletons, and is followed by the nucleus translocation and gene expression‎.324 Since abnormal YAP/TAZ pathway is strongly associated with various tumors,324 it is suggested that its oncogenic mechanotransductive signaling may be a general trait implicated in early malignant transformation. The interaction between mutations and mechanical signaling during tumorigenesis warrants further investigations.

Tissue architecture restraint

Tissue structure is shaped by collective mechanical characteristics of individual cells, as well as their interactions with neighboring counterparts, stromal cells and the ECM. The maintenance of three-dimensional structural balance relies on stable number and arrangement of cells, which is also an important tumor-suppressive mechanism. Since there are tight interconnections and limited space in solid tissues, cell proliferation and elimination generate mechanical stress by the resistance of surroundings, thereby providing feedback to regulate cell behaviors.325 When over-proliferative cells cause density increase and compression, dense responsive signals are activated to suppress proliferation and eliminate redundant cells.326 Differential sensitivity to mechanical signals triggers cell competition.327 The mutations that endow cells with insensitivity to compression would be preserved (Fig. 7a). For example, when subjected to compression, Scribble mutant Madin-Darby canine kidney cells tended to undergo apoptosis due to p53 activation by ROCK and p38 pathways.328 On the contrary, RasV12 mutant cells downregulated ERK in neighboring wild-type cells via competition, triggering apoptosis of wild-type cells.329

Fig. 7

Tissue architecture restraint for clone expansion and alterations in tumorigenesis. a Cell density sensing mechanisms trigger apoptosis in cells sensitive to compaction, whether mutant or wild-type. b Mutations that disrupt the ECM and enable anchorage-independent survival allow cells to move to the lumen and expand. Additionally, the loss of cell-cell junctions can unleash the proliferative potential of mutations in situ instead of through translocation. c Mutant intestinal crypts are more likely to split rather than merge, increasing their number but still keeping overall balance through spreading and decreasing local crypt density. The Kras mutation speeds up this splitting, to a degree that cannot be counteracted by dispersal, leading to tumorigenesis. d When the homeostatic tissue architecture is disrupted, the mutant cells mediate a dysregulated tumor structure. This manifests as alterations in cell-cell junctions, cell-ECM adhesions and cytoskeleton rearrangement. The initial tissue curvature, as well as the stiffness of the basal membrane and suprabasal cells, all affect the nascent tumor morphogenesis. In addition, the EMT mediated by EFNB1-EPHB4 interactions in epithelial cells harboring TP53 mutations occurs alongside early malignant morphogenesis. MMP metalloprotease, EMT epithelial-to-mesenchymal transition. Created with BioRender.com

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Furthermore, cell-cell junctions and cell-ECM adhesions are other important factors to arrest oncogenic growth and maintain homeostasis326,330(Fig. 7b). A well-organized acinar structure formed by a non-transformed human mammary epithelial cell line, MCF10A, remained quiescent in the presence of sporadic oncogenic mutations with proliferative potential until they expressed matrix metalloproteinases and disrupted cell-matrix adhesions. This disruption resulted in the translocation of mutant cells into the lumen, releasing more space for expansion.330 In addition, although detachment from the ECM alleviated the space limitation, the loss of the survival signal provided by the ECM would also lead to decreased fitness and apoptosis.330,331 Only cells that achieve anchorage-independent survival could continue to expand.330 Furthermore, the extrusion of mutant cells is also regulated by cell-cell junctions. Disruption of cell-cell junctions leaded to the transformation of the proliferative cells from lumen translocation to proliferation in situ.330

Some tissues have microstructures, which impose another barrier to the expansion of mutant clones.37,332,333 As mentioned earlier, the expansion of mutant stem cells is typically limited to a single intestinal crypt. Further expansion requires crypt fission, but it is a rare event for normal adult tissues, at approximately one fission every 27 years.334 Additionally, there are concurrent crypt fusions to maintain crypt density.335 Some mutations can break the balance and speed up crypt fission333,336 (Fig. 7c). This may account for discrepant elevations in the frequency of crypt fission without a concurrent rise in crypt fusion.334 Alternatively, dispersal of intestinal crypts occurs to counteract rising crypt density. However, the rate of crypt fission in Kras mutant crypts is too fast to be accommodated through dispersal, resulting in an increase in the local density of crypts, which increases the risk of polyps and tumor formation.334

