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

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 Apc(min/+) 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 Apc(min/+) mice in which HIF-2α was activated in the intestine. Consistent with this result, bromodeoxyuridine incorporation indicated that cellular proliferation was increased in colon tumors following HIF activation. Further analysis showed 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 show that a chronic increase in HIF-2α in the colon initiates protumorigenic signaling, which may have important implications in developing preventive and therapeutic strategies for colon cancer.

Figure 1. HIF-2α modulates intestinal tumorigenesis in Apc^min/+ mice

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

Figure 2. HIF-2α promotes carcinoma formation in Apc^min/+ mice

(A) H & E staining of representative duodenum and colon from 3-month-old Vhl^F/F/Apc^min/+ and Vhl^ΔIE/Apc^min/+ mice. (B) H & E staining of representative colon from 3-month-old Vhl^ΔIE/Apc^min/+ mice with an adenoma and progression to early stage carcinoma. (C) H & E staining of representative colon carcinoma from 6-month-old Vhl^ΔIE/Apc^min/+ mice. (D) Incidence of carcinoma in 3- and 6-month Vhl^F/F/Apc^min/+, Vhl^ΔIE/Apc^min/+, Vhl^F/F/Hif-2α^F/F/Apc^min/+, and Vhl^ΔIE/Hif-2α^ΔIE/Apc^min/+ mice.

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

(A) Western blot for cleaved caspase 3 (cCasp 3), cleaved keratin 18 (K18), and GAPDH in colon organoid cultures from Vhl^F/F, Vhl^ΔIE, Vhl^F/F/Hif-2α^F/F, and Vhl^ΔIE/Hif-2α^ΔIE 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.

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

(A) qPCR analysis of cyclin D1 (Ccnd1) in normal and tumor colon tissues from Vhl^F/F/Apc^min/+, Vhl^ΔIE/Apc^min/+, and Vhl^ΔIE/Hif-2α^ΔIE/Apc^min/+ 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 Vhl^F/F/Apc^min/+ and Vhl^ΔIE/Apc^min/+ 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 Vhl^F/F/Hif-2α^F/F/Apc^min/+ and Vhl^ΔIE/Hif-2α^ΔIE/Apc^min/+ 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 Vhl^F/F/Apc^min/+ mice. Each bar represents the mean value ± S.D. (n=4).

Figure 5. DMT-1 expression in colon tumors

(A) Global gene expression profiling in colon RNAs isolated from 5-week-old Vhl^F/F (n=4), Vhl^F/F/Apc^min/+(n=3), Vhl^ΔIE (n=3) and Vhl^ΔIE/Apc^min/+ 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 Vhl^F/F (n=6), Vhl^F/F/Apc^min/+ (n=5), Vhl^ΔIE/Apc^min/+ (n=5) and Vhl^ΔIE/Hif-2α^ΔIE/Apc^min/+ 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 Vhl^F/F/Apc^min/+, Vhl^ΔIE/Apc^min/+, Vhl^F/F/Hif-2α^F/F/Apc^min/+ and Vhl^ΔIE/Hif-2α^ΔIE/Apc^min/+ mice. N, normal tissue; T, tumor tissue.

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

(A) Tumor numbers in the colon from Vhl^F/F/Apc^min/+ (n=11), and Vhl^ΔIE/Apc^min/+ 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 Vhl^F/F/Apc^min/+ and Vhl^ΔIE/Apc^min/+ mice treated with control or low-iron diet for 2 months.