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. Author manuscript; available in PMC: 2013 May 1.
Published in final edited form as: Cancer Res. 2012 Mar 14;72(9):2285–2293. doi: 10.1158/0008-5472.CAN-11-3836

Hypoxia-inducible factor-2α activation promotes colorectal cancer progression by dysregulating iron homeostasis

Xiang Xue 1, Matthew Taylor 1, Erik Anderson 1, Cathy Hao 1, Aijuan Qu 4, Joel K Greenson 3, Ellen M Zimmermann 2, Frank J Gonzalez 4, Yatrik M Shah 1,2,*
PMCID: PMC3342485  NIHMSID: NIHMS365107  PMID: 22419665

Abstract

Hypoxia-inducible factor (HIF), a key modulator of the transcriptional response to hypoxia, is increased in colon cancer. However, the role of HIF in colon carcinogenesis in vivo remains unclear. In this study, we found that intestinal epithelium-specific disruption of the von Hippel-Lindau tumor suppressor protein (VHL) resulted in constitutive HIF signaling, and increased HIF expression augmented colon tumorigenesis in the Apcmin/+ intestinal tumor model. Intestine-specific disruption of Vhl increased colon tumor multiplicity and progression from adenomas to carcinomas. These effects were ameliorated in mice with double disruption of Vhl and HIF-2α. Activation of HIF signaling resulted in increased cell survival in normal colon tissue, however tumor apoptosis was not affected. Interestingly, a robust activation of cyclin D1 was observed in tumors of Apcmin/+ mice in which HIF-2α was activated in the intestine. Consistent with this result, BrdU incorporation indicated that cellular proliferation was increased in colon tumors following HIF activation. Further analysis demonstrated that dysregulation of the intestinal iron absorption transporter divalent metal transporter-1 (DMT-1) was a critical event in HIF-2α-mediated colon carcinogenesis. These data provide a mechanistic basis for the widely reported link between iron accumulation and colon cancer risk. Together, our findings demonstrate that a chronic increase in HIF-2α in the colon initiates pro-tumorigenic signaling which may have important implications in developing preventive and therapeutic strategies for colon cancer.

Keywords: hypoxia-inducible factor-2α, colon cancer, Apcmin/+, iron homeostasis, divalent metal transporter-1

Introduction

Hypoxic microenvironment is a hallmark for solid tumors (1). In response to hypoxia, tumor cells activate genes that are critical in angiogenesis, cell survival, cell proliferation, and glucose metabolism(2). Hypoxia-induced signal transduction is transcriptionally mediated by hypoxia-inducible factor (HIF), a member of the Per-ARNT-Sim family of basic helix-loop-helix transcription factors that bind hypoxia response elements (HREs) at target gene loci under hypoxic conditions(3–5). Functional HIF is a heterodimer that comprises a constitutive subunit, aryl hydrocarbon nuclear translocator (Arnt, also known as HIF-1β) and a hypoxia-inducible alpha subunit(3, 4). Stabilization of the α-subunit is regulated by a family of oxygen and iron-dependent prolyl hydroxylase (PHD) enzymes. PHD enzymes hydroxylate the α-subunit enabling the binding of the von Hippel-Lindau tumor suppressor protein (VHL) coupled to the E3 ubiquitin ligase complex, which leads to proteasomal degradation of HIF-α subunits(6, 7).

Two highly homologous and transcriptionally active subunits have been identified, HIF-1α and HIF-2α(8, 9). Both are expressed in many of the same cells and regulate overlapping and distinct sets of genes critical in the adaptation to hypoxic environments, including cancer development(10). In cancer-derived cell lines and in renal carcinomas, HIF-1α and HIF-2α have opposing roles in cell proliferation. HIF-1α decreases cell proliferation whereas HIF-2α induces proliferation via an increase in c-Myc activity(11–16). HIF-1α and HIF-2α are overexpressed in a variety of tumor tissues including colon cancer(17, 18). However, the role of HIF-α in colon carcinogenesis is not completely clear.

The present study demonstrates that Apcmin/+ mice with an intestine-specific activation of HIF signaling via disruption of Vhl using a villin-cre recombinase developed mainly colorectal tumors, with carcinomas seen in 8 of 10 (80%) mice followed for 6 months. Disruption of both Vhl and Hif-2α in intestinal epithelial cells prevented colon tumors indicating a HIF-2α-dependent mechanism. Through global gene expression analysis in the Apcmin/+ mice and human colorectal tumor samples, a HIF-2α-dependent dysregulation of colon iron homeostasis was observed. Increase in local iron levels have been implicated in the progression colon carcinogenesis (19–25), and the present data demonstrate the increase in tumor iron exacerbated cell proliferation and was critical in colon tumor formation following HIF-2α activation. Together, these data reveal a novel role for HIF-2α in initiating a coordinated process that is critical in the progression of colon cancer.