Alongside overriding the structural restrictions of normal tissues, early tumor morphogenesis is shaped by cell proliferation, abnormal mechanics of transformed cells, and their microenvironment337,338 (Fig. 7d). Ras mutation induces MCF10A transformed cells to aggregate from two-dimensional (2D) to 3D structure through differential localization of E-cadherin at the top and bottom layers, reduction of adhesion to ECM, and redistribution of epithelial tension regulators. Neighbor structures of the lesion are also implicated. In tubular epithelia, whether lesion growth occurs outwards or inwards to the ductal lumen results from the balance between cellular tension of the lesion and the resistance of the tissue curvature.338 In stratified epithelium, the assembly of the basal membrane and the stiffness of superbasal layers also play a significant role in shaping tumors. Tumor budding is promoted by well-remodeled and soft basement membrane in SmoM2 induced basal cell carcinomas (BCCs). By contrast, in HRasG12V induced squamous cell carcinoma, stiffness from basal membrane and superbasal stratification promotes a folding architecture, which is more likely to develop an invasive tumor.339 However, molecular mechanisms underlying the gradual oncogenic tissue disorganization are not well understood. Based on spatial transcriptomic technology, our laboratory recently deciphered spatiotemporal expression‎ patterns and identified key molecules driving the stepwise tissue destruction in esophageal tumorigenesis. Transformed cells interacted with each other through EFNB1-EPHB4 and triggered cell proliferation and EMT by SRC/ERK/AKT signaling, which were possibly instigated by ΔNP63 overexpression‎ due to a TP53 mutation.259

Cancer risk prediction and intervention strategies

Molecule-based cancer risk prediction

A better understanding of molecular and phenotypic determinants of malignant transformation facilitates cancer prevention, while the first step is to conduct risk assessment. Traditionally, it relies mainly on histopathological identification of precancerous lesions and combined demographic risk factors to identify individuals at high-risk of developing cancer, whereas predictive values are generally low. Only a small proportion of pathologically identified precancerous lesions progress to invasive tumors, inducing overdiagnosis and unnecessary interventions.340 In addition, as we have discussed above, some precancerous molecular alterations can emerge precedent or independent of morphological abnormalities. Therefore, molecular drivers identified in early tumorigenesis can be exploited to improve the efficacy of risk stratification, and further improve targeted surveillance and early interception.

Detection of germline mutations to eval‎uate inherited cancer susceptibility is widely explored, as exemplified by BRCA1 and BRCA2 pathogenic variations for breast and ovarian cancers.341 In the past few decades, large-scale case-control association studies across cancer types have facilitated the identification of cancer-risk loci and the development of polygenic risk scores for risk prediction.342 The combinations of polygenic risk scores and other known risk factors, including family history, lifestyle and reproduction, have been shown to accurately predict life-long risks of breast cancer.343 On the other hand, the pervasive existence of cancer driver mutations in normal tissues not only provides opportunities but also places higher demands for somatic molecule-based risk prediction, requiring accurately distinguishing between those as normal background and those as a consequential cancer signal during tumorigenesis. For example, in Barrett’s esophagus, TP53 mutation and 17p LOH are relatively more specific predictors of progression to esophageal adenocarcinoma,59,344 with the TP53 mutation even capable of predicting progression in samples with no dysplasia.345 Furthermore, a predictive panel of multiple driving mutations will have better performance than the TP53 mutation alone.346,347,348,349 Another case of point is in CHIP, where it has been used in combination with hematologic and biochemical indicators to develop three independent risk prediction models for progressions to different myeloid neoplasms, including AML, myelodysplastic syndromes, and myeloproliferative neoplasms in a cohort of 454,340 UK Biobank participants, enabling early prediction of tumor occurrence in normal individuals.350 Given that CIN and CNAs are already present in specific precancerous diseases and accumulate throughout malignant progression, such as in Barrett’s esophagus, CNAs may also be a potential strategy for predicting risk of cancer.351,352,353,354 A genomic instability-based model was reported to distinguish patients with Barrett’s esophagus at high-risk of progression, among which 50% patients in the high-risk group were predicted 8 years before transformation of high-grade dysplasia or cancer.351 Furthermore, based on evolutionary measurement, genetic clonal diversity and clonal expansion are explored as predictive indicators of malignant evolution in colon, esophagus, and blood, potentially being a more universal method for various cancers.355,356,357,358,359 DNA methylation is also a promising type of risk prediction marker, demonstrating value in risk prediction for Barrett’s esophagus progression and gastric cancer formation.360,361,362,363 Liquid biopsy tests of circulating cell-free DNA fragments and/or their methylation patterns have gained widespread attention due to their non-invasiveness, low cost, and viable implementation. Tests for tumor DNA methylation have been validated in detecting multiple advanced cancers, whereas it appears to perform poorly in early-stage tumors.364,365 Advances in technology and more precises predictive panels are required to enhance this promising testing tool for use in premalignant stages.

