저산소 환경과 암세포 발생, 성장의 관계 - 논문 4편 꼭 읽어야

[문형철]

beyond reason

진정한 치유는 기쁨, 감사, 축복 540 치유의 에너지 장에서 일어난다

현대의학 선배탐구자들에게 감사와 존경을

--> 이 과정에 저산소 환경이 관여하고 세포 DNA 돌연변이, 손상이 일어나 암이 발생하고 전이가 생김. 

henze2010.pdf

Cell Cycle. 2010 Jul 15;9(14):2749-63. Epub 2010 Jul 30.

Feedback regulators of hypoxia-inducible factors and their role in cancer biology.

Henze AT1, Acker T.

Author information

1

Institute of Neuropathology, Giessen, Germany.

Abstract

Malignant tumors are characterized by regions of low oxygen concentration (hypoxia). The hypoxic tumor microenvironment contributes to tumor progression by activating a set of adaptive responses via the key transcriptional regulators HIF-1alpha and HIF-2alpha. These factors have been traditionally linked to an aggressive tumor phenotype by promoting processes essential for tumor growth, such as angiogenesis, glycolysis, metastasis and invasion, as well as differentiation and self renewal. Notably, the complex HIF pathway also initiates anti-tumorigenic mechanisms that lead to cell cycle arrest or cell death, indicating the need for a stringent control of the extent and the direction of the hypoxia response. The importance of this control for tumor cell survival is illustrated by the intricate regulation of HIF activity at the mRNA, protein and epigenetic level by a complex network of positive and negative feedback regulators. 

- 악성종양은 저산소 환경이 특징임. 저산소 종양환경은 HIF(hypoxic inducible factor, 저산소 유발인자)에 의해 활성화되어 종양의 진행에 영향을 줌. 

- 이러한 HIF들은 종양성장을 위한 필수물질(과정)인 혈관신생(angiogenesis), 당분해(glycolysis), 전이와 침윤(metasis and invasion) 그리고 분화와 self renewal을 촉진하여 악성종양으로 만듬. 

- complex HIF pathway는 세포사멸을 일으키는 anti-tumorigenic 기전을 개시함. 

- 암세포 성장조절을 위한 중요성이 그림으로 그려짐. 

We propose that these feedback regulators help to flexibly adjust and adapt HIF activated responses to the fluctuating oxygen concentrations within tumors during acute and chronic hypoxia and to curtail the tumor-suppressing components of the HIF pathway. Moreover, feedback regulation of HIF induces a switch from HIF-1 alpha to HIF-2 alpha driven responses under chronic hypoxia which may have essential functions in the regulation of tumor cell differentiation and tumor stem cell maintenance. Given their central role in cancer biology, HIF feedback regulators may represent an attractive and novel anti-tumor therapy target to overcome cell death resistance in tumors.

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- Hs-CRP 수치가 1mg/L이하일때 보다 3mg/l이상일때 모든 암발생, 사망위험이 각각 38%, 61%(남자), 29%, 24%(여자) 올라간다는 연구도 있음 

J Epidemiol. 2011; 21(3): 161–168. 

Published online 2011 May 5. Prepublished online 2011 Feb 26. doi: 10.2188/jea.JE20100128

PMCID: PMC3899404

PMID: 21368452

High-Sensitivity C-Reactive Protein and Cancer

Seounghee Lee,1,2 Jae-Won Choe,1 Hong-Kyu Kim,1 and  Joohon Sung2

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Abstract

Background

High-sensitivity C-reactive protein (hs-CRP) is a commonly used inflammatory marker. The association between hs-CRP and cancer is less consistent than that between hs-CRP and cardiovascular diseases. This study explored the association between hs-CRP and cancer, using a large database of Korean health examination records.

Methods

A total of 80 781 Koreans who visited the health promotion center of a general hospital were included. There were 729 cases of cancer of any primary site during a 3-year period. Subjects with a known cancer or a condition capable of affecting hs-CRP were excluded.

Results

Serum hs-CRP was significantly higher in cancer cases (2.9 mg/L) than in non-cases (1.4 mg/L; P < 0.0001). With the lowest hs-CRP category (<1 mg/L) as reference, the crude odds ratios (ORs) for cancer were 1.36 (95% confidence interval [CI] = 1.16–1.62) for the second highest category (1–3 mg/L) and 2.49 (95% CI = 2.02–3.07) for the highest category (>3 mg/L), and the adjusted ORs for cancer were 1.16 (95% CI = 0.95–1.42) for the second highest category and 1.94 (95% CI = 1.51–2.51) for the highest category. After excluding cancer cases detected within 1 year after the check-up, the associations remained, although the reduced number of cancer cases (n = 88) attenuated the significance of the associations.

Conclusions

Serum hs-CRP was positively associated with the risk of cancer, although causality cannot be inferred in this cross-sectional study. The results support the hypothesis that chronic inflammation plays a role in cancer.

Key words:  high-sensitivity C-reactive protein, cancer, inflammation

HIF1-alpha expressing cells induce a hypoxic-like response in neighbouring cancer cells

• Hannah Harrison, 
• Henry J. Pegg, 
• Jamie Thompson, 
• Christian Bates & 
• Paul Shore 

BMC Cancer volume 18, Article number: 674 (2018) Cite this article

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Abstract

Background

Hypoxia stimulates metastasis in cancer and is linked to poor patient prognosis. In tumours, oxygen levels vary and hypoxic regions exist within a generally well-oxygenated tumour. However, whilst the heterogeneous environment is known to contribute to metastatic progression, little is known about the mechanism by which heterogeneic hypoxia contributes to cancer progression. This is largely because existing experimental models do not recapitulate the heterogeneous nature of hypoxia.

The primary effector of the hypoxic response is the transcription factor Hypoxia inducible factor 1-alpha (HIF1-alpha). HIF1-alpha is stabilised in response to low oxygen levels in the cellular environment and its expression‎ is seen in hypoxic regions throughout the tumour.

Methods

We have developed a model system in which HIF1-alpha can be induced within a sub-population of cancer cells, thus enabling us to mimic the effects of heterogeneic HIF1-alpha expression‎.

Results

We show that induction of HIF1-alpha not only recapitulates elements of the hypoxic response in the induced cells but also results in significant changes in proliferation, gene expression‎ and mammosphere formation within the HIF1-alpha negative population.

Conclusions

These findings suggest that the HIF1-alpha expressing cells found within hypoxic regions are likely to contribute to the subsequent progression of a tumour by modifying the behaviour of cells in the non-hypoxic regions of the local micro-environment.

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Background

Tumour hypoxia greatly influences breast cancer progression [1,2,3] and is generally linked to poor prognosis in breast cancer patients due to increased proliferation, cell survival, invasive/metastatic ability, therapy-resistance and altered cancer stem cell activity [4,5,6,7,8,9]. The main mediator of signalling in these poorly oxygenated areas is Hypoxia inducible factor 1-alpha (HIF1-alpha) which is stabilised at low oxygen levels activating numerous downstream pathways [10].

Areas of hypoxia form as tumours outgrow their blood supply and exist within otherwise well-oxygenated tumours. The hypoxic landscape is very dynamic and oxygen levels fluctuate and hypoxic areas shift as the tumour progresses and new blood supplies are formed [11]. How the hypoxic tumour micro-environment contributes to cancer progression and metastasis is not fully understood and this is in part due to a lack of experimental models in which the heterogeneous nature of hypoxia can be modelled. Currently, in vitro, hypoxia is modelled in hypoxic incubators set at low oxygen levels (e.g. 1%) [6] or by treating cells with a chemical mimetic, such as Dimethyloxalylglycine (DMOG) or cobalt chloride (CoCl2) [12]. These methods expose the whole population of cells to hypoxia, which is a situation that is unlikely to exist in tumours.

We have therefore developed a novel model in which heterogeneous expression‎ of HIF1-alpha can be induced in a sub-population of cells. Uniquely, this model enables analysis of changes in HIF1-alpha expressing cells and their effect on surrounding cells in which HIF1-alpha is not expressed. Using this model we demonstrate that, in addition to changes in proliferation, gene expression‎ and mammosphere forming cell activity in the HIF1-alpha expressing cells, significant changes also occur within the HIF1-alpha negative population. These findings demonstrate that HIF1-alpha expressing cancer cells have a profound effect on the co-cultured HIF1-alpha negative cells. Similar effects are likely to be seen in tumours with hypoxic, HIF1-alpha positive cells having influence upon the normoxic cells surrounding them. These changes to the local micro-environment may be important in disease progression and metastasis.

Methods

Cell lines

MCF7 and MDA-MB-231 were purchased from American Type Culture Collection (ATCC® HTB-22™ and HTB-26™). Cell lines were established for use in cell culture and no ethical approval was required for their use. Lines were authenticated by multiplex-PCR assay using the AmpF/STR system (Applied Biosystems) and confirmed as mycoplasma free. Monolayers were grown in complete medium (DMEM/10% FCS/2 mmol/L L-glutamine/PenStrep) and maintained in a humidified incubator at 37 °C at an atmospheric pressure of 5% (v/v) CO2/air.

Inducible cell line production

A yellow fluorescent protein tagged destabilising domain YFP-DD was removed from pBMN YFP-DHFR (DD) (a gift from Thomas Wandless, Addgene plasmid # 29326) and cloned into HA-HIF1-alpha P402A/P564A-pcDNA3 (a gift from William Kaelin, Addgene plasmid # 18955). The new vector, mHIF-YFP-DD-pcDNA3 was used to transfect MCF7 and MDA-MB-231 using Lipofectamine 2000 according to manufacturer’s instructions. Following selection with G418 cells were FACS sorted as single cells to produce stable, clonal cell lines. Proteins fused with YFP-DD are made unstable and although they are transcribed and translated continually, they are not seen to be expressed. Trimethoprim (TMP, Cay-16,473, Cayman) was used to induce stabilisation of the fusion.

Hypoxic and hypoxia mimetic cell culture

Cells we incubated for 48 h in the SCI-tiveN hypoxic workstation (Ruskinn) in 1% O2, 5% CO2 and 94% N2 in a humidified environment at 37 °C. Cells were plated, cultured, and harvested within the workstation to maintain hypoxia at all times. For cobalt chloride (CoCl2) induction, 100 μM CoCl2 was added to fresh culture medium and cells were incubated for 48 h in a humidified incubator at 37 °C at an atmospheric pressure of 5% (v/v) CO2/air. Following hypoxic culture or exposure to CoCl2, induction of HIF1-alpha was verified by Western blot (Additional file 1: Figure S4).

Conditioned medium

Conditioned medium (CM) was collected and spun at 1000 g for 10 min to remove cell debris. CM was stored at -20 °C for no longer than 2 weeks. CM was mixed 50:50 for fresh complete medium before being used to treat cells. The HIF1 inhibitor YC-1 was added to monolayer culture at a concentration of 10 mmol/L YC-1 (Cayman Chemicals).

Mammosphere culture

Mammosphere culture was carried out as previously described [13], and spheres greater than 50 μm were counted on day 5. Briefly, cells were plated in non-adherent culture as single cell suspensions at a concentration of 2500 cells/6 well plate. Cells were cultured in DMEM:F12 supplemented with B27 (Thermo Fisher) for 5 days.

Co-culture

Parental and inducible cells were mixed at a ratio of 50:50 and cultured in the presence of TMP for 48 h. Cells were then separated based upon expression‎ of YFP using the FACS Aria. When comparing to mono-culture all cells were passed through the sorter to account for any pressure/sorting effect.