Materials and Methods

Animals and diets

The floxed or compound floxed mice hemizygous for villin-cre were mated to each other to generate VhlF/F, VhlΔIE, VhlF/F/Hif-2αF/F, VhlΔIE/Hif-2αΔIE mice and are described previously(26–28). Apcmin/+ were purchased from The Jackson Laboratory. To investigate the role of HIF-2α in colorectal cancer, VhlΔIE and VhlΔIE/Hif-2αΔIE mice were crossed with Apcmin/+ mice to generate VhlF/F/Apcmin/+, VhlΔIE/Apcmin/+, VhlΔIE/Hif-2αΔIE/Apcmin/+, and VhlF/F/Hif-2αF/F/Apcmin/+ mice. All mice were 129S6/SvEv background and maintained in standard cages in a light and temperature-controlled room and were allowed standard chow and water ad libitum. For low-iron study, the 6-week-old VhlΔIE/Apcmin/+ and VhlF/F/Apcmin/+ mice were given iron-replete AIN93G diet containing 350 ppm of iron or iron-deficient AIN93G diet (less than 5 ppm of iron) for 8-weeks (Dyets, Bethlehem, PA). All animal studies were carried out in accordance with Institute of Laboratory Animal Resources guidelines and approved by the University Committee on the Use and Care of Animals at the University of Michigan (UCUCA approval number: 10299).

Histology, immunohistochemistry, and immunofluorescence

For BrdU staining, animals were sacrificed following a 2-hour treatment of 100 mg/kg of BrdU (Sigma, St Louis, MO). Paraffin-embedded tissue sections (5-µm) were deparaffinized in xylene, and rehydrated in ethanol gradient. Immunohistochemical analysis was performed with antibodies for BrdU (Bu20a, eBioscience Inc., San Diego, CA) followed by detection using Alexa Fluor® 488 goat anti-mouse IgG (Molecular Probes Inc., Eugene, OR). TUNEL assay (Roche Diagnostics, Indianapolis, IN) was performed according to the supplier’s instructions. Briefly, deparaffinized sections were labeled with TdT and biotinylated dUTP, and then were examined under a fluorescence microscope (29). Tissue iron detection was performed in paraffin embedded sections stained with Perls Prussian blue and enhanced with DAB and H2O2 (30). Histological analysis was done on hematoxylin and eosin stained paraffin sections and microscopically analyzed by a gastrointestinal pathologist.

Quantitative Real-Time RT-PCR (qPCR)

RNA was isolated from fresh or frozen tissue using Isol-RNA lysis reagent (3 Prime, Gaithersburg, MD). After quantification with NanoDrop 2000 (NanoDrop products, Wilmington, DE), RNA with a purity of ~ 2.0 (260/280 ratio) was reverse-transcribed using M-MLV Reverse Transcriptase (Fisher Scientific, Waltham, MD). cDNA was quantified using SYBR green dye and run on a 7900HT Fast Real Time RT-PCR system (Life Technologies, Carlsbad, CA) (primers listed in Supplemental Table 1). Ct values were normalized to β-actin and expressed as fold difference from controls.

Western blot analysis

Tissues were homogenized and lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.1% SDS) for whole cell extracts. Proteins were separated and transferred to nitrocellulose membranes using standard methods. Membranes were incubated with antibodies against cleaved keratin 18 (K18) (kindly provided by Prof. Bishr Omary, University of Michigan), total caspase 3, cleaved caspase 3 (Cell Signaling Technology Inc., Beverly, MA), HIF-2α (Novus Biologicals, Littleton, CO), DMT-1 (alpha Diagonostic, San Antonio, TX) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) followed by incubation with appropriate HRP-conjugated secondary antibodies (Cell Signaling Technology Inc.). Blots were detected using the Enhanced Chemiluminescence Detection kit (Thermo Scientific, Wayne, MI).