Rapid development of high-throughput omics technologies in recent years has facilitated explorations of numerous biomarkers, and predictive panels based on transcriptomics, proteomics and metabolomics have been developed for specific tumors (Table 4). Based on serum metabolomics, lung adenocarcinoma and its preneoplasia can be distinguished from benign lesions by a metabolic panel.173 A gut microbiome-based panel has also shown efficacy in distinguishing CRC and adenoma from normal tissues, and further research is needed to verify its predictive role in disease progression.202,203 Based on multiplexed ion beam imaging by time of flight and tissue transcriptomics, Risom et al. mapped a spatial cellular landscape of ductal carcinoma in situ (DCIS) and delineated spatial and functional coordinated changes in stromal components from DCIS to invasive breast cancer, including myoepithelium, fibroblasts, and immune cells. Based on the features, they developed a risk prediction model for breast cancer invasion, which is largely dependent on myoepithelium and stroma. Intriguingly, disruption of myoepithelium indicates low risk of progression, which is contrary to the traditional belief that an intact myoepithelial barrier protects from tumor invasion, and the mechanism has not yet been detected.366 Altogether, multidimensional molecular features in the transition of tumors could be utilized to develop predictive assays. Nevertheless, most studies to date are based on small cohorts and sometimes lack validation cohorts, requiring further validations before being introduced into clinic.

Table 4 Molecular markers for cancer risk prediction

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Intervention strategiesChemoprevention

Chemoprevention refers to the use of synthetic or natural substances to reduce the risk of developing cancers (Table 5). The most popular chemoprevention strategy is endocrine therapy for breast cancer prevention. Indeed, endocrine therapies have been widely attempted for breast and prostate cancer prevention, by inhibiting binding of sex steroids and their receptors to block downstream gene regulation and tumor cell growth.367 Females with high-risk breast cancer are recommended to use selective estrogen receptor modulators, such as tamoxifen and raloxifene, or aromatase inhibitors, which inhibit aromatization of androgens and decrease the level of estrogens, but specific adverse events need considerations, including fracture, thrombosis, endometrial cancer, and cataract.368,369 In placebo-controlled randomized trials, tamoxifen can reduce the incidence of breast cancer by 31%, while raloxifene, aromatase inhibitors, exemestane and anastrozole, reduce it by 56% and 55%, respectively.369 They may also be effective in preventing DCIS.369 Similarly, 5α-reductase inhibitors, such as dutasteride and finasteride, have been attempted for prostate cancer prevention by inhibiting the synthesis of dihydrotestosterone, the most potent endogenous androgen.370,371,372 Although they have demonstrated an overall reduction in prostate cancer risk, the efficacy in high-grade prostatic tumor prevention requires further confirmation.370,371,372