Proliferation assay

Cells were plated on glass cover slides for 24 h. ClickIT™ EdU kit Plus EdU Alexa Fluor™ 647 Imaging Kit (C10640, Thermo) was used according to manufacturer’s instructions. Briefly cells were cultured with EdU for 45 min to allow incorporation of fluorescently labelled EdU into newly synthesized DNA therefore marking the proliferating cells. Cells were then fixed, permeablised and labelled with ClickIT-647.

Nuclear/cytoplasmic separation

Cells were resuspended in 400 μl of ice cold Buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF) with the addition of complete mini-EDTA-free protease inhibitor cocktail (Roche) and incubated at 4 °C for 15 min. Cells were lysed with 10% NP-40 (Sigma) before centrifuging at 4 °C and removal of the cytoplasmic extracts in the supernatant. The pellet was then resuspended in ice cold Buffer B (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT and 1 mM PMSF) containing protease inhibitors and vortexed vigorously for 45 min at 4 °C. Nuclear proteins were collected from supernatant following centrifugation at 4 °C. Both the nuclear and cytoplasmic extracts were stored at − 80 °C for future use.

Immunocytochemistry

Cells were grown in monolayer on coverslips for 48 h +/− 10 μM Trimethoprim (TMP). Medium was removed and cells were fixed and permeablised with 4% formaldehyde and 0.1% Triton for 20 min at room temperature. Non-specific binding was blocked using 1% BSA before antibody staining: rabbit anti-GFP (ab290, Abcam), Phalloidin-TRITC (P1951, SIGMA) or Phalloidin-iFluor 647 (ab176759, Abcam), Goat anti-Rabbit Alexa Fluor 488 (A11008, Thermo) and Goat anti-Mouse Cyanine3 (A10521, Thermo). Coverslips were mounted with ProLong™ Gold Antifade Mountant with DAPI (P36940, Thermo).

Image analysis

Images were collected on a Zeiss Axioimager.D2 upright microscope using a 10× objective and captured using a Coolsnap HQ2 camera (Photometrics) through Micromanager software v1.4.23. Specific band pass filter sets for DAPI, FITC and Cy5 were used to prevent bleed through from one channel to the next. Images were then processed and analysed using Fiji ImageJ (http://imagej.net/Fiji/Downloads) [14] which is freely available online.

Western blotting

Protein was separated on an SDS–PAGE and transferred to Hybond-C Extra nitrocellulose membrane. Primary antibodies included: HIF-1a (610,959, BD Biosciences) and Lamin B1 (ab16048, Abcam).

Densitometry was conducted using ImageJ software, which is freely available at http://rsb.info.nih.gov/ij/.

Quantitative reverse transcription PCR

RNA was extracted using the Qiagen RNAeasy kit according to manufacturer’s instructions and quantified on the Nanodrop spectrophotometer (Thermo). Real time one step qRT-PCR was carried out using the QuantiTect SYBR® Green RT-PCR Kit (Qiagen) according to manufacturer’s instructions before analysis on the 7900 PCR machine (Applied Biosystems).

Statistical analysis

Data is represented as mean +/− SEM. Statistical significance was measured using parametric testing, assuming equal variance, in the majority of experiments with standard t tests for 2 paired samples used to assess difference between test and control samples.

Results

Inducible HIF1-alpha cancer cell lines

To determine the effects of HIF1-alpha expressing cells on the surrounding HIF1-alpha negative cells we developed a model culture system in which HIF1-alpha can be induced in a sub-population of cells. To achieve this we fused a YFP-tagged destabilising domain (YFP-DD) to HIF1-alpha, thus enabling the stabilising effect of hypoxia on HIF1-alpha to be mimicked by the addition of an inducer molecule [15, 16]. The DD fusion protein is stable in the presence of Trimethoprim (TMP) but when TMP is removed the protein rapidly degrades (Fig. 1a). In this model YFP-DD was fused to a mutant form of HIF1-alpha (mHIF) which is stable in normoxia [17]. This fusion was stably expressed in MCF7 (ER+) and MDA-MB-231 (ER-) breast cancer cell lines [MCF7-mHIF-YFP-DD and 231-mHIF-YFP-DD] and inducible expression‎ was verified by Western blot for HIF1-alpha performed on nuclear extracts following 48 h of culture with 10 μM TMP (Fig. 1b, Additional file 1: Figure S1 shows an unedited blot). Control lines were produced expressing YFP-DD only (MCF7-YFP-DD and 231-YFP-DD, data not shown).

Fig. 1

a Schematic detailing the inducible system used in our novel cell line model. A mutant form of HIF1-alpha (mHIF) which is stable in normoxia is fused to a YFP-tagged destabilising domain. The fusion is readily transcribed and translated but is unstable and broken down in the absence of Trimethoprim (TMP). When TMP is added the fusion is stabilised and expressed. b Stabilisation and expression‎ following addition of 10 μM TMP was assessed by Western blot of nuclear extracts. Western blots were performed on single membranes cut in two with upper section labelled for mHIF and lower section labelled for Lamin B1. c To activate downstream signalling HIF1-alpha must interact with HIF1-beta to translocate into the nucleus. Successful nuclear translocation was verified by immunocytochemistry. Scale bar equal to 25 μm. d qRT-PCR was performed on control (YFP-DD) and inducible (mHIF-YFP-DD) cells in the presence or absence of 10 μM TMP. Gene expression‎ was seen to change in the inducible cells suggesting HIF1-alpha activation of signalling. e Expression‎ of mHIF-YFP-DD was measured over time following addition of TMP (green bars) and following wash out of TMP (red bars) in both MCF7 and 231 lines. Significantly increased expression‎ was seen in both lines within 1 h and the signal had returned to normal after 8 h. f Expression‎ of mHIF-YFP-DD was measured over time following addition of a single dose of TMP at Day 0. Expression‎ remained elevated over 4 days

Full size image

To assess localisation and control of induction, MCF7-mHIF-YFP-DD and 231-mHIF-YFP-DD cells were cultured with 10 μM TMP for 48 h. Cells were then fixed and stained with an anti-GFP antibody to identify HIF1-alpha positive cells and Phalloidin-TRITC and DAPI to define the cytoplasm and nucleus respectively. Nuclear localisation, which is required for active HIF1-alpha signalling, was not affected by the increased size resulting from fusion to YFP-DD as the protein was present in the nucleus of induced cells (Fig. 1c). Activation of HIF1-alpha target genes was subsequently determined using qRT-PCR for three known genes. Significant increases in VEGF, EPO and CAIX were seen following the addition of 10 μM TMP (Fig. 1d). No change was observed in the control lines.

Induction occurred quickly with a significant increase in expression‎ after 1 h in MCF7 cells and 30 min in MDA-MB-231. Expression‎ reached its highest level by 24 h in both lines (Fig. 1e). Following removal and wash out of TMP, expression‎ rapidly decreased returning to un-induced levels after 8 h. If TMP was not removed, induction remained high over 4 days following a single treatment with 10 μM TMP (Fig. 1f). Taken together these data show that the stabilising effect of hypoxia on HIF1-alpha can be mimicked by the addition of TMP in these cell lines.

Stabilising HIF1-alpha with TMP mimics known hypoxic responses

We next determined if stabilising HIF1-alpha by the addition of TMP simulated the response known to be induced by hypoxia (1% O2) and a chemical hypoxia mimetic (100 μM CoCl2). qRT-PCR was performed on a panel of known HIF1-alpha target genes (VEGF, GLUT1, EPO, CAIX and TGFA) after 48 h induction with TMP, hypoxia or CoCl2. In all three conditions target genes were induced to similar levels (Fig. 2a). TMP had no effect on gene expression‎ (data not shown).

Fig. 2

a qRT-PCR was performed for cells from hypoxic culture (parental 21% vs 1% O2), hypoxia mimetic culture (parental +/− CoCl2) and our HIF1-alpha inducible system (mHIF-YFP-DD +/− TMP). A similar pattern was seen in all conditions. b Following 48 h in hypoxic culture (parental 21% vs 1% O2), hypoxia mimetic culture (parental +/− CoCl2) or our HIF1-alpha inducible system (mHIF-YFP-DD +/− TMP) cells were plated in mammosphere culture. A comparable response was seen in all conditions. c Conditioned medium was collected from cells cultured in hypoxia (parental 21% vs 1% O2) or our HIF1-alpha inducible system (mHIF-YFP-DD +/− TMP) and was used to treat parental cells (MCF7 and MDA-MB-231) for 48 h. These cells were then plated in mammosphere culture. The conditioned medium induced the same hypoxic response as the culture condition from which it came regardless of the cells being treated

Full size image

Hypoxic culture is known to have a contrasting effect on the cancer stem cell (CSC) population in breast cancer depending upon the disease sub-type [6]. In breast cancers which express estrogen receptor (ER), CSC activity increases following exposure to hypoxia. In contrast, there is a significant decrease in CSC activity in ER-negative tumours. This effect was previously shown to be directly down-stream of HIF1-alpha as siRNA targeting HIF1-alpha blocked the effect of hypoxic culture completely [6]. To assess whether the same effects were seen in our HIF1-alpha expressing cells, mammosphere formation was assessed following 48 h of culture with TMP. Mammosphere number was increased in MCF7-mHIF-YFP-DD cells and decreased in 231-mHIF-YFP-DD cells as is seen in hypoxic culture and following CoCl2 exposure (Fig. 2b).

Secreted factors are known to play a major role in the regulation of hypoxia in tumour signalling both locally and to distant sites [18]. We aimed, therefore, to assess whether the CSC response seen following hypoxic culture or following HIF1-alpha induction in our model was controlled via secreted signals. Conditioned medium (CM) taken from cells cultured in hypoxia had the same effect on mammosphere formation as hypoxic culture itself regardless of the cell type being treated; CM taken from MCF7 (ER+) cells caused an increase in mammosphere number in both MCF7 and MDA-MB-231 whilst CM taken from MDA-MB-231 (ER-) caused a decrease in mammosphere formation in both MDA-MB-231 and MCF7 cells (Fig. 2c). This effect was blocked with addition of the HIF1-alpha inhibitor YC-1 during production of CM (Additional file 1: Figure S2). The same paracrine effects were seen when using CM collected from inducible lines; CM collected from MCF7-mHIF-YFP-DD and 231-mHIF-YFP-DD treated with TMP resulted in altered mammosphere formation in both MCF7 and MDA-MB-231 (Fig. 2c). Our data suggest that the HIF1-alpha induced mammosphere response is mediated by paracrine signals secreted by the hypoxic or HIF1-alpha expressing cells. The secreted factor (s) is different depending on the cell type releasing it and its effect is not cell type specific as CM from either cell line induced the same response in both parental lines. This supports the hypothesis that the hypoxic cancer cells within a tumour can influence the local micro-environment by affecting the different cell types surrounding them.

Expression‎ of HIF1-alpha in a sub-population of cancer cells induces significant changes in surrounding cells

To simulate the heterogeneous nature of HIF1-alpha expression‎ that is seen within a population of tumour cells we generated co-cultures by mixing wild-type MCF7 or MDA-MB-231 cells with HIF1-alpha inducible cells. TMP was added to co-cultures for 48 h before cells were isolated by flow sorting to separate HIF1-alpha positive (YFP positive) from negative cells (Additional file 1: Figure S3). These cultures were subsequently used to examine the effects of the HIF1-alpha positive cells on the surrounding parental sub-population.