Primary colon organoid and other cell culture

For organoid whole-colon cultures, colons were opened longitudinally and were rinsed gently and quickly placed in culture medium, RPMI-1640 plus 10% heat inactivated fetal bovine serum (FBS) and 1% antibiotic/antimycotic (1 units/mL of penicillin, 1 µg/mL of streptomycin, and 2.5ng/mL of amphotericin B) (Life Technologies, Carlsbad, CA). The colons were cut into 5- to 7-mm pieces and incubated in the culture medium described above for 2-hour at 37 °C, 5% CO2/21% O2. After incubation, tissues were homogenized and lysed in RIPA buffer to perform Western blot analysis as described above. HCT116 cells were obtained from ATCC and maintained at 37 °C in 5% CO2 and 21% O2. Cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotic/antimycotic. Early passaged (P10) stable HIF-2α and parental HCT116 cells were generated by transfection of a normoxically stable HIF-2α or pIRES puro 3 empty vector with lipofectamine 2000 (Life Technologies) according to manufacturer's instructions and selected by 2 µg/mL puromycin. The cells were maintained in growth media described above containing 1 µg/mL of puromycin.

PolyHema Apoptosis Assay

Cell adhesion to proper extracelluar matrix is essential for epithelial cell survival. Failure of attachment leads to a form of apoptosis termed anoikis (31). Poly-hydroxyl-ethyl-meth-acrylate (PolyHema) is a polymer which can form a surface that prevents cell adherence when coated on cell culture plate(32). Twenty-four well plates were coated with 250 µL/well PolyHema (20 mg/mL) to achieve nonadherent conditions. HIF-2α overexpressing or parental HCT116 cells were plated at a concentration of 1×104 cells/mL in DMEM supplemented with 10% FBS and 1% antibiotic/antimycotic into PolyHema coated plates or non-coated control plates. After 24- or 48-hour culture, 125 µL 5 mg/mL Thiazolyl Blue Tetrazolium Bromide (MTT) (Sigma) reagent was added to each well and incubated for 30 minutes. DMSO was added and absorbance was measured at 570 nm.

cDNA microarray analysis

RNA was extracted from colon mucosal scrapings from 5 weeks old VhlF/F, VhlΔIE, VhlF/F/Apcmin/+ and VhlΔIE/Apcmin/+ mice. Isolated RNA with RNA integrity number values >6.5 examined by Agilent 2100 bioanalyzer (Agilent Technologies, Inc., Santa Clara CA) was amplified, reverse transcribed, labeled, and hybridized to mouse 430 2.0 Affymetrix GeneChips (Affymetrix, Santa Clara, CA), and data were analyzed as previously described(28). The full data set is available on the GEO database accession number GSE36091.

Statistics

Results are expressed as mean ± S.D. P values were calculated by independent t-test, One-way ANOVA, Dunnett’s t-test, and two-way ANOVA. p<0.05 was considered significant.