Table 5 Potential agents for cancer prevention

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Given the important roles of inflammatory responses in tumorigenesis, anti-inflammatory regimens for cancer prevention are of great interest. Nonsteroidal anti-inflammatory drugs, especially aspirin, have shown preliminary efficacy in the prevention of various cancers, including those of the central nervous system, breast, esophagus, stomach, head and neck, liver, bile duct, colorectum, endometrium, lung, ovaries, prostate, and pancreas.373,374 Evidence for aspirin in preventing CRC is the most definitive. However, due to its severe adverse event of gastrointestinal bleeding, it is currently only recommended for Lynch syndrome and patients with removed familial adenomatous polyposis but is not routinely recommended for healthy individuals.375,376,377 Targeting key pro-carcinogenic inflammatory factors, such as IL-1, IL-6, and TNF-α, may enable more precise cancer prevention. In the cardiovascular CANTOS trial, the intervention arm using canakinumab, an IL-1β monoclonal antibody, significantly reduced lung cancer incidence.378 However, the costs and fatal adverse events of cytokine targeting therapy necessitate careful consideration for preventive applications.

Metformin is the first-line treatment for type 2 diabetes mellitus, primarily targeting molecules involved in energy metabolism, such as mitochondrial complex I, MAPK, and mTOR. It also plays a role in reducing insulin levels, enhancing insulin sensitivity and exerting effects on immune cells.379,380 In cell competition models, metformin reverses insulin resistance or enhances aerobic glycolysis, eliminating the competitive advantage of mutant cells, suggesting its potential inhibitory effect on tumor initiation.184,381 Since a preliminary retrospective case-control study in Scotland was reported in 2005, the preventive use of metformin for tumors has been supported by several observational studies.382 A randomized controlled trial in Japan confirmed the protective role of metformin from adenoma and polyp recurrence383; however, there is a lack of further evidence from intervention trials for the reduced risk of various cancers with metformin use.384 It is hypothesized that personalized regimens of metformin may be necessary, in order to particularly target tumors that are dependent on oxidative phosphorylation, as metformin primarily targets mitochondrial respiration. Additionally, due to metabolic reprogramming of tumor cells after metformin treatment, combination therapy targeting metabolic pathways on which tumor cells depend may enhance metformin efficacy.380 Another focus of metabolic regulation is statins, a class of drugs used to treat lipid disorders. As inhibitors of 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase (HMGCR), statins are associated with reduced overall cancer risk, and liver, prostate, lymphoma, and CRC risks.385,386,387,388,389,390,391,392,393 Statins inhibit de novo cholesterol synthesis via the meval‎onate pathway and promote the removal of plasma low-density lipoprotein cholesterol by acting on low-density lipoprotein receptors. Mechanistically, statins exert anti-tumor effects by promoting cell death, regulating angiogenesis, and modulating immunity.394 In early stages of colorectal tumorigenesis, they can also modulate gut microbiota.395 Treatment with statins increases microbial tryptophan availability in the gut, promoting the growth of Lactobacillus reuteri, which converts tryptophan into indole-3-lactic acid and regulates Th17 cells to inhibit tumor formation. More evidence is required to support their clinical use.

A deeper understanding of molecular events in early tumorigenesis offers insights into novel interventional targets. Accurately identifying the cell state at the root or pivotal transitional point along the pathway of malignant transformation is essential for searching for potential targets within these cells. For instance, compared with wild-type luminal epithelium, the mammary epithelium in individuals carrying BRCA2 mutations exhibits an increased number of ERBB3lo luminal epithelial cells, which potentially serve as the cells of origin for both ER+ and ER- breast cancers.396 mTORC1 signaling is significantly upregulated in these cells. Short-term treatment with a mTORC1 inhibitor substantially curtailed tumorigenesis in a preclinical model, thus uncovering a potential strategy for BRCA2 breast cancer prevention.396 In tobacco-induced LUAD, considering that the identified progenitor cell subset harbors KRAS mutation, it is logical to hypothesize that KRAS mutation inhibitor can play a role in intercepting the earliest tumorigenesis.253 Similarly, as TP53 mutations are widespread in normal tissues and are identified as the early key driver events in various tumor initiation, there are many explorations for TP53 targeting strategies. Methods such as blocking murine double minute 2 (MDM2), as well as restoring dysfunctional p53 are being attempted.397 It is expected to identify more promising targets and accordingly develop robust agents in the future.