Following co-culture and separation, MCF7 and MDA-MB-231 cells were plated into non-adherent culture to assess the effects on mammosphere formation. Induction of HIF1-alpha in mono-culture resulted in increased and decreased mammosphere formation as expected in MCF7 and MDA-MB-231 respectively (Fig. 3a). Importantly we observed that TMP induction of HIF1-alpha in MCF7 co-cultures also resulted in a significant increase in mammosphere formation in the non-induced sub-population of cells (Fig. 3a). In MDA-MB-231 co-cultures a reduction in mammospheres was observed. Moreover, qRT-PCR analysis of the HIF1-alpha target genes also revealed a significant increase in their expression‎ in the parental, co-cultured cells (Fig. 3b). This included an increase in TGF-alpha expression‎ in the parental cell lines following co-culture, a change which is not seen in the HIF1-alpha positive inducible cells grown in either mono- or co-culture or in those cultured in hypoxia (1%) or CoCl2 culture. All genes responded similarly to HIF1-alpha induction in the HIF1-alpha expressing cells in both mono- and co-cultures (Fig. 3b). To determine the effects of HIF1-alpha induction on the proliferative capacity of neighbouring cells, ClickIT Edu was used to assess changes in proliferation after HIF1-alpha induction in co-cultured cells. HIF1-alpha negative cell proliferation was altered in MCF7 and MDA-MB-231 when grown in co-culture with HIF1-alpha expressing cells; MCF7 cells proliferated more quickly whilst MDA-MB-231 cells grew more slowly (Fig. 3c). HIF1-alpha has been shown to reduce proliferation in cancer cells [19] and, as expected, the HIF1-alpha positive cells proliferated more slowly than the parental cells in monoculture of both lines. Interestingly, the HIF1-alpha expressing cells proliferated more quickly when grown in co-culture and in the case of MDA-MB-231 there was no significant difference between the parental and inducible cells in this case. This change suggests a bi-directional effect of co-culture on proliferation suggesting a great deal can be learned from this system.

Fig. 3

Following 48 h in the presence of TMP in either mono-culture (MC) or co-culture (CoC), mHIF-YFP-DD and parental cells were plated into mammosphere culture or used to make RNA for qRT-PCR. a Mammosphere formation increased or decreased in the mHIF-YFP-DD expressing lines as expected in MCF7 and 231 cells respectively. Interestingly, mammosphere forming cell number was also affected in the both parental cell lines following co-culture. b Gene expression‎ changes were seen within the HIF1-alpha inducible cells as expected. Changes were also visible, however, in the parental cells grown in co-culture. c Representative photomicrographs showing EdU stain in mono- and co-culture, Green anti-GFP, Blue DAPI and Red EdU. Scale bar equal to 25 μm. Proliferation, assessed by counting cells positive for EdU, was increased in mHIF-YFP-DD and parental cells cultured in co-culture

Full size image

Taken together, these data reveal that induction of HIF1-alpha expression‎ in a sub-population of cancer cells has a significant effect on gene expression‎, the mammosphere forming population and the proliferative capacity of the surrounding cancer cells. Thus our model system has revealed a complex interplay between HIF1-alpha positive and negative cancer cells that are likely to have a significant effect on the development of a tumour.

Discussion

The inducible system described here allows HIF1-alpha, the main regulator of hypoxia, to be switched on and off within a sub-population of cells. In our model HIF1-alpha stabilises and degrades within a relatively short period of time and, although these kinetics may not exactly replicate those seen in vivo, this will allow us to model fluctuations in expression‎ of the protein and enable us to gain a more realistic view of its effects on cancer cells within a tumour. All of the experiments were performed in normoxic conditions enabling the specific effects of HIF1-alpha induction to be determined in the absence of further confounding changes due to hypoxia.

Using a co-culture model we have demonstrated that expression‎ of HIF1-alpha is sufficient to induce the same expression‎ changes in important target genes as commonly observed in hypoxia and CoCl2 exposure. Also, the induction of HIF1-alpha causes the same contrasting effect on mammosphere forming cells in MCF7 and MDA-MB-231 cells that result from hypoxic and CoCl2 culture [6]. This model system revealed that when cultured together with parental cells, there is a significant effect on mammosphere formation in this HIF1-alpha negative population. Mammosphere formation increases in parental MCF7 and decreases in MDA-MB-231 when cultured in the vicinity of HIF1-alpha expressing cells. As MS are thought to be representative of the CSC population [13] this is potentially of great importance as it implies that the CSC population in tumours may be altered both within and outside of the hypoxic zones. This would mean that the effects of hypoxia could be far more wide reaching than initially assumed. Further verification of changes to the CSC population are required before any solid conclusions can be drawn. These HIF1-alpha induced changes in the parental cell population could not have been identified using hypoxic chambers and chemical mimetics.

Our model allows us to separate and investigate the effects of the main hypoxia regulator, HIF1-alpha, from other factors which are involved in the hypoxic response within tumours, expression‎ of HIF2-alpha for example. This allows us to see more clearly the effects of HIF1-alpha and may allow us to further elucidate the role of this protein in the hypoxic and non-hypoxic zones of a tumour. Studying the effects of these other factors is of course important and work is underway to investigate them in our model system.

Co-culture of our inducible cells with the parental lines also showed gene expression‎ changes within the parental HIF1-alpha-negative cells. Changes included an increase in TGF-alpha expression‎ which was not seen in the HIF1-alpha positive inducible cells or in those cultured in hypoxia (1%) or with CoCl2. TGF-alpha is a known target of HIF1-alpha [20] but it was not activated within our HIF1-alpha induced or hypoxic cultured cells. TGF-alpha has been previously suggested as a marker of poor outcome at follow-up and specifically for lymph node metastasis [21], whether this is linked to hypoxia is yet to be seen but this kind of change in the bulk of the tumour may explain how HIF1-alpha induction by hypoxia in the primary tumour can increase metastasis even though this recurrence often happens many years after removal of the primary tumour.

Proliferation rates were seen to be affected in both the parental and HIF1-alpha expressing cells during co-culture experiments. This bi-directional signalling cannot be identified using conventional culture systems. This data further suggests that reduced oxygen may affect not only the cells within the hypoxic region, which are expressing HIF1-alpha, but also the bulk of the tumour and supports the need for a culture system in which HIF1-alpha negative cells are represented alongside HIF1-alpha expressing cells as this change would not be seen in conventional culture.

This new inducible co-culture system allows HIF1-alpha induction within a sub-population of cancer cells and so facilitates the analysis of the complex interplay between HIF1-alpha positive and negative cells, which are found within hypoxic and normoxic tumour cells. We also note that stabilisation of HIF1-alpha is fast and the ligand can be washed out of the system with expression‎ falling quickly. Thus the system also allows the effects of the dynamic changes in HIF1-alpha seen in tumour hypoxia to be analysed. This is important as it will allow us to model the fluctuating hypoxia observed in tumours [11]. Finally, co-culture with other cell types from the local micro-environment is also possible and this may reveal more complexity in the hypoxia induced response to HIF1-alpha in tumours.

Conclusions

This model offers the opportunity to study, in detail, the complex interactions between HIF1-alpha expressing cells and those around them. This may offer insight into the effects HIF1-alpha expressing cells have on their local micro-environment leading to altered metastasis and disease progression. Our data shows a clear bi-directional effect within the co-culture environment which could not be identified previously.

This unique model can easily be adapted to produce inducible co-cultures with other genes, which are important in other environments and diseases, meaning it offers a great deal of potential to the wider scientific community.

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• Published: 15 January 2015

The role of hypoxia-inducible factor-2 in digestive system cancers

• J Zhao, 
• F Du, 
• G Shen, 
• F Zheng & 
• B Xu 

Cell Death & Disease volume 6, pagee1600(2015)Cite this article

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Abstract

Hypoxia is an all but ubiquitous phenomenon in cancers. Two known hypoxia-inducible factors (HIFs), HIF-1α and HIF-2α, primarily mediate the transcriptional response to hypoxia. Despite the high homology between HIF-1α and HIF-2α, emerging evidence suggests differences between both molecules in terms of transcriptional targets as well as impact on multiple physiological pathways and tumorigenesis. To date, much progress has been made toward understanding the roles of HIF-2α in digestive system cancers. Indeed, HIF-2α has been shown to regulate multiple aspects of digestive system cancers, including cell proliferation, angiogenesis and apoptosis, metabolism, metastasis and resistance to chemotherapy. These findings make HIF-2α a critical regulator of this malignant phenotype. Here we summarize the function of HIF-2 during cancer development as well as its contribution to tumorigenesis in digestive system malignancies.

Facts

• Hypoxia is a common phenomenon in digestive system cancers.

• HIF-2α is an essential mediator of the cellular oxygen-signaling pathway.

• HIF-2 plays important roles in digestive system cancers and upregulates genes involved in cell proliferation, angiogenesis and apoptosis, metabolism, metastasis and resistance to chemotherapy.

• HIF-2 inhibitors can suppress the expression‎ of HIF-2 target genes to reduce angiogenesis and metastasis.

Open questions

• Studies assessing the HIF-2α roles in digestive system cancers are scarce, with inconsistent outcomes. Therefore, the actual roles of HIF-2α are still unclear.

• The definite mechanisms of HIF-2 activity during digestive system cancers remain elusive. How does HIF-2 take part in tumorigenesis of digestive system cancers?

• The exploitation of HIF-2α inhibitors faces many challenges.

Introduction to HIF-2α

HIF-2 is the second member of the hypoxia-inducible factor (HIF) family that includes three proteins: HIF-1, HIF-2 and HIF-3. Its amino-acid sequence shares ∼48% homology with that of HIF-1.1, 2, 3 HIF-2 is a heterologous dimeric protein complex that consists of two subunits: HIF-2α and HIF-1β. Both HIF-2α and HIF-1β belong to the superfamily of transcription factors bHLH-PAS. HIF-2α, also named endothelial PAS domain protein-1 (EPAS1), member of pas superfamily 2 (Mop2), hypoxia-inducible factor 2α subunit or Hif-2-α, was identified in 1997.1, 2, 3, 4 It binds to and activates transcription from the HIF-1α response element derived from the 3-prime flanking region of the EPO gene. Hypoxic conditions stimulate HIF-2α activation.5 Like HIF-1, HIF-2 is primarily regulated by specific prolyl hydroxylase-domain enzymes (PHDs) that initiate its degradation via the von Hippel–Lindau protein (pVHL) tumor suppressor protein.6 Under normoxia, PHDs hydroxylate two conserved proline residues (Pro 405 and 531) within HIF-2α, using oxygen, α-ketoglutarate and iron as cofactors. HIF-2α hydroxylation facilitates the binding of pVHL to the HIF-2α ODD7 (Figure 1). The pVHL protein constitutes the substrate recognition module of an E3 ubiquitin ligase complex comprising elongin C, elongin B, cullin-2 and ring-box 1 that directs HIF-2α polyubiquitylation and proteasomal degradation.8With the inhibition of PHD activity and elimination of pVHL binding under hypoxia, HIF-2α enters the nucleus and heterodimerizes with HIF-1β. Subsequently, HIF-2α binds to a conserved DNA sequence known as the hypoxia-response element (HRE) and transactivates a variety of hypoxia-responsive genes.9 As the transcriptional activator of erythropoietin, HIF-2 controls intracellular hypoxic responses throughout the body.