Results

Activation of HIF-2α potentiates colon carcinogenesis following APC mutation

Mice with an intestine-specific disruption of Vhl (VhlΔIE) activate HIF signaling, whereas no induction of HIF signaling is observed in littermate controls (VhlF/F)(28, 33). To assess the role of HIF in intestinal tumorigenesis, these mice were crossed to the Apcmin/+ mice (Supplemental Figure 1A). Apcmin/+ mice have a nonsense mutation at codon 850 of the murine Apc gene leading to a truncated protein (34). Adenomatous polyposis coli (APC) is a tumor suppressor protein highly relevant in colon cancer; over 80% of patients with sporadic colon cancer have a somatic mutation of the Apc gene(35), and mutations are observed in early and late colitis associated neoplasms(36–38). In patients with Apc mutations, the predominant cancer is colon cancer and very rarely (≈10 fold less) are small intestinal tumors observed(39). However, Apcmin/+ mice develop mostly small intestinal tumors(34). Consistent with previous data, VhlF/F/Apcmin/+ mice developed predominantly small intestinal tumors. In the small intestine, tumors were found in 17/17 3-month-old VhlF/F/Apcmin/+ mice, with a tumor multiplicity of 32.24, whereas 4/17 VhlF/F/Apcmin/+ mice developed tumors in the colon with a tumor multiplicity of 0.59. Interestingly, VhlΔIE mice crossed to the Apcmin/+ background had a dramatic shift in the tumor localization to the colon in 3-month-old mice (Figure 1A–1C). Small intestinal tumors were significantly decreased in the 3-month-old VhlΔIE/Apcmin/+ compared to VhlF/F/Apcmin/+ mice (tumor incidence: 11/15 mice; tumor multiplicity: 2.80). However, a significant increase in tumor incidence and numbers were observed in the colon of 3-month-old VhlΔIE/Apcmin/+ mice (tumor incidence: 15/15 mice; tumor multiplicity: 13.87). Consistent with this, Western blotting analysis demonstrated an increase of HIF-2α protein expression in both normal colon and tumor tissues from VhlΔIE/Apcmin/+ mice (Supplemental Figure 2). To assess whether the changes in tumor number and incidence were dependent on HIF-2α, a double knockout mouse model of Vhl and Hif-2α on an Apcmin/+ background (VhlΔIE/Hif-2αΔIE/Apcmin/+) was generated and compared to littermate controls (VhlF/F/Hif-2αF/F/Apcmin/+) (Supplemental Figure 1B). Interestingly, small intestinal tumors in VhlΔIE/Hif-2αΔIE/Apcmin/+ mice were significantly repressed compared to the VhlF/F/Hif-2αF/F/Apcmin/+ mice. These data were similar to that observed in the VhlΔIE/Apcmin/+ mice indicating that VHL-mediated decrease in small intestinal tumorigenesis is independent of HIF-2α (Figure 1B and 1C). However, the increase in the colon tumorigenesis in VhlΔIE/Apcmin/+ mice was completely ablated following compound knockout of Vhl and Hif-2α in the Apcmin/+ background, demonstrating that HIF-2α plays a critical role in the development of colon cancer (Figure 1B and 1C). These results were further confirmed in the 6-month-old VhlΔIE/Apcmin/+ and VhlΔIE//Hif-2αΔIE/Apcmin/+ mice (Figure 1D and 1E). Compared to 3-month-old VhlF/F/Apcmin/+ mice, the tumor multiplicities in 6-month-old VhlF/F/Apcmin/+ mice were increased in the small intestine (tumor multiplicity: 62.50, tumor incidence: 6/6 mice) and colon (tumor multiplicity: 1.33, tumor incidence: 3/6 mice). However, small intestinal tumors were still significantly decreased in the 6-month-old VhlΔIE/Apcmin/+ (tumor incidence: 10/10 mice; tumor multiplicity: 15.10) compared to 6-month-old VhlF/F/Apcmin/+ mice. The tumor incidence and numbers observed in the colon of 6-month-old VhlΔIE/Apcmin/+ mice (tumor incidence: 10/10 mice; tumor multiplicity: 14.50) were not further increased compared to 3-month-old VhlΔIE/Apcmin/+ mice. However, they were still significantly increased compared to 6-month-old VhlF/F/Apcmin/+ mice. Assessing the double knockout mice (VhlΔIE/Hif-2αΔIE/Apcmin/+) at 6-months further demonstrated that the decrease in small intestinal tumorigenesis is independent of HIF-2α, whereas the increase in colon carcinogenesis is completely dependent on HIF-2α.

Figure 1. HIF-2α modulates intestinal tumorigenesis in Apcmin/+ mice.

Figure 1

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(A) Representative colons from VhlF/F/Apcmin/+ (n=24) and VhlΔIE/Apcmin/+ mice (n=25). Tumor counting in the small intestine (B) and colon (C) from 3-month-old VhlF/F/Apcmin/+ (n=17), VhlΔIE/Apcmin/+ (n=15), VhlF/F/Hif-2αF/F/Apcmin/+ (n=13) and VhlΔIE/Hif-2αΔIE/Apcmin/+ mice (n=11). Tumor counting in the small intestine (D) and colon (E) from 6-month-old VhlF/F/Apcmin/+ (n=7), VhlΔIE/Apcmin/+ (n=10), VhlF/F/Hif-2αF/F/Apcmin/+ (n=6), and VhlΔIE/Hif-2αΔIE/Apcmin/+ mice (n=6). *p<0.05 and ***p<0.001; NS, not significant.

Activation of HIF-2α increases tumor progression in the Apcmin/+ mice

All tumors assessed in the small intestine or colon from 3- and 6-month-old VhlF/F/Apcmin/+ mice demonstrated well-organized glandular structures and were classified as adenomas (Figure 2A). However, 2 out of 15 tumors from 3-month-old VhlΔIE/Apcmin/+ mice demonstrated early signs of carcinoma formation (Figure 2B and 2D). In 6-month-old VhlΔIE/Apcmin/+ mice, 8 out of 10 mice had colon tumors that displayed complex glands with cribriform architecture and desmoplastic stroma, and thus were classified as carcinomas (Figure 2C and 2D). To assess if the increase in colon cancer progression was HIF-2α-dependent, tumor analysis was performed on 6-month-old VhlΔIE/Hif-2αΔIE/Apcmin/+ mice (Figure 2D). Although adenomas were observed, no tumors progressed to carcinomas. These data demonstrate that activation of HIF-2α is critical for the progression of colon tumors.