Immunoprevention

Immunoprevention involves modulating the host immune system to elicit early anti-tumor immune responses, eliminating tumor cells at precancerous stages. The most classic example of immunoprevention is vaccination against carcinogenic pathogens, such as human papillomavirus for preventing and treating cervical intraepithelial neoplasia,398,399 and HBV for preventing hepatocellular carcinoma.400,401,402,403 In addition to viral-based vaccines, vaccines targeting tumor antigens have been attached great attention in recent years. The targets can be tumor-associated antigens abnormally overexpressed in tumor cells compared to normal tissues, such as carcinoembryonic antigen (CEA) and HER2, or aberrantly post-translationally modified antigens, such as Mucin 1 (MUC1).404 Among them, HER2-based vaccines have preliminarily shown success in interception of DCIS in clinical trials.405,406 In addition, since aberrant hypoglycosylation of MUC1 occurs in precancerous lesions of multiple epithelial cancers, there are many attempts of vaccines targeting MUC1 in clinical trials for various cancers.404 However, it was recently reported that there were limited effects of MUC1 vaccine in preventing colonic adenomas in a randomized controlled trial. Individuals with advanced adenoma received MUC1 peptide vaccine within 1 year after adenoma removal. Despite eliciting significant antigen-specific immune responses, adenoma recurrence did not significantly decrease.407 Therefore, further study is still required to improve vaccine efficacy for preventive usage. Another strategy is to target tumor-specific driver mutations that have already occurred in precancerous stages, such as KRAS and EGFR mutations for lung cancer prevention.408,409,410 These tumor-specific antigens are likely to be more immunogenic with better responses, but clinical trials are required to verify their efficacy and safety. Clinical trials on the mutant KRAS-targeted long peptide vaccine for high-risk pancreatic cancer recipients and EGFR-targeted vaccine for high-risk lung cancer recipients are currently underway (NCT05013216, NCT04298606).

As described above, immunosuppressive microenvironment has emerged at an early stage of tumorigenesis; accordingly, immunomodulatory strategies are being attempted for tumor prevention. For precancerous actinic keratosis of the skin, a combination of calcipotriol and 5-fluorouracil was adopted in a randomized controlled trial, which can induce squamous cell expression‎ of thymic stromal lymphopoietin, thereby mobilizing anti-tumor immunity. Compared to using 5-fluorouracil alone, the combination showed significant lesion reduction, accompanied by upregulation of thymic stromal lymphopoietin, HLA-II, natural killer cell group 2D ligand expression‎, as well as CD4 T cell infiltration.411 Long-term follow-up indicated that the effects of immunomodulation persisted three years later, with a decrease in the incidence of CSCC.412 PD-L1 and PD-1 upregulation has been observed in precancerous lesions of the oral cavity413,414 and lung tumors,415 suggesting that PD-1 monoclonal antibodies are an ideal early immunoprevention strategy. Currently, relevant clinical trials are underway (NCT03347838, NCT03603223). In a preliminary trial to eval‎uate the safety and clinical response of anti-PD-1 therapy among patients with high-risk proliferative verrucous leukoplakia, 12 patients (36%) (95% CI, 20.4%-54.8%) had a > 80% decrease in size and degree of dysplasia after receiving nivolumab, suggesting potential clinical activity for nivolumab in high-risk proliferative verrucous leukoplakia.416