Figure 1

The structural domains of HIF-1α and HIF-2α. Both of them contain basic helix-loop-helix (bHLH), per-Arnt-SIM (PAS), oxygen-dependent degradation domain (ODD) and C-terminal transactivation domains (CADs). Prolyl hydroxylases (PHDs) hydroxylate proline residues 402 and 564, and 405 and 531, respectively, in ODD of HIF-1α and HIF-2α, under normoxic conditions, targeting it for degradation by the proteosome, respectively. Numbers refer to amino-acid similarity between human HIF-1α and HIF-2αin the defined domains

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HIF-2α is expressed in endothelial cells, the parenchyma and interstitial cells of multiple organs.1, 10, 11 Although HIF-2α is stabilized at higher O2pressure compared with HIF-1α in vitro, it was not detected under normoxic conditions in various organs.11, 12, 13 However, HIF-2α was shown to be markedly induced under hypoxia in all organs investigated, including brain, heart, lung, kidney, liver, pancreas and intestine.11

Hypoxia is a common phenomenon in many tumors. The HIF-1α and HIF-2α proteins are expressed in most types of human tumors, including breast, colon, ovarian, pancreatic, prostate, renal and hepatocellular cancers.14, 15, 16 Compared with surrounding normal tissues, HIF-1α or HIF-2α (or both) have been detected at higher levels in the majority of primary human cancers and their metastases.14, 15, 16, 17 HIF-2α mediates the adaptive response to decreased O2 availability at the cellular and organismal level.

HIF-2 Is Different from HIF-1

The HIF transcription factors mediate the primary transcriptional responses to hypoxic stress in normal and transformed cells. Both HIF-1α and HIF-2α are O2-labile α-subunits and heterodimeric complexes composed of bHLH-PAS proteins. In addition, both of them can bind ARNT and mediate transcription. However, increasing evidence has indicated that HIF-2 is distinct or even opposite in many ways from HIF-1.12, 18, 19, 20 To ease understanding, we here delineate the differences between HIF-2 and HIF-1 in three parts. In fact, all the disparities are cause-and-effect relationships and should be conceived as part of a whole in the cellular processes.

First, HIF-2α expression‎ has been shown to differ from that of HIF-1α(Table 1) in various tissues. In contrast to HIF-1α that appears to be expressed in nearly all cell types, HIF-2α expression‎ is restricted to specific types, including endothelial cells, glial cells, type II pneumocytes, cardiomyocytes, kidney fibroblasts, interstitial cells of the pancreas and duodenum and hepatocytes. In the umbilical cord, HIF1α is expressed in smooth muscle cells that surround blood vessels, whereas HIF-2α was detected in endothelial cells that line blood vessel walls.1, 2, 3 HIF-1α was observed by cytoplasmic staining in addition to nuclear accumulation; HIF-2α is confined to the cell nucleus and expressed only under hypoxic stimulation. Although HIF-1α is expressed in renal tubules and neuronal cells, HIF-2α was detected in nonparenchymal cells including glomerular, kidney peritubular endothelial cells and fibroblasts, and brain endothelial and glial cells.11Furthermore, HIF-2α expression‎ has been shown to differ from that of HIF-1α in many cancers. In addition, HIF-2 is expressed in tumor-associated macrophages, although HIF-1α distribution tends to be more restricted to perinecrotic regions in renal cell carcinoma (RCC).14

Table 1 Different expression‎ of HIF-1α and HIF-2α

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Another difference concerns the oxygen conditions needed for expression‎. HIF-1α was shown by immunohistochemistry to be already expressed under normoxic baseline conditions, unlike HIF-2α.11 More severe hypoxia (6% O2) is required to induce HIF-1α compared with HIF-2α in liver and kidney.11 HIF-1α is most active during short periods (2–24 h) of intense hypoxia or anoxia (<0.1% O2), whereas HIF-2α was shown to be active under mild or physiological hypoxia (<5% O2) in both SK-N-BE(2)c and KCN-69n neuroblastoma cell lines.12 These findings indicate that HIF-2 plays a critical role in driving the hypoxic response, whereas HIF-1 controls the initial response to hypoxia in certain contexts.12, 20

The two proteins also differ by the time needed for activation and duration of activity. HIF-1α induction in kidney and liver was shown to be transient; however, HIF-2α expression‎ is sustained.11 Moreover, HIF-1α was transiently stabilized and primarily mediated acute responses, whereas HIF-2α gradually accumulated and managed prolonged hypoxic gene activation under hypoxia (1% O2) in neuroblastoma. The activity of HIF-2α persists even after 48–72 h of hypoxia in neuroblastoma cell lines.12

Second, based on their distinct expression‎ patterns, HIF-2α plays different roles in many respects compared with HIF-1α (Table 2). It was found that the majority of hypoxia-induced genes contained HIF-1-binding sites, and gene expression‎ was shown to be dependent on HIF-1α expression‎. HIF-2α binding was redundant for many genes, but knockdown of HIF-2α levels did not affect gene expression‎.21 In fact, emerging studies have shown that HIF-1α and HIF-2α regulate different target genes, with non-redundant and even opposite biological functions. HIF-1α plays more important roles in endothelial cell proliferation, migration and vessel sprouting; HIF-2α plays a more significant role in controlling vascular morphogenesis, integrity and assembly.22 Overexpression‎ of HIF-2α was shown to enhance the expression‎ of endothelial tyrosine kinase receptor Tie2, in contrast to HIF-1α.1 HIF-2α or other hypoxia-induced factors cannot compensate for the loss of HIF-1α, whereas hypoxic induction of HIF-1α target genes is attenuated in Hif-1α-deficient endothelial cells.23 Interestingly, HIF-1α decreased IL-8 expression‎ whereas HIF-2α overexpression‎ increased the mRNA and protein levels of this cytokine in HMEC-1 cells.24 HIF-2αalso seems to have a stronger transactivation activity than HIF-1α on vascular endothelial growth factor (VEGF) promoter.2, 13, 25, 26 Moreover, HIF-2 is the dominant HIF regulating VEGF and other angiogenic factors in mouse liver hemangiomas.27 HIF-1α facilitates cell growth in vitro and tumorigenesis in vivo, whereas HIF-2 showed opposite effects in colon cancer cells.28 Surprisingly, HIF-2α also has distinct or even opposite functions with HIF-1α in some cancers. In RCC, HIF-2α, but not HIF-1α, promotes tumor growth in xenograft models. Overexpression‎ of stable HIF-1α inhibits tumor growth in 786-O RCC cells,29, 30 whereas overexpression‎ of stable HIF-2α-expressing pVHL restores xenograft growth to the level of parental VHL-null cells.30, 31, 32HIF-2α expression‎ was shown to increase with the degree of dysplasia in preneoplastic kidney lesions of patients with VHL disease, whereas HIF-1α expression‎ decreased, indicating that HIF-2α plays a role in the transformation of dysplastic cells.30, 33 A similar phenomenon was also observed in hepatocellular carcinoma (HCC) and colorectal cancers as described, though the data did not allow firm conclusion. HIF-1αdeficiency inhibited overall tumor growth, whereas deficiency of HIF-2α stimulated tumor growth in colorectal cancers xenograft.28 HIF-1αand HIF-2α are differentially regulated in vivo and reflected distinctive protein expression‎ patterns and stabilization mechanisms in HCC.15, 34HIF-2α also appears to have a more general role in promoting tumorigenesis. Subcutaneous teratomas generated from ES cells with the HIF-2α cDNA ‘knocked in’ into the Hif-1α locus exhibited a fourfold mass increase compared with WT RCC 786-O cells controls, mainly because of increased proliferation.35 HIF-2α was shown to be stable under prolonged and mild hypoxia conditions in neuroblastoma, and may promote angiogenesis even in tumors experiencing minimal hypoxic stress.12 Loss of HIF-1α in endothelial cells reduces NO synthesis, retards tumor cell migration through endothelial layers and restricts tumor cell metastasis; however, loss of HIF-2α resulted in opposite effect in each case.36 Deficiency of HIF-2α increased tumor growth and progression, whereas HIF-1α deletion had nearly no effect on tumor burden and progression in a KRAS-driven lung tumor model.37 HIF-2α signaling, but not HIF-1α, may contribute to tumor angiogenesis via autocrine transforming growth factor-β1 (TGF-β1) production under nonhypoxic conditions in prostate tumor cells.38 For cancer stem cells (CSCs), though hypoxia and HIFs may contribute to the maintenance of putative cancer ‘stem’ cells, HIF-2α seemed also to play a distinct role from HIF-1α, through unique mechanisms. A recent study revealed that HIF-2α regulates Oct4 directly upstream and binds hypoxic regulatory elements in the promoter of murine Oct4, which is a stem cell regulatory protein; this is not the case for HIF-1α.39 HIF-2αmay have a specific function in glioblastoma stem cells: it was shown to be selectively expressed in the CD133+ subpopulation of glioblastoma cells, whereas HIF-1α expression‎ is widespread among both tumorigenic and nontumorigenic cells.40

Table 2 Different functions and related target genes between HIF-1 and HIF-2

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Third, HIF-2α and HIF-1α regulate key downstream genes, including c-Myc, p53, mTOR and β-catenin, conversely (Figure 2). For example, HIF-1α and HIF-2α showed opposite effects on c-Myc interaction with its transcription cofactors. HIF-1α induction was demonstrated to inhibit c-Myc transcriptional activity and suppress cell proliferation.24, 41, 42However, HIF-2α induction promotes cell cycle progression by enhancing c-Myc function.24, 43 In addition, HIF-2α-mediated suppression of p53 is central to maintenance of the enhanced stemness phenotype in human embryonic stem cells. Unlike HIF-2α, HIF-1αexpression‎ declined markedly following reoxygenation in the model44, 45(Figure 3). HIF-2α may regulate the focal adhesion kinase family interacting protein of 200 kD gene and stimulate mTORC1 to promote cellular proliferation in O2-deprived cells.46 On the contrary, HIF-1αinduces DDIT4 gene expression‎ and represses mTORC1 by promoting the release of sequestered TSC2 from 14-3-3 proteins.47, 48 A recent study found that HIF-1 negatively regulates Wnt/β-catenin signaling by sequestering β-catenin from β-catenin/T-cell factor (TCF); instead, HIF-2α interacts with β-catenin at a different site and assembles with β-catenin/TCF to facilitate gene transcription.49

Figure 2

HIF-1 and HIF-2 regulate conversely some key downstream genes. HIF-2 increases c-Myc, mTOR and β-catenin activity and decrease p53 activity, whereas HIF-1 exert opposite effects

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Figure 3

(a) HIF-1α upregulates P53 activity whereas HIF-2α downregulates P53 activity under hypoxia directly (full line) or indirectly (dotted line). (b) HIF-1αand HIF-2α regulate distinctively c-Myc interacting with its transcription cofactors. HIF-1α diminishes the association with MAX and SP1, and then decreases MYC activity, whereas HIF-2α combines with MAX and SP1 complex, thereby increasing MYC activity

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HIF-2α in Digestive System Cancers

HIF-2α is frequently detected in most types of solid tumors, including head and neck, renal, bladder, glial, breast, ovarian, prostate and renal cancers.14, 15, 25, 28, 50, 51, 52, 53 An increasing number of studies reporting HIF-2 in the context of digestive system cancer have emerged in recent years and conveyed that this protein plays important roles in digestive system cancers. Interestingly, its prognostic roles are explicit in pancreatic and gastric cancers but confused in colon and hepatocellular cancers (Table 3). HIF-2 upregulates several genes involved in a variety of tumorigenesis events: cell proliferation, angiogenesis, metabolism, metastasis and resistance to chemotherapy (Figure 4).