Figure 2. HIF-2α promotes carcinoma formation in Apcmin/+ mice.

Figure 2

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(A) H & E staining of representative duodenum and colon from 3-month-old VhlF/F/Apcmin/+ and VhlΔIE/Apcmin/+ mice. (B) H & E staining of representative colon from 3-month-old VhlΔIE/Apcmin/+ mice with an adenoma and progression to early stage carcinoma. (C) H & E staining of representative colon carcinoma from 6-month-old VhlΔIE/Apcmin/+ mice. (D) Incidence of carcinoma in 3- and 6-month VhlF/F/Apcmin/+, VhlΔIE/Apcmin/+, VhlF/F/Hif-2αF/F/Apcmin/+, and VhlΔIE/Hif-2αΔIE/Apcmin/+ mice.

HIF-2α regulates cell survival in the colon

Analysis of apoptosis in colon tumors between VhlF/F/Apcmin/+ and VhlΔIE/Apcmin/+ was performed using TUNEL assay. No significant difference was observed in tumors from VhlF/F/Apcmin/+ and VhlΔIE/Apcmin/+ mice (Supplemental Figure 3). Interestingly, a dramatic increase in cell survival was observed in normal colon epithelial cells of VhlΔIE mice compared to VhlF/F mice. Intestinal epithelial cells undergo a spontaneous form of apoptosis termed anoikis as the differentiated cells reach the villus tips and are shed(40). Capturing this by TUNEL or caspase staining is difficult. Therefore, the apoptotic response of primary colon organoids generated from VhlF/F and VhlΔIE mice were compared. The colons from VhlF/F and VhlΔIE mice were isolated and snap frozen immediately or cultured for 2-hour. Upon culturing, the colon undergoes a dramatic induction of anoikis-induced apoptosis as seen with increased expressions of cleaved keratin 18 and cleaved caspase 3 in colons isolated from VhlF/F mice (Figure 3A). However, in colons cultured from VhlΔIE mice, significant decreases in cleaved keratin 18 and cleaved caspase 3 expressions were observed (Figure 3A). In contrast, cleaved keratin 18 and cleaved caspase 3 expressions were partially restored in colons cultured from VhlΔIE/Hif-2αΔIE mice (Figure 3A). To further confirm these results, a stable HIF-2α overexpressing colon-derived HCT116 cell line was generated. As expected, in parental HCT116 cells, HIF-2α was not detectable whereas in the stable overexpressing cells, a dramatic increase in HIF-2α expression was observed (Figure 3B). Cell survival was assessed in an anoikis-induced apoptotic assay. Cells were incubated in poly-hema coated plates to inhibit attachment, leading to increased apoptosis. Parental cells demonstrated decreased survival rate following 24- and 48-hour of incubation. The survival rate was significantly increased in HCT116 cells overexpressing HIF-2α (Figure 3C) with a concordant decrease in the activation of caspase 3 following 24-hour of incubation on poly-hema coated plates (Figure 3D). Although no significant difference is observed in tumor apoptosis (Supplemental Figure 3), activation of HIF-2α induces cell survival that may play an important role in colon tumor progression.

Figure 3. HIF-2α increases cell survival in the colon.

Figure 3

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(A) Western blot for cleaved caspase 3 (cCasp 3), cleaved keratin 18 (K18), and GAPDH in colon organoid cultures from VhlF/F, VhlΔIE, VhlF/F/Hif-2αF/F, and VhlΔIE/Hif-2αΔIE mice incubated for 0 h or 2 h at 37 °C. (B) Western Blot analysis for HIF-2α and GAPDH from HCT116 stably transfected with empty vector (EV) or HIF-2α plasmid. (C) Cell survival rate of HCT116 cells stably transfected with EV or HIF-2α plasmid in an anoikis-induced apoptosis assay. Survival rates were compared to uncoated control plates. Each bar represents the mean value ± S.D. (n=3). *p<0.05 and **p<0.01, versus EV. (D) Western Blot analysis for cCasp3, total caspase 3 (Casp3), and GAPDH from HCT116 cells stably transfected with EV or HIF-2α plasmid 24 h following induction of anoikis.