Lifestyle and dietary interventions

Lifestyle and dietary interventions are low-cost, low-risk, and accessible preventive strategies. There are various advocated healthy lifestyles against cancers, including avoiding and ceasing exposure to carcinogens such as tobacco, alcohol, and UV, as well as adopting healthy diets and engaging in regular exercise.417,418,419,420 In terms of dietary interventions, multiple healthy dietary patterns, such as the Mediterranean diet, vegan diets, and various healthy diet guideline indices have been proposed.417 Their core tenets are avoiding carcinogenic dietary components, controlling total calories, and increasing proportions of beneficial constituents.421 However, most evidence is based on epidemiological associations from population studies, and many confounding factors cannot be excluded.421 Exploring the molecular mechanisms behind specific nutrients in healthy diets for cancer prevention can not only strengthen the evidence supporting existing dietary interventions, but also yield insights for developing novel and scientifically grounded strategies. Low calorie intake and various fasting regimens are confirmed to inhibit nutrient sensing pathways and activate nutrient scarcity sensors to regulate cellular stress responses, modulating tumor cell activity and anti-tumor immune response.422 Another popular regimen is ketogenic diet, which means to intake low carbohydrates, high fat, and moderate protein to enhance ketone metabolism. Some clinical trials have confirmed its therapeutic effects in patients with breast cancer undergoing chemotherapy or radiotherapy.423,424,425 Although there is a lack of clinical evidence to support the preventive usage of ketogenic diet, its benefits have been shown in preclinical models. Ketogenic diet induced-β-hydroxybutyrate could bind the Hcar2 receptor on intestinal stem cells and activate tumor suppressive TF Hopx to inhibit cell proliferation and exert anti-cancer effects.426 Furthermore, since oral supplement of β-hydroxybutyrate alone could achieve an anti-tumor effect, it may be served as an alternative regimen for the ketogenic diet, possibly addressing the issue of low compliance with strategies that change the overall dietary pattern.426 Specific diets may also act as prebiotics or probiotics.427 The most popular one is high-fiber diets, which are associated with a lower risk of multiple cancers.428,429,430,431,432,433 Dietary fiber can be fermented into short-chain fatty acids by microbes to regulate microbe composition and diversity, protect intestinal mucus barrier, and prevent bacterial translocation, thereby modulating systemic metabolism, immunity, and inflammation.434,435,436,437 A small randomized cross-over trial confirm‎s that supplementing fermentable fiber inulin and inulin-propionate ester, which is aimed at delivering short-chain fatty acids to the colon, can modulate gut microbes, metabolism, and inflammation, thereby improving insulin resistance.438 Recently, the BE GONE trial confirmed similar findings through the supplementation of beans, a fiber-rich food. Soybeans can act as prebiotics, regulating gut microbes, inflammation, and metabolism, improving biomarkers of metabolic obesity and colon cancer.439 However, direct evidence from fiber intervention trials for cancer prevention is still lacking. Another study held an opposite conclusion. It found that high-dose soluble fiber could dysregulate gut microbiota and metabolites, leading to enrichment of potentially pathogenic bacteria and depletion of probiotics, and prompt colorectal tumorigenesis.440 Specific effects of high-fiber diets are still warranted to be further explored.

Apart from adjusting dietary structure and macronutrient intake, direct supplementation of specific anti-tumor nutrients and metabolites is a more implementable strategy. Given the epigenetically tumor-suppressive effects of α-ketoglutarate demonstrated in mouse models, dietary supplementation of its precursor molecule glutamine may be a potential preventive strategy.177 Other dietary supplements, including marine omega-3 fatty acids sourced from fish and seafood,441 as well as the plant-derived natural alkaloid berberine,442 have demonstrated preliminary efficacy in preventing CRCs. Further large-scale clinical trials and long-term follow-up are required. On the other hand, various vitamins have been proposed for tumor prevention, and some of them have illustrated promising applications. Nicotinamide, which belongs to vitamin B3 family, plays a role in inhibiting oxidation and DNA damage.443,444 A Phase III clinical trial showed that it can effectively reduce the risk of non-melanoma skin cancers and actinic keratoses in high-risk populations.445 However, it failed to show a preventive effect in immunocompromised individuals following organ transplantation, possibly due to DNA damage resulting from the use of immunosuppressive drugs.446 In addition, low-dose acitretin, a vitamin A derivative, has been demonstrated to have a preliminary preventive effect on skin cancer in organ-transplanted recipients. Renal transplanted patients with actinic keratosis received acitretin therapy (20 mg/d) for 1 year and there was an improvement of actinic keratosis in all patients.447 Mechanistically, acitretin exerts an anti-tumor effect by increasing the number of epidermal Langerhans’ cells and enhancing skin immune monitoring.447 Other examples of effective preventive strategies include vitamin D for DCIS and high-dose folic acid for recurrent colorectal adenoma.448,449 However, these clinical trials are limited by their small scale and short-term follow-up. Apart from the examples mentioned above, there is a notable scarcity of successful cases in interventions using other vitamins and micronutrients.450,451 This underscores the need for more high-quality retrospective and prospective studies to eval‎uate the potential impacts of micronutrients. Such studies should be conducted in conjunction with preclinical research that demonstrates molecular mechanisms, thereby facilitating the identification of compounds suitable for future dietary interventions.