Table 3 HIF-2 activation in digestive system cancer

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Figure 4

Mechanisms underlying HIF-2α effect on tumorigenesis in digestive system cancer. HIF-2α regulates genes that modulate key aspects of tumorigenesis, including proliferation, metabolism, angiogenesis, metastasis and resistance to chemotherapy

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Proliferation

Studies have suggested that HIF-2 can regulate cell proliferation through multiple mechanisms, although the outcomes are not identical for all cancers and even controversial in some cases. HIF-2 was shown to be necessary and sufficient to maintain tumor growth in VHL-deficient RCC cells.31, 32 HIF-2 was shown to control cellular proliferation through modulation of c-Myc activity and cyclin D1 in RCC cells.30, 54 In addition, new mechanisms for HIF-2 were described in digestive system cancer.

A recent study showed that HIF-2 plays opposite roles in different digestive tract cancer cell lines through Sirt1. Sirt1 inhibited HCT116 cell growth but promoted HepG2 cell growth. The cell type-dependent actions of Sirt1 on cell growth may be attributable to its opposite effects on HIF-2α activity in different cell lines.18 PrPc (cellular prion protein) is a glycosylphosphatidylinositol anchored membrane protein with various physical functions. PrPc is highly expressed in colorectal adenocarcinomas. It is involved in regulating Glut1 expression‎ through the Fyn-HIF-2α pathway.55

By maintaining appropriate levels of both Smad4 and β-catenin, HIF-2αmodulates the Wnt signaling pathway during mPanIN progression, with the oncogenic protein Ras expressed in the pancreas. Loss of Hif-2α in mice instead led to markedly higher number of mPanIN lesions.56 The average amounts of HIF-2α at both protein and mRNA levels were significantly lower in tumor than peritumoral tissues in HCC, and the decreased levels were associated with lower overall survival.57 Bax and Bak were expressed at higher levels in HCC SMMC-7721 cells with high HIF-2α expression‎ relative to controls.57 Both Bax and Bak are pro-apoptotic BCL-2 proteins and would facilitate the release of cytochrome c. The latter triggers cleaved caspase 3, which is also a key executer of apoptosis detected at higher levels in cells overexpressing HIF-2α.58, 59 These data confirmed that high levels of HIF-2α in HCC cells cause cell growth arrest through apoptosis. Furthermore, it was suggested that HIF-2α induces apoptosis through a novel TFDP3/E2F1 pathway involving both p53-dependent and -independent modes. Hence, high HIF-2α expression‎ in HCC was shown to be correlated with a good outcome.57. Menrad et al.60 found that knockdown of HIF-1α or HIF-2α increased cell viability as well as spheroid size and decreased caspase-3 activity. Indeed, HIF-2α-knockdown cells upregulated HIF-1αand enhanced Bcl-XL and BNIP3 expression‎. BNIP3 binds to the Beclin-1/Bcl-XL complex, thereby releasing Beclin-1 that subsequently induces autophagosome formation.61, 62 However, the role of HIF-2α in the development of cancer is unclear, and inconsistencies abound in the existing literature. Bangoura et al.15 reported that HIF-2α is overexpressed in pericarcinoma tissues in HCC and significantly correlated with tumor grade and reduced survival in patients with HCC. HIF-2α/EPAS1 itself, to some extent, affects patient survival.15

HIF-2α is overexpressed in colon cancer,14, 17 and its activation in vivodirectly upregulates COX2 expression‎ and facilitates colon tumorigenesis. HIF-2α activates COX2/mPGES-1/PGE2 signaling to facilitate colon tumorigenesis.63 In addition, HIF-2α exerts its proliferative effects through modulating the EGFR, IGF1R and ERK/Akt signaling pathways in colorectal carcinoma, and its inhibition prevents growth and tumorigenesis of colorectal cancer in vivo.64 However, an opposite outcome was reported, with HIF-2α-selective knockdown having no effect on cellular proliferation in colorectal cells, although colony formation doubled in soft agar assays.28

A research has also investigated the relationship between HIF-2α and microRNA (miRNA) in digestive system cancers. HIF-2α was shown to mediate miR-210 and c-Myc to participate in neoplasma. MicroRNA-210 is a direct transcriptional target of HIF-2α and its upregulation led to a switch from Mnt to c-Myc expression‎ during cholestatic cholangiocarcinogenesis in vivo.65

At present, inconsistent conclusions have been reached for the role of HIF-2 in digestive system cancers, especially in HCC and colorectal carcinoma. The function of HIF-2α appears to be cell-type dependent. The different expression‎ patterns and levels of HIF-2 as well as cell-specific cofactors may affect its activity. Indeed, the organism environment is very complex and HIF-2 could be influenced by many other factors. For instance, HCC is often complicated with hepatitis B or C and hepatocirrhosis that may also promote a hypoxic response that stabilizes HIFs in oxygen tension.66, 67 Therefore, HIF-2 may also be involved in these diseases, leading to a complicated network in certain organs.

Metabolism

HIF-2α plays a critical role in metabolism, modulating the expression‎ of cytochrome c oxidase isoforms so as to maximize the efficiency of the electron transport chain. Defects in this response lead to impaired ATP production and elevated oxidant production in hypoxia.68 HIF-2α/ARNT targets (e.g., SOD2) also protect cellular and mitochondrial components during oxidative stress in mice.69

Cancer cells shift glucose metabolism from oxidative route to glycolytic pathway, which involves decreased mitochondrial respiration and increased lactate production, even in the presence of oxygen.70, 71 HIF-αtarget genes are involved in the regulation of numerous pathways important for tumor metabolism.72

HIF-2α depletion downregulated the expression‎ of heme oxygenase (HMOX1) genes that exert antioxidant functions in RCC.73 In colon cancer cells, absence of oncogenic KRAS or both HIF-1α and HIF-2α led to decreased cardiolipin level and inefficient mitochondrial respiration; ACSL5 induction is directly responsible for maintaining cardiolipin level and efficient mitochondrial respiration in cancer. Colon cancer cells with oncogenic KRAS mutation and expressing both HIF-1α and HIF-2αwere shown to maximize ATP production and minimize ROS generation, probably through the induction of enzymes important for mitochondrial cardiolipin synthesis.74

Poorer survival of HIF-2α and wild-type TP53 was associated with carbonic anhydrase 9 (CA9) stromal-positive colorectal adenocarcinomas. Furthermore, tumors expressing HIF-2α or CA9 in their stroma show poorer prognosis in wild-type TP53 tumors compared with mutant malignancies. It is plausible that p53 is involved in the metabolic switch to glycolysis, when oxidative phosphorylation is impaired during hypoxia;75 p53 may simply correlate with defects in another pathway such as the BNIP3 cell death pathway that substitutes for TP53 loss in a similar manner during carcinogenesis.76 CYP (cytochrome P450) 3A4 mediates exogenous-drug transportation and oxidative metabolism. We have observed that HIF-2α modulates the recruitment of pregnane X receptor (PXR) to the PXR response element in the CYP3A4 (cytochrome P450 3A4) gene promoter region in gastric cancer BGC-823 cells (Jiuda Zhao, unpublished data).

Angiogenesis

Angiogenesis plays an important role in tumorigenesis and cancer progression. Hypoxia is the prime driving force for tumor angiogenesis, and HIFs play a key role in this process. Growing evidence supports that HIF-2α is involved in angiogenesis.77 HIF2 expression‎ was directly correlated with microvessel density and cyclooxygenase 2 expression‎ in colorectal carcinoma, indicating its potential role in angiogenesis of colorectal carcinomas.53 Selective knockdown of HIF-2α resulted in more pronounced decrease of VEGF and MVD levels compared with HIF-1α repression in colon cancer xenograft studies. HIF-2α exerts these unique effects on colonic tumorigenesis through cyclin G2 (CCNG2) and angiopoietin-like 4 (ANGPTL4), a secreted protein of the angiopoietin-like family induced by hypoxia.28 HIF-2 upregulates the expression‎ of VEGF that binds VEGFR and stimulates tumor angiogenesisin in HCC.77 Transfection of HIF-2α siRNA into HCC cells was shown to downregulate the expression‎ of VEGF, cyclin D1, HIF-2αand TGF-α, inhibiting the activation of EGFR.78 Conditional inactivation of HIF-2α resulted in suppressed development of VHL-associated liver hemangiomas; in addition, angiogenic gene expression‎ in hepatocytes is predominantly regulated by HIF-2 and not HIF-1. It suggests that HIF-2 is the dominant HIF in the pathogenesis of VHL-associated vascular tumors. Hemangioma formation does not require HIF-1α, but is dependent on ARNT, suggesting that HIF-2α may play an essential role in VHL-associated vascular tumorigenesis.79 These observations indicate that HIF-2α is an important mediator of angiogenesis in digestive tract cancers.

Metastasis

Hypoxia is an important microenvironmental factor that induces cancer metastasis. HIF activation correlates with metastasis in multiple tumors and can promote metastasis through the regulation of key factors governing tumor cell metastatic potential, including E-cadherin, LOX, CXCR4, SDF-1 and TWIST in RCC, breast and head/neck cancers.80, 81, 82, 83, 84, 85

Some studies have indicated that HIF-2α also participates in digestive tract cancer metastasis by regulating the JNK signaling pathway, BNIP3, PAI-1 and CSCs. In gastric cancer, HIF-1α and HIF-2α levels are higher in metastasis samples compared with non-metastatic ones. Hypoxia (1% O2, 8 h) was shown to induce HIF-1α and HIF-2α expression‎ in gastric cancer cell lines. Small interfering RNA (siRNA) against HIF-1α and HIF-2α in gastric cancer cells significantly inhibited hypoxia-induced adhesive and invasive abilities. HIF-2α is therefore involved in metastasis and invasion of gastric cancer cells under hypoxia, in a mechanism involving the JNK signaling pathway.86 In primary colorectal cancer, overexpression‎ of HIF-2α together with BNIP3 was linked to local invasion and lymph node metastasis.87

The role of HIF-2α in HCC is not entirely understood. Some studies have demonstrated that HIF-2α is associated with tumor metastasis, whereas others hold the opposite opinion. A study found that HIF-2α is overexpressed in HCC compared with noncancerous lesions, and its levels were significantly correlated with tumor grade, venous invasion, intrahepatic metastasis, necrosis and capsule infiltration.15 Two HREs at −3.6 kb of the 5'-flanking promoter region of the PAI-1 gene can function as cis-acting elements to regulate PAI-1 gene induction by hypoxia in mouse hepatoma cells. The interaction of these HREs with the HIF-1 or HIF-2 protein mediates the transcription of the PAI-1 gene that is involved in invasion and metastasis.88 However, Yang et al.reported recently that high HIF-2α expression‎ was found in only 13.5% (17/126) of tumors compared with 47.6% (60/126) of peritumoral tissues in HCC samples. There was no relationship between HIF-2α and capsular infiltration or portal vein invasion in HCC patients.34

Another mechanism by which HIF-2 controls metastasis is through modulation of CSCs. It is widely accepted that metastasis originates from CSCs with tumor-initiating capabilities that allow most disseminated tumor cells to reconstitute growing, heterogeneous secondary tumors.89 Blocking HIF-1α or HIF-2α activity results in dramatically decreased CSC proliferation and self-renewal in hematological malignancies and glioma stem cells.40, 90, 91, 92 Limited research has indicated that HIF-2α also plays roles in metastasis of digestive tract cancers by regulating the CSCs. Interestingly, HIF-2α co-stained with CD133 in pancreatic ductal adenocarcinoma (PDA), suggesting that tumor hypoxia is associated with the expression‎ of CSC markers in PDA. Hypoxia-driven EMT occurs in tumor tissues from pancreatic cancer patients, with CSC-positive tumor cells present in hypoxic tumor microenvironments. Only stem-like cells acquire high migratory potential and thus may be responsible for invasion and metastasis.93

HIF-2 and Resistance to Chemotherapy

The hypoxic environment plays a critical role in promoting resistance to anticancer drugs.94, 95 Previous studies have indicated that HIF-2 responds rapidly to decreased oxygen levels to activate the expression‎ of a broad range of genes that promote resistance.