HIF-2α regulates cell proliferation in colon cancer

Since apoptosis could not fully explain the increase in colon tumor formation in the VhlΔIE/Apcmin/+ mice, the contribution of cell proliferation to HIF-2α induced colon cancer was assessed. Cyclin D1 expression was assessed in VhlΔIE/Apcmin/+ and VhlF/F/Apcmin/+ mice by qPCR analysis. No significant difference of cyclin D1 expression was observed in the normal colon tissues of VhlF/F/Apcmin/+ and VhlΔIE/Apcmin/+ mice. However, cyclin D1 was significantly induced in the colon tumors of VhlΔIE/Apcmin/+ mice compared to tumors isolated from VhlF/F/Apcmin/+ mice (Figure 4A). Consistent with cyclin D1 expression, BrdU incorporation revealed no significant difference in the normal colon tissues of VhlF/F/Apcmin/+ and VhlΔIE/Apcmin/+ mice (Figure 4B and C). However, the numbers of BrdU positive proliferating epithelial cells in the colon tumors of VhlΔIE/Apcmin/+ mice were significantly higher than those in tumors of VhlF/F/Apcmin/+ mice (Figure 4B and 4C). Elevated tumor proliferation was ablated in the colon tumors of VhlΔIE/Hif-2αΔIE/Apcmin/+ mice (Figure 4D). These data demonstrate that HIF-2α increases tumor cell proliferation, which may be critical for the increase in colon tumor formation and progression.

Figure 4. HIF-2α increases cell proliferation in the colon tumors.

Figure 4

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(A) qPCR analysis of cyclin D1 (Ccnd1) in normal and tumor colon tissues from VhlF/F/Apcmin/+, VhlΔIE/Apcmin/+, and VhlΔIE/Hif-2αΔIE/Apcmin/+ mice. Each bar represents the mean value ± S.D. (n=4). Expression was normalized to β-actin. (B) BrdU staining and (C) quantification in normal and tumor colon tissues from 6-month-old VhlF/F/Apcmin/+ and VhlΔIE/Apcmin/+ mice 2-hour following 100 mg/kg BrdU intraperitoneal injection. Each bar represents the mean value ± S.D. (n=4). (D) BrdU staining and quantification in normal and tumor colon tissues from VhlF/F/Hif-2αF/F/Apcmin/+ and VhlΔIE/Hif-2αΔIE/Apcmin/+ mice 2-hour following 100 mg/kg BrdU intraperitoneal injection. N, normal tissue; T, tumor tissue; NS, not significant; *p<0.05, **p<0.01 versus normal tissue; ## p<0.01 versus VhlF/F/Apcmin/+ mice. Each bar represents the mean value ± S.D. (n=4).

Divalent metal transporter-1 (DMT-1) and iron uptake represent a cellular target and regulator of HIF-2α-induced colon cancer

To identify the precise molecular mechanisms that could contribute to the increase in colon carcinogenesis, microarray gene expression analysis was performed on colon RNA isolated from 5-week-old VhlF/F and VhlΔIE, VhlΔIE/Apcmin/+ and VhlF/F/Apcmin/+ mice. The data identified 469 genes that were significantly increased/decreased in both VhlΔIE and VhlΔIE/Apcmin/+ compared to their littermate controls and 29 genes that were significantly increased/decreased in both VhlF/F/Apcmin/+ and VhlΔIE/Apcmin/+ compared to their littermate Apc wild-type controls (Figure 5A). The top ten genes that were regulated in a HIF-2α-dependent manner were further assessed utilizing ONCOMINE (Supplemental Table 2), a cancer microarray data-mining platform(41). Those genes that were increased in the colons of VhlΔIE/Apcmin/+ mice and in human colon cancer samples were identified. This analysis demonstrated a specific increase in DMT-1 expression in colon adenomas and carcinomas isolated from patients compared to normal controls in 3 independent studies(42–44) (Figure 5B). DMT-1 is the major apical iron transporter in the intestine(45–47). It is essential for iron absorption and its expression in the small intestine is directly regulated by HIF-2α(26). Colon tumors have been shown to accumulate iron(23). Moreover, mouse models of colon cancer and population-based studies demonstrate a direct correlation between an increase in intestinal iron and increased incidence of colon cancer(24, 25). These studies suggest that dysregulation of local iron homeostasis is critical in colon carcinogenesis. Consistent with these results, measuring tissue iron using enhanced Prussian iron staining demonstrated a significant increase in iron accumulation in colon tumors compared to normal tissue (Figure 5C). To assess whether HIF-2α was critical for DMT-1 regulation during colon tumorigenesis, DMT-1 expression was measured by qPCR. DMT-1 was significantly induced in the colons of VhlF/F/Apcmin/+ mice compared to wild-type littermates (VhlF/F), which was further potentiated in VhlΔIE/Apcmin/+ mice compared to wild-type littermates in a HIF-2α-dependent manner (Figure 5D). Similar expression patterns were also noted in the small intestine (Supplemental Figure 4). Western blotting analysis further demonstrated that DMT-1 protein expression was increased in a HIF-2α-dependent manner in colon tumors (Figure 5E).