Conclusion and future perspectives

Driven by genetic and epigenetic alterations along with environmental signaling, transformed cells not only acquire cell-intrinsic proliferative advantages, but also actively remodel their environment to support their aberrant behaviors during early tumorigenesis. Encouragingly, apart from mutagenesis, many determinants of tumorigenesis are reversible, and understanding the molecular mechanisms underlying early malignant evolution provides significant translational opportunities. Cancer prevention aims to identify high-risk individuals and implement early interventions with high efficacy, low adverse events, and the potential to cure. Since many targetable aberrative pathways in advanced tumors have also been found in the earliest stages, including those affecting the cell cycle, anti-apoptosis, metabolic remodeling and immune evasion, classic anti-tumor agents might be repurposed for earlier interventions. However, extensive clinical trials to verify their efficacy and safety are warranted, and the balance of expenses and benefits should be considered.

Tumors originate from individual cells, presenting significant challenges in capturing these rare cell subsets. Advances in next-generation sequencing, single-cell, and spatial omics have revolutionized the study paradigm of tumorigenesis. At an extremely high resolution, precursor clones of various tumors have been identified, and co-evolutionary dynamics of the transformed cells and their microenvironment are being depicted. Furthermore, integrative analyses of paired omics modalities, such as genome, epigenome, and transcriptome, have been preliminarily applied to map the early tumorigenesis events,117,135,302,452 offering insights into the ordering and interplays among multiple evolutionary drivers, as well as their roles in regulating cellular phenotype. As multiplexing spatial and single-cell multi-omics technologies continue to enhance their throughput, resolution, and accuracy, coupled with innovations in bioinformatics tools to analyze unprecedented high-dimensional data,453 it is anticipated that multi-omics approaches can be leveraged to achieve a more comprehensive understanding of the complex biological processes of tumorigenesis.

To date, many studies primarily infer evolutionary trajectories computationally from multisampling of cancer specimens. However, this approach is limited to capturing only the dominant malignant clones and their major driver events. There is a loss of information regarding dynamic precancerous clonal competition and selection, since other precancerous clones may have been swept out in advanced tumors. Therefore, the importance of employing multiple sampling strategies to cover various stages of the malignant continuum is being increasingly recognized. Specifically, acquiring both cancerous and non-cancerous clones with shared ancestors simultaneously can optimize phylogenetic analysis results, depicting both the malignant evolutionary dynamics and the fate of remaining non-cancerous clones with partially shared mutations. This approach highlights the additional changes necessary for evolution into a malignant phenotype and their sequence among various driver events.454 Given that there are some premalignant stages that do not progress to invasive tumors, it is emphasized that rational cohort design in longitudinal studies to distinguish premalignant lesions from regression to progression can indicate key mechanisms that ultimately drive tumorigenesis. Yet, significant challenges remain in ensuring patient compliance and completely removing precancerous lesions during initial sampling, which usually aims at prevention and may interrupt natural disease progression.455 Alternatively, inducing autochthonous tumors in animal models or organoids offers an alternative way to study the early evolutionary processes. By prospectively introducing driver events informed by prior knowledge, and integrating lineage tracing with in vivo imaging techniques, real-time clone dynamics and their temporal evolutionary trajectory are visible, further facilitating the study of biological functions of specific perturbations in early tumorigenesis. At this point, Yao et al. recently reported their protein level reporter system, which is capable of tracing mutant p53 protein accumulation, a cancer-specific event as well as a potential mark for early transformed cells.456 The system sensitively identified rare precancerous cells in noncancerous tissues, and further facilitated characterization of cellular phenotypes underlying transformation, as well as the identification of potential interventional targets.456 In the future, a deeper understanding of the ordering and interactions of the driver molecular events, and their dynamic evolution under varying local and systemic environmental pressures and during specific tumorigenic phases, will help us gain more insights into tumor prevention, diagnosis, and early intervention.

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