In the case of colon cancer cells, oxaliplatin increased HIF-2αaccumulation and enhanced cell growth; indeed, HIF-2α may mediate, at least partly, oxaliplatin resistance in SW1116 cells.96 Disruption of HIF-1α or HIF-2α gene, or both, further improved the tumor response to sunitinib therapy in human colon cancer cells.97 Upregulation of HIF-2α induced by sorafenib was shown to contribute to resistance of hypoxic HCC cells by activating the TGF-α/EGFR pathway.79

To better understand the roles of HIF-2 in chemotherapy resistance, our laboratory studied the relationship between HIF-2α and multiple drug resistance (MDR1). We found that overexpression‎ of HIF-2αinhibits the PXR transcriptional activity and reduces the expression‎ of PXR downstream genes, whereas increasing hypoxia elicited MDR1 and CYP3A4 expression‎. Moreover, overexpression‎ of HIF-2α significantly reduced the pharmacological effects of Paclitaxel, Mitomycin C, Imatinib and Sorafenib on gastric cancer BGC823 cells, and the corresponding IC50 values increased markedly (Jiuda Zhao, unpublished data). Therefore, HIF-2 may serve as a biomarker for a better understanding of chemoresistance in digestive system cancer through MDR1 gene induction.

Taken together, these experimental and clinical data delineate an important role for HIF-2 in chemotherapy resistance through multiple mechanisms and provide a basis for reversing drug resistance with HIF-2 inhibitors.

HIF-2 and Tumor Inhibition

As HIF complexes are involved in cancer cell adaptation to hypoxic tumor microenvironments, inhibitors of the HIF signaling pathway have been developed and used to inhibit the expression‎ of HIF target genes and reduce angiogenesis and metastasis.98 The ability to selectively inhibit HIF activity would be beneficial to clinical treatment.99 A large collection of compounds have been reported to inhibit HIF transcriptional activity, either directly or indirectly.100, 101, 102 For example, downregulation of the oncoprotein human double minute 2 reduced the levels of HIF-1 and HIF-2α in a p53- and VHL-independent manner.103 The JNK inhibitor SP 600125 abolished hypoxia-induced HIF-1α and HIF-2α expression‎, and inhibited the adhesive and invasive abilities of gastric cancer cells.86 Two new cembrane diterpenes were found to selectively inhibit HIF-2α and modulate downstream effectors of tumorigenesis.104

In addition, agents that inhibit reactive oxygen species generation, such as superoxide dismutase mimetics, have also been shown to decrease HIF levels.105 Inducing PHD enzyme activity with derivatives of α-ketoglutarate is also a method to target hypoxic areas of tumors, although it reduces signaling through both HIF-1α and -2α.46Overexpression‎ of the tumor suppressor protein pVHL inhibits HCC growth in mice by downregulating HIF-1α and HIF-2α.106

To date, only few inhibitors have been shown to selectively target HIF-2α. Furthermore, although laboratory effects of chemical and biologic inhibitors of HIF-2α on tumor cells are being investigated, clinical grade HIF-2α-specific inhibitors have yet to be developed.

Conclusion and Perspectives

In conclusion, multitudinous studies have provided persuasive evidence that HIF-2 plays important roles in many critical aspects of digestive system cancers. It is involved in cellular proliferation, metabolism, angiogenesis, metastasis and resistance to drugs. Clinical data also indicated that HIF-2 overexpression‎ is associated with prognosis of digestive system cancers. The HIF-2 pathway may therefore constitute a useful biomarker for assessing disease states as well as developing cancer treatments.

However, multiple questions regarding HIF-2 need to be promptly answered. First, inconsistent associations between HIF-2 and digestive tract cancers have been reported. The study conclusions do not corroborate each other, sometimes for the same organ. This suggests that HIF-2-specific gene activating functions respond differently to various stimuli, with gene- or tissue-specific regulatory mechanisms involving additional transcription factors. Moreover, the limited sample size in different studies and different cell line status in some studies may also affect the results. Second, although data have been amassed delineating the mechanisms and consequences of increased HIF-2 activity during cancer progression, its definite mechanisms remain elusive in digestive system cancers. The role of HIF-2α in the pathogenesis of cancers is extensively studied in RCC, but far too few studies have assessed HIF-2α in digestive system cancers. Third, the exploitation of HIF-2α inhibitors is still challenging. Most inhibitors target nxxxxxxxxonselectively both HIF-1α and HIF-2α. HIF targeting involves the overlapping but distinct biological roles of the HIF-α subunits. Compounds that promote the binding of IRP1 to the 5′ UTR of HIF-2αmRNA decrease HIF-2α hypoxic induction, but also repress HIF-1αsynthesis via an independent mechanism.107 Discriminating between the α-subunits and their relative contributions to different digestive system cancers will be important for appropriately testing compounds for use in therapy. The intricate interplay between HIF-1 and HIF-2 should also be studied further. In addition, although another HIF family member, HIF-3, can also dimerize with ARNT and bind to HREs in vitro,108 its role in the hypoxic regulation of target gene expression‎ and interaction with HIF-1 and HIF-2 in vivo is not well understood. We believe that further studies are warranted to elucidate the important role of HIF-2 in digestive system cancers. There is still a long way to go before one can confidently promote the clinical application of HIF-2 targeting therapy for digestive system cancers.

Abbreviations

CA9:

carbonic anhydrase 9

CSC:

cancer stem cell

CYP3A4:

cytochrome P450 3A4

EPAS1:

endothelial PAS domain protein-1

HCC:

hepatocellular carcinoma

HIF:

hypoxia-inducible factor

HRE:

hypoxia-response element

MDR1:

multiple drug resistance

PrPc:

cellular prion protein

PDA:

pancreatic ductal adenocarcinoma

PHD:

prolyl hydroxylase-domain enzyme

pVHL:

von Hippel–Lindau protein

PXR:

pregnane X receptor

RCC:

renal cell carcinoma

VEGF:

vascular endothelial growth factor

References

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The emerging role of hypoxia, HIF-1 and HIF-2 in multiple myeloma

• S K Martin, 
• P Diamond, 
• S Gronthos, 
• D J Peet & 
• A C W Zannettino 

Leukemia volume 25, pages1533–1542(2011)Cite this article

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• 72 Citations

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Abstract

Hypoxia is an imbalance between oxygen supply and demand, which deprives cells or tissues of sufficient oxygen. It is well-established that hypoxia triggers adaptive responses, which contribute to short- and long-term pathologies such as inflammation, cardiovascular disease and cancer. Induced by both microenvironmental hypoxia and genetic mutations, the elevated expression‎ of the hypoxia-inducible transcription factor-1 (HIF-1) and HIF-2 is a key feature of many human cancers and has been shown to promote cellular processes, which facilitate tumor progression. In this review, we discuss the emerging role of hypoxia and the HIFs in the pathogenesis of multiple myeloma (MM), an incurable hematological malignancy of BM PCs, which reside within the hypoxic BM microenvironment. The need for current and future therapeutic interventions to target HIF-1 and HIF-2 in myeloma will also be discussed.

Introduction

Multiple myeloma (MM) is a hematological malignancy of unknown etiology, which affects plasma cells (PCs) in the bone marrow (BM). The key clinical features of MM include the production of a monoclonal paraprotein, amyloidosis, renal insufficiency, anemia, increased BM angiogenesis, osteolytic bone lesions, severe bone pain, spontaneous fractures and hypercalcemia.1 Despite recent therapeutic advances, MM remains incurable, has a high rate of relapse and drug resistance, and patients currently have a median overall survival of 44 months.2

Hypoxia is a dysregulation of the homeostatic balance between oxygen delivery and oxygen consumption, which causes cells or tissues to become deprived of sufficient oxygen. Associated with a number of short- and long-term pathologies, hypoxia is a key feature of the tumor microenvironment and has been shown to drive tumor progression and treatment resistance.3, 4, 5, 6, 7 A hypoxic tumor environment most commonly arises when rapidly proliferating cells outgrow the existing vasculature, or in response to transient limitations in blood perfusion. Importantly, these situations apply strong selective pressure to tumor cell subsets that are able to adapt quickly to reduced oxygen availability. Consequently, cancers that adapt to hypoxia are highly aggressive, resistant to treatment and associated with a poor prognosis.

Hypoxia and cancer

In solid tumors, the uncontrolled growth of malignant cells creates a chronic, diffusion-limited state of hypoxia in which the innermost cells become progressively distanced from the host vasculature. Consequently, although solid tumors contain a heterogeneous range of oxygen pressures ranging from approximately 5% O2 in the well-vascularized areas to complete anoxia (no oxygen) in the necrotic areas, the average oxygen pressure generally lies in the hypoxic range at approximately 1% O2.8, 9, 10 Acute or fluctuating hypoxia can also result as a consequence of transient limitations in blood perfusion—for example, severe structural abnormalities in tumor microvessels and disturbances in microcirculation, which cause cyclical cessation and re-establishment of blood supply.

Over the years, several different approaches have been used to measure tumor oxygen levels, including electron paramagnetic resonance oximetry, nuclear magnetic resonance spectroscopy and PET (reviewed by Hockel and Vaupel10). The current ‘gold standard’ approach involves taking direct polarographic measurements using microsensor techniques;11 however, this is invasive and not suitable for all situations. In fact, all of the current approaches have their own benefits and limitations, and no single method is considered universally applicable. Instead, researchers must weigh up factors such as the invasiveness of the procedure, the degree of resolution required and cost to determine which is best suited to a particular experiment. As discussed in detail elsewhere,12, 13 contemporary studies commonly measure tumor hypoxia using indirect, radiobiological or immunohistochemical approaches—radiobiological assessments measure the fraction of the clonogenic cells in tumors that are hypoxic, and immunohistochemical approaches (perhaps the most widely used) identify hypoxic adducts in tumor preparations using antibodies to the well-characterized hypoxia marker, pimonidazole, or surrogate markers of hypoxia such as the hypoxia-inducible transcription factors (HIFs) factors, which are induced by hypoxia.

It is well-established that hypoxia promotes tumor progression and metastasis by selecting for aggressive cell clones and upregulating the expression‎ of genes associated with proliferation, angiogenesis, survival, extracellular matrix remodeling, cell migration and invasion, and cancer stem cell maintenance.14, 15 In line with this, the expression‎ of hypoxia-inducible gene products or low pO2 in the primary tumor is associated with invasive growth, metastasis, resistance to treatment and poor overall survival.16, 17, 18 Experimental tumor models have also shown that tumors containing a high proportion of hypoxic cells metastasize more frequently than genetically equivalent control tumors containing low numbers of hypoxic cells.19, 20, 21, 22

Adaptation to hypoxia: the HIFs

Owing to the fundamental importance of oxygen for metabolism and survival, cells have evolved intricate response mechanisms to sense and respond to hypoxia by activating a variety of cell- and tissue-specific transcription programs. Although these mechanisms are believed to be evolutionarily conserved in all mammalian cells, not all cells respond to hypoxia in the same manner, or to the same degree, owing to differing oxygen requirements and sensitivities to hypoxic stress.23 Adaptive responses to hypoxia are regulated by several transcription factors, including HIF-1, HIF-2, ETS-1, cAMP response element binding protein, activator protein-1 and nuclear factor-κB.24, 25, 26, 27, 28, 29, 30, 31 Of these, the most important regulators of cellular responses to hypoxia are the HIFs.