Figure 5. DMT-1 expression in colon tumors.

Figure 5

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(A) Global gene expression profiling in colon RNAs isolated from 5-week-old VhlF/F (n=4), VhlF/F/Apcmin/+(n=3), VhlΔIE (n=3) and VhlΔIE/Apcmin/+ mice (n=5). (B) DMT1 gene expression levels in three independent human colon cancer studies were analyzed using ONCOMINE. **p<0.01,***p<0.001 versus normal colon tissue. (C) Enhanced Prussian blue staining for iron in normal colon and colon cancer tissue. Arrows indicate iron staining. (D) qPCR analysis of Dmt1 in colon epithelial cells from VhlF/F (n=6), VhlF/F/Apcmin/+ (n=5), VhlΔIE/Apcmin/+ (n=5) and VhlΔIE/Hif-2αΔIE/Apcmin/+ mice (n=3). Expression was normalized to β-actin. *p<0.05,***p<0.001 (E) Western Blot analysis for DMT-1 and GAPDH from normal and tumor colon tissues of 3-month-old VhlF/F/Apcmin/+, VhlΔIE/Apcmin/+, VhlF/F/Hif-2αF/F/Apcmin/+ and VhlΔIE/Hif-2αΔIE/Apcmin/+ mice. N, normal tissue; T, tumor tissue.

Low-iron treatment reduces HIF-2α-induced colon tumor formation

To verify that the increase in iron contributed to the increase in colon carcinogenesis, VhlΔIE/Apcmin/+ and VhlF/F/Apcmin/+ mice were placed on control or low-iron diet. As expected, the VhlΔIE/Apcmin/+ mice on control diet demonstrated increased colon tumor numbers compared to littermate controls (Figure 6A). The increase in colon tumors was completely ablated in VhlΔIE/Apcmin/+ mice on low-iron diet for 2 months (Figure 6A). Analysis of BrdU incorporation revealed no significant difference in the normal colon tissues of VhlF/F/Apcmin/+ and VhlΔIE/Apcmin/+ mice treated with control diet. The number of BrdU positive proliferating epithelial cells in the colon tumors of VhlΔIE/Apcmin/+ mice was dramatically higher than that in tumors of VhlF/F/Apcmin/+ mice, whereas low-iron diet treatment greatly decreased the number of BrdU positive epithelial cells in the tumor colon tissues of both VhlF/F/Apcmin/+ and VhlΔIE/Apcmin/+ mice (Figure 6B). These results demonstrate that tumoral iron is essential for colon cancer progression and a critical pathway by which HIF-2α signaling contributes to colon tumorigensis.

Figure 6. Low-iron diet decreases HIF-2α-mediated intestinal tumorigenesis and cellular proliferation.

Figure 6

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(A) Tumor numbers in the colon from VhlF/F/Apcmin/+ (n=11), and VhlΔIE/Apcmin/+ mice (n=10) treated with control or low-iron diet for 2 months. **p < 0.01; NS, not significant. (B) BrdU staining of colon tumors from VhlF/F/Apcmin/+ and VhlΔIE/Apcmin/+ mice treated with control or low-iron diet for 2 months.

Discussion

Hypoxia is an adaptive response in many solid tumors. However, its role in tumor development in vivo is still not clear. The present study found that the activation of HIF signaling through HIF-2α enhanced colon cancer incidence and progression in the Apcmin/+ mice. The Apc tumor suppressor is a gatekeeper gene mutated in a majority of patients with familial, sporadic, and colitis-associated colon cancer(35–38). However, Apcmin/+ mice develop mostly small intestinal tumors and colon tumors are observed with low incidence and multiplicity(34). HIF-2α activation in the intestines of VhlΔIE/Apcmin/+ mice dramatically increased colon tumor multiplicity and incidence. Moreover, at 6 months of age the majority of colon tumors observed were histologically defined as carcinomas, whereas no tumors identified in the VhlF/F/Apcmin/+ mice progressed further than adenomas, demonstrating that HIF-2α may be a critical transcription factor involved in colon cancer progression.

Interestingly, small intestinal tumors observed in the VhlΔIE/Apcmin/+ mice were rare and this decrease in tumorigenesis was independent of HIF-2α expression. Despite the profound decrease in tumorigenesis, no changes in small intestinal proliferation were observed between VhlΔIE/Apcmin/+ and VhlF/F/Apcmin/+ mice (Supplemental Figure 5). Moreover, the VhlΔIE/Apcmin/+ mice had increased DMT-1 expression in the small intestine, suggesting that decreased small intestinal tumorigenesis by the HIF-2α-independent mechanism is downstream of the iron accumulation effects. HIF-1α was shown to have an antagonistic role in cell growth and VHL has several HIF-independent functions(11, 48–52), and these pathways are currently being assessed in the small intestine. There is some evidence that colon tumors in the Apcmin/+ do not form because of the large tumor load of the small intestine compromise the mice, therefore the slower forming colon tumors do not develop. However, in the VhlΔIE/Hif-2αΔIE/Apcmin/+ mice, the decrease in small intestinal tumors, did not lead to a refractory increase in colon tumors. Confirming that the increase in colon tumors in the VhlΔIE/Apcmin/+ mice is mediated by HIF-2α activation. The VhlΔIE/Apcmin/+ mice, through HIF-2α-dependent and -independent mechanisms, can recapitulate downstream events following Apc mutation that is observed in sporadic, familial, and colitis-associated colon cancer, and is an optimal pre-clinical animal model to study colon cancer.

The mechanisms that decrease small intestinal tumorigenesis are currently not known. However, the increases in colon tumor incidence, multiplicity, and progression in VhlΔIE/Apcmin/+ mice was completely ablated following double knockout of Vhl and Hif-2α in the Apcmin/+ background, demonstrating that HIF-2α plays a critical role in the development of colon cancer following Apcmin/+ mutation. Activation of HIF-2α in the intestine increased epithelial cell survival. This was measured using colon organoids that may be in undergoing apoptosis very early following the culture of the colons and using colon cancer-derived cells lines. Therefore, the anoikis detected may not be a physiological response and a more detailed analysis of HIF-2α mediated cell survival is needed. However, the present study clearly demonstrates a role of HIF-2α in tumor proliferation. To understand the mechanisms involved in the increase in colon tumorigenesis, the HIF-2α-dependent gene expression profile was analyzed by the ONCOMINE database(41). The HIF-2α target gene DMT-1 was highly induced in colon adenomas and carcinomas compared to normal colon tissue isolated from patients. DMT-1 is a critical intestinal iron transporter and its overexpression can lead to iron accumulation(45, 46, 53, 54). Iron accumulation is a critical factor in colon carcinogenesis and studies have shown that both body iron stores and dietary iron intake are positively associated with subsequent risk of colon cancer(22). In mouse models of colon cancer, iron-enriched diets increase the development of colon tumors(21). Iron activates free radical formation, which have a pleiotropic effect in carcinogenesis, including increase in cell survival and proliferation, which is consistent with the present study. Together the data suggest that HIF-2α mediated dysregulation of intestinal iron transport is critical for the increase in colon tumors observed in the VhlΔIE/Apcmin/+ mice. Therefore inhibition of HIF-2α activation in colon tumors may represent a potential strategy for the prevention and treatment of colon cancer. Moreover, our data suggest that iron chelators will be beneficial for patients with colon cancer. It is important to note that HIF-2α activates a large battery of genes in the intestine and its mechanistic role in tumor development is most likely through multiple pathways. However, in the present study we clearly demonstrate that the activation of HIF-2α contributes to the increase in cell proliferation, dysregulation of local iron homeostasis, and eventually an increase in the incidence of colon cancer.

Supplementary Material

1

Acknowledgments

We thank Dr. Bishr Omary for providing cleaved K18 antibody. The University of Michigan Cancer Center Microarray Core for Affymetrix gene array services and analysis.

Grant Support

This study was supported by grants to Y.M.S from the National Institutes of Health (CA148828), The University of Michigan Gastrointestinal Peptide Center, and Jeffrey A. Colby Colon Cancer Research and the Tom Liu Memorial Funds of the University of Michigan Comprehensive Cancer Center. F.J.G was supported by the Intramural Research Program of the National Cancer Institute.

Footnotes

Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed.

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