The HIFs are heterodimeric complexes composed of an inducible α-subunit (HIF-1α, HIF-2α or HIF-3α) and a constitutively-expressed β-subunit (aryl hydrocarbon nuclear translocator, or ARNT). Of the three HIF family members, HIF-1 and HIF-2 are the most well-characterized. HIF-1 and HIF-2 have been shown to be the master regulators of oxygen homeostasis and they have pivotal roles in both embryonic development and postnatal physiology.25, 32, 33, 34, 35 As yet, the role of the more distantly related HIF-3 remains unclear, as at least one of the HIF-3α isoforms (known as IPAS-1) appears to suppress hypoxia-induced gene expression‎.36 To date, over 70 hypoxia-responsive HIF-1 and/or HIF-2 target genes have been identified, which encode proteins involved in angiogenesis, erythropoiesis, glycolysis, iron transport, cell proliferation and survival, and vascular remodeling.3, 4, 5, 6

As summarized in Figure 1, the stability and transactivation of HIF-1 and HIF-2 is tightly controlled by post-translational regulation of the α-subunit.37, 38, 39, 40, 41 Under aerobic (normoxic) conditions, HIF-α proteins are rapidly degraded by the ubiquitin–proteasome pathway (t1/2<5 min) following prolyl hydroxylase (PHD)-dependent hydroxylation of residues P402 and P564 within the oxygen-dependent degradation domain.42 Under normoxic conditions, HIF-α proteins also undergo asparaginyl hydroxylation by the factor inhibiting HIF (FIH) enzyme, which represses HIF transcriptional activity by blocking the recruitment of the CBP/p300 coactivator.43 The absolute requirement for oxygen for PHD and FIH activity serves as the primary cellular oxygen sensor in these processes, and when oxygen levels become deficient, their activity is inhibited. Under hypoxic conditions, HIF-α hydroxylation is abrogated and proteasomal degradation is inhibited, thereby allowing HIF α-subunits to dimerize with the constitutively expressed β-subunit, ARNT.44 Following the recruitment of additional transcriptional coactivators, the active HIF complex binds to hypoxia-response elements (5′-RCGTG-3′) in target genes and initiates transcription programs to mediate adaptive cellular responses to hypoxia.6, 44, 45, 46, 47, 48

Figure 1

The oxygen-dependent regulation of the HIF complex. Under normoxic conditions, oxygen-dependent enzymes inhibit HIF complex formation by hydroxylating the asparagine and proline residues in the HIF-α subunits. These modifications prevent the binding of coactivators and promote VHL recruitment and proteasomal degradation, respectively. Under hypoxic conditions, enzyme activity is inhibited, resulting in stable, transcriptionally active HIF. HIF, hypoxia-inducible transcription factor; VHL, von Hippel–Lindau.

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In addition to environmental hypoxia, a number of studies have also shown that HIF-1α and HIF-2α protein levels can be elevated by genetic abnormalities (for example, loss-of-function mutations in von Hippel–Lindau (VHL), p53 and phosphatase and tensin homolog;49, 50, 51 gain-of-function mutations in Myc, Ras and Raf52, 53, 54), signaling malfunctions (for example, hyperactivated PI3K/Akt and Src55, 56), growth factors and hormones,57, 58, 59, 60, 61 and reactive oxygen species.62, 63

To date, aberrant expression‎ of HIF-1α and/or HIF-2α has been documented in cancers of the brain, colon, lung, breast, prostate, kidney, pancreas, cervix, bladder and ovary,64, 65, 66, 67, 68, 69 and overexpression‎ of both HIFs has been found to indicate a highly aggressive disease phenotype associated with poor prognosis.70, 71, 72, 73, 74, 75, 76 Whereas examination of aberrant HIF expression‎ has largely been restricted to solid tumors, recent studies have reported that HIF-1α and HIF-2α are also overexpressed in hematological malignancies such as diffuse large B-cell lymphoma,77, 78 chronic lymphocytic leukemia79 and acute lymphoblastic leukemia.80

Although HIF-1α and HIF-2α show 48% amino-acid sequence homology and have similar protein structures and oxygen-dependent mechanisms of regulation, there are a growing number of physiological and mechanistic differences between these proteins, which indicates that they possess distinct, non-redundant roles.81, 82, 83, 84 Of note, whereas both HIF-1 and HIF-2 bind to the same hypoxia-response element DNA consensus sequence in target gene promoters,85, 86 they regulate both common and unique target genes.25, 87, 88 While not entirely understood, this may, in part, be explained by the restricted expression‎ of HIF-2α compared with the ubiquitous expression‎ of HIF-1α;65, 86, 89, 90, 91, 92 differing affinities for PHD- and FIH-dependent regulation of HIF-α protein stability and transcriptional activity; and differential recruitment of isoform-specific transcriptional coactivators and/or repressors to the HIF-1 and HIF-2 complexes.93, 94, 95

Several oxygen-dependent and oxygen-independent mechanisms of HIF-α protein degradation can dictate the selective recruitment of either HIF-1α or HIF-2α to ARNT to mediate cell- and tissue-specific adaptation to hypoxia. As discussed, HIF-α proteins are hydroxylated under normoxic conditions by the PHD enzymes, of which there are four known isoforms (termed PHD1, PHD2, PHD3 and PHD4). Each PHD isoform shows varying affinities for HIF-α hydroxylation, with PHD2 showing the highest affinity for HIF-1α hydroxylation96 and PHD3 showing the highest affinity for HIF-2α hydroxylation.97 In addition, regulators of HIF-1α and HIF-2α protein stability such as human hypoxia-associated factor (HAF) and murine integration site-6 confer isoform-specific degradation of these proteins independently of oxygen tensions. HAF (also known as SART1800)98 is a novel E3 ligase, which specifically binds to and ubiquitinates HIF-1α (but not HIF-2α) to direct its proteasomal degradation under normoxic conditions.99 Whereas having no clear homology with other ubiquitin ligases, integration site-6 has been described as an HIF-2α-specific E3 ligase, which degrades HIF-2α through an unidentified ubiquitin ligase and the 26S proteasome.100 As recently reviewed by Majmundar et al.,101 the regulation of the HIF pathway is further complicated by the identification of a growing list of environmental cues, such as microRNAs, redox homeostasis and cellular metabolites, which are known to modulate the HIF pathway.

It is becoming increasingly apparent that HIF-1 and HIF-2 contribute to tumorigenesis in a complex, context-dependent manner. For example, whereas HIF-1α expression‎ positively correlates with decreased patient mortality in many cancers, HIF-2α expression‎ is a negative prognostic factor in many of these same cancers (reviewed by Bertout et al.102). In VHL-deficient renal cell carcinoma, overexpression‎ and knockdown studies have shown that HIF-2α promotes tumor growth whereas HIF-1α inhibits tumor growth,103 and in some cancers, HIF-1 binds to and stabilizes p53 to mediate hypoxia-induced apoptosis, whereas HIF-2 suppresses p53 and promotes chemoresistance.104, 105 Therefore, tumor behavior and drug response can vary significantly according to aberrant expression‎ of HIF-1α and/or HIF-2α in different cancers.

The BM microenvironment

While the BM microenvironment has long been considered to be physiologically hypoxic,106, 107 a lack of direct non-invasive techniques has hindered accurate in situ measurement of BM oxygen levels. Using polarographic oxygen electrodes to measure intra-medullary oxygen tensions in the femoral head of surgical patients during fracture fixation, Watanabe et al.108 detected an average BM oxygen level of 15 mm Hg. Using a cannula inserted into the femoral long bones of anesthetized dogs, Tondevold et al.109 reported intra-medullary oxygen levels of 23–27 mm Hg. In recent studies, immediate assessment of oxygen levels in human BM aspirates showed an average oxygen tension of 54 mm Hg,110 which, while still considered hypoxic, is markedly higher than reported by others.

Using a surrogate marker of hypoxic adducts, pimonidazole, recent murine studies have shown that the BM in fact contains a heterogeneous O2 distribution and that distinct BM compartments contain different oxygen tensions.111, 112, 113 The ‘endosteal niche’ located at the bone–BM interface is strongly hypoxic, marked by high levels of HIF-1α expression‎ and pO2 tensions below 10 mm Hg. By contrast, the central ‘vascular niche’ is comparatively less hypoxic and contains low levels of HIF-1α expression‎, owing to its close proximity to endothelial sinuses and oxygenated blood circulation.112 It has been postulated that these endosteal and vascular niches have a key role in supporting the function of hemopoietic stem cells, with the hypoxic endosteal niche maintaining hemopoietic stem cells in an immature, quiescent state and the more oxygenated vascular niche promoting the maturation and proliferation of hemopoietic stem cells.114 Given the presence of distinct hypoxic compartments in the BM, this raises the intriguing possibility that tumor cell behavior within the BM may be dependent on the particular niche in which they reside; this, however, awaits further investigation.

Hypoxia and MM

While numerous studies have examined the role of hypoxia in solid tumor progression, there is a paucity of published data regarding the role of hypoxia and the HIF transcription factors in hematological malignancies such as MM. Initial interest in this area stemmed from the well-established association between angiogenesis and MM disease progression, and the knowledge that hypoxia is a critical regulator of angiogenesis in other pathologies. Using the 5T2MM mouse model of MM, Asosingh et al.115 showed that, while the BM of both normal and myelomatous animals is hypoxic, a heavily infiltrated MM BM is significantly less hypoxic owing to an increase in BM angiogenesis. They also showed that the native hypoxic BM environment selects for tumor-initiating CD45+ MM PC populations which are resistant to apoptosis, suggesting that the hypoxic BM environment has a role in the establishment of MM following MM PC migration to the BM from secondary lymphoid organs.115

This was the first study to show the important role played by the hypoxic BM microenvironment in MM disease progression, and was pivotal in highlighting the importance of examining the role of hypoxia in the etiology, pathogenesis and treatment of MM. Intriguingly, a more recent study using the 5T33MM murine model of MM found that the myelomatous BM environment is actually more hypoxic than the normal BM microenvironment.113 These findings are perhaps more analogous to what has been observed in a solid tumor setting, in that low oxygen levels are often never fully restored by angiogenesis owing to the leakiness of the tumor vasculature and its inability to deliver an adequate blood supply to an ever-expanding tumor mass.116 However, while further studies are required to clarify whether the MM BM microenvironment is more/less hypoxic than the normal BM compartment (and whether these discordant observations relate to the MM models or hypoxia detection methodologies used), it is important to note that both studies showed that the MM BM microenvironment is hypoxic and an important microenvironmental stimulus for MM disease etiology and pathogenesis.

In the human setting, a recent study by Colla et al.110 measured oxygen levels in human BM aspirates collected from healthy, monoclonal gammopathy of undetermined significance and MM patients, and detected average oxygen tensions of 54, 56 and 52 mm Hg, respectively. While the differences between these groups were not statistically significant, all three showed hypoxic BM oxygen levels and is in keeping with the findings of the murine studies.

Extending these findings further, two recent studies have examined HIF-1α and HIF-2α protein expression‎ in BM biopsy specimens taken from MM and monoclonal gammopathy of undetermined significance patients at clinical presentation. In studies emanating from our laboratory,117 HIF-1α was found to be widely expressed by many cell types within the BM, whereas HIF-2α expression‎ was restricted to CD68+ macrophages and CD138+ MM cells, suggesting that the induction of aberrant HIF-2α expression‎ is associated with the malignant transformation of MM cells. In the second, much larger study,118 strong HIF-1α and HIF-2α expression‎ was observed in 33 and 13.2% of MM cases, respectively, and a significant association was observed between the expression‎ of HIF-1α and HIF-2α in MM cells. As already mentioned, aberrant expression‎ of either HIF-1α or HIF-2α has been documented in many solid tumors and when either or both are found to be overexpressed, it indicates a highly aggressive tumor with a much poorer prognosis. As yet, the association between aberrant HIF expression‎, and patient survival and prognosis has not been examined in MM.

Intriguingly, examination of temporal modulation of HIF-1α and HIF-2α expression‎ in MM cell lines in response to hypoxia has showed that the HIF-1α protein is rapidly upregulated by hypoxia, whereas induction of HIF-2α is a relatively delayed response.117 From this, it has been proposed that in MM cells, HIF-1 predominantly coordinates immediate, ‘up-front’ responses to hypoxia, whereas HIF-2 coordinates responses to more prolonged, chronic hypoxia, as has been reported previously in other cell types.93, 119 As yet, the biological implications of the different expression‎ patterns and induction kinetics of HIF-1α and HIF-2α in MM remain unknown; however, in solid tumors, aberrant HIF-2α expression‎ is often associated with a worse prognosis than HIF-1α (reviewed by Lofstedt et al.,120 and Patel and Simon121).

To date, the most well-documented biological consequence of aberrant HIF expression‎ in MM is angiogenesis. Studies have shown that there is a positive correlation between HIF-1α and HIF-2α expression‎, and the level of BM angiogenesis and expression‎ of vascular endothelial growth factor (VEGF) and VEGF receptor in patient biopsy specimens.118Moreover, forced overexpression‎ of either HIF-1 or HIF-2 in MM cells significantly enhances MM-induced angiogenesis in an in vivoxenograft model.117 Gene expression‎ profiling of CD138+ MM PCs isolated from MM patients and cultured under normoxic or hypoxic conditions has also shown a significant upregulation in the expression‎ of angiogenic genes such as interleukin-8 (IL-8) and VEGF by hypoxia. Conversely, small interfering RNA-mediated knockdown of HIF-1α in MM cell lines significantly reduces MM-induced angiogenesis in vitro.110 A number of anti-MM drugs, such as adaphostin, lenalidomide and bortezomib, have also been shown to mediate their antiangiogenic effects, at least in part, through inhibition of HIF-1.122, 123, 124, 125Moreover, preliminary studies suggest that HIF-1α is constitutively activated in endothelial cells isolated from MM patients but not monoclonal gammopathy of undetermined significance or healthy donors.126

Given that hypoxia promotes numerous aspects of tumor progression in solid malignancies (for example, proliferation, survival, metabolism and migration), it is likely that future studies will find that other aspects of MM biology are strongly influenced by hypoxia. For example, although a direct connection between hypoxia and osteolytic bone disease in MM has yet to be established, recent studies suggest that an increase in the size and number of bone-resorbing osteoclasts could be attributable, in part, to hypoxia and HIF-1.127

As mentioned earlier, in addition to being induced by environmental hypoxia, aberrant HIF expression‎ in MM cells can arise through genetic abnormalities such as loss of tumor-suppressor activity. While research in this area remains ongoing, Colla et al.128 have shown that, compared with normal PCs, CD138+ MM PCs have reduced expression‎ of the tumor suppressor ING-4 (inhibitor of growth family member-4). Using ING-4 small interfering RNAs, they showed that ING-4 directly regulates HIF-1 expression‎ in MM cells, which in turn stimulates the production of angiogenic cytokines and increases angiogenesis. Hatzimichael et al.129 have also reported that the HIF-selective prolyl hydroxylase, EGLN3, undergoes epigenetic CpG methylation in 33% of MM patients, which was the first evidence of epigenetic modulation of an HIF-specific PHD in human cancer. While loss of other tumor suppressors such as VHL, p53 and phosphatase and tensin homolog has also been shown to induce HIF expression‎ in other tumor cell types,49, 50, 51 this has yet to be confirmed in MM.

The helix–loop–helix transcription factor, c-Myc, is a proto-oncogene, which is de-regulated in 30–50% of patients with advanced MM.130 Of note, recent studies have documented an important relationship between HIF-1 and c-Myc in MM PCs, which results in the upregulation of HIF-1α independent of hypoxia.124 Under normoxic conditions, deregulated c-Myc promotes constitutive HIF-1 protein activation, promotes aberrant VEGF production by MM PCs and angiogenesis, and is associated with a poor prognosis in MM patients. This study also showed that drugs such as bortezomib, doxorubicin, adaphostin and lenalidomide interfere with the c-Myc/HIF-1α axis in MM PC lines, which provides further insight into the mechanism of action of many of these agents, which are currently used clinically to treat MM.

Therapeutic targeting of hypoxia and the HIFs in MM

The increasing importance of HIF-1α and HIF-2α in tumorigenesis (and indeed, numerous other disease states) raises the possibility that agents which specifically inhibit these transcription factors, would provide significant therapeutic benefit. Importantly, based on the complex interplay between HIF-1 and HIF-2 in tumorigenesis, it is likely that combined inhibition of HIF-α isoforms will be appropriate in certain disease settings, whereas HIF-1α- or HIF-2α-specific therapies may be better suited in others. While transcription factors have traditionally been considered to be poor drug targets owing to difficulties associated with small-molecule inhibition of protein–protein or protein–DNA interactions,131 a number of strategies for direct targeting of hypoxic tumor cells have been developed. These strategies include small-molecule therapeutics and antisense inhibitors, which interfere with HIF-1 synthesis, stability or transactivation, hypoxia-responsive viral vectors and hypoxia-activated pro-drugs. Furthermore, a growing number of conventional and novel anticancer agents has been shown to indirectly inhibit HIF activity (summarized in Table 1).

Table 1 Therapeutic inhibitors of hypoxia and HIFs

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The specific HIF-1 inhibitor, PX-478, has shown to be effective against a range of tumors in SCID mice132 and is currently undergoing early-phase clinical trial in treating advanced solid tumors or lymphoma. PX-478 inhibits HIF-1 protein translation and HIF-1 de-ubiquitination, which leads to increased HIF-1 poly-ubiquitination and degradation. Also undergoing phase-I clinical trials is EZN-2968, which is an antisense RNA inhibitor that binds specifically to and inhibits the expression‎ of HIF-1α mRNA, and reduces HIF-1α mRNA and protein levels, leading to decreased expression‎ of HIF-regulated genes in vitroand in vivo.133

Hypoxia-activated pro-drugs combine a potent DNA-alkylating agent with a hypoxia-activated molecular trigger. These agents are designed to deliver cytotoxic agents to hypoxic tumor zones and minimize drug-related toxicities in normoxic tissues.134 In several preclinical tumor models, TH-302 has shown potent hypoxia-dependent cytotoxicity, and early dose-escalation studies show it to be well-tolerated in humans.135Of note, recent studies show that TH-302 induces G0/G1 cell-cycle arrest and triggers apoptosis in MM cell lines in vitro and in the 5T33MM in vivo model of MM.113

It remains to be seen whether HIF-targeting drugs will have serious side effects. As erythropoietin is an important HIF-inducible gene, it is possible that prolonged HIF inhibition would lead to oxygen deprivation in normal tissues. BM cells also could be potentially vulnerable to hypoxia inhibition, given their residence in a naturally hypoxic environment close to the endosteal surface of bone. Nevertheless, the emerging role of hypoxia and the HIF transcription factors in MM disease progression suggests that the development of drugs which specifically target hypoxic zones, could provide significant therapeutic benefit for MM patients.

Concluding remarks

Despite recent treatment advances resulting from the introduction of agents such as thalidomide, lenalidomide and bortezomib, MM remains incurable. Furthermore, owing to the complexity of interactions between tumor cells and their microenvironment, and the involvement of an array of chemokines, cytokines, growth factors and enzymes, elucidation of the mechanisms responsible for MM disease progression remains incomplete. Hypoxia is a common feature in cancer, which affects many important biological properties of tumor cells. The intention of this review was to highlight the emerging role of hypoxia and the HIF transcription factors in MM biology. By relating an extensive body of literature regarding the role of hypoxia in the solid tumor setting to what is currently known about the kinetics of MM disease progression, we have formed a hypothetical model of the role of hypoxia in MM disease progression (Figure 2). In the initial stages of disease establishment, we postulate that the hypoxic BM microenvironment108, 109, 110, 112 supports the initial survival and growth of the MM cells.136 As the MM PCs establish themselves within the endosteal niche of the BM, they become exposed to even greater levels of hypoxia,137 which activates HIF-1 and/or HIF-2, and stimulates the production of angiogenic factors and angiogenesis to increase oxygen delivery to the tumor cells, thereby facilitating tumor growth. In support of this, elevated HIF expression‎ has been observed in the early stages of tumor development prior to histological evidence of angiogenesis and invasion, and this is thought to contribute to the angiogenic switch.64 As MM PCs evolve, they acquire a range of chromosomal mutations which can upregulate the expression‎ of the HIF transcription factors independently of hypoxia (for example, aberrant c-Myc and suppressed ING-4, VHL, p53 and phosphatase and tensin homolog activity49, 50, 51, 124, 128). Together, these chromosomal abnormalities and hypoxia-induced angiogenesis will accelerate disease progression into the active, progressive phase of MM leading to symptomatic disease. When this occurs, patients will show increased levels of HIF-1α and/or HIF-2α expression‎,117, 118 BM angiogenesis,110, 117, 118 and osteolytic bone disease. While HIF-1α expression‎ may begin to decrease following the activation of the angiogenic switch, owing to an increase in BM angiogenesis and tumor oxygenation,115 we anticipate that HIF-2α expression‎ will remain elevated until the ‘end stage’ based on the fact that HIF-2 is not as efficiently degraded in response to increasing oxygen tensions.25, 93 This chronic activation of HIF-2 will continue to drive the expression‎ of pro-angiogenic, osteoclastogenic and cell survival genes to further promote disease progression.

Figure 2

The proposed role of hypoxia in the pathogenesis of MM. In response to chromosomal mutations and chronic exposure to hypoxia, the induction of HIF-1α and HIF-2α expression‎ selects for a subpopulation of ‘angiogenic MM PCs’ () capable of triggering the angiogenic switch. In addition to genes associated with angiogenesis, the HIFs activate genes associated with osteoclast recruitment and activation, and cell-cycle regulation, which further drive the transition from indolent MM to active MM. HIF, hypoxia-inducible transcription factor; MM, multiple myeloma; PC, plasma cell.

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In conclusion, a number of recent studies have provided valuable insight into the functional role of hypoxia in MM disease progression, and have shown that hypoxia, HIF-1 and HIF-2 represent potential therapeutic targets in MM. With a greater understanding of the hypoxic microenvironment in which myeloma PCs reside, it is likely that more efficient ways to target hypoxic zones and inhibit hypoxia-driven disease processes will be identified, ultimately providing the much-needed therapeutic advances for MM patients.

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