The renin-angiotensin system mediates epidermal growth factor receptor-vitamin D receptor cross-talk in colitis-associated colon cancer

Abstract

Purpose

We previously showed that epidermal growth factor receptor (EGFR) promotes tumorigenesis in the azoxymethane/dextran sulfate sodium (AOM/DSS) model, whereas vitamin D (VD) suppresses tumorigenesis. EGFR-vitamin D receptor (VDR) interactions, however, are incompletely understood. VD inhibits the renin-angiotensin system (RAS), whereas RAS can activate EGFR. We aimed to elucidate EGFR-VDR cross-talk in colorectal carcinogenesis.

Experimental Design

To examine VDR-RAS interactions, we treated Vdr+/+ and Vdr/− mice with AOM/DSS. Effects of VDR on RAS and EGFR were examined by Westerns, immunostaining and real time PCR. We also examined the effect of vitamin D3 on colonic RAS in Vdr+/+ mice. EGFR regulation of VDR was examined in hypomorphic Egfr^Waved2 (Wa2) and Egfr^wildtype mice. Ang II-induced EGFR activation was studied in cell culture.

Results

Vdr deletion significantly increased tumorigenesis, activated EGFR and βcatenin signaling and increased colonic RAS components: including renin and angiotensin II. Dietary VD3 supplementation suppressed colonic renin. Renin was increased in human colon cancers. In studies in vitro, Ang II activated EGFR and stimulated colon cancer cell proliferation by an EGFR-mediated mechanism. Ang II also activated macrophages and colonic fibroblasts. Compared to tumors from Egfr^Waved2 mice, tumors from Egfr^wildtype mice showed up-regulated Snail1, a suppressor of VDR, and down-regulated VDR.

Conclusions

VDR suppresses the colonic RAS cascade, limits EGFR signals and inhibits colitis-associated tumorigenesis, whereas EGFR increases Snail1 and down-regulates VDR in colonic tumors. Taken together, these results uncover a RAS-dependent mechanism mediating EGFR and VDR cross-talk in colon cancer.

INTRODUCTION

Inflammation is recognized as an essential promoter of malignant transformation (1). Ulcerative colitis (UC), an inflammatory bowel disease (IBD) of the colonic epithelium, is associated with increased colon cancer risk (2). The duration and severity of inflammation modulate this risk (2). Since diagnosis of early colon cancer in UC is challenging and the prognosis for invasive disease limited, increasing efforts have focused on chemoprevention. Vitamin D is a potential chemopreventive agent in IBD-associated colon cancer (3). This pro-hormone is converted to active 1α,25-dihydroxyvitamin D3 [1,25(OH)₂D₃] by hepatic 25-hydroxylase and renal and extra-renal 1α-hydroxylase. 1,25(OH)₂D₃ binds the vitamin D receptor (VDR) to transduce biological signals in diverse tissues, including the colon (4).

The azoxymethane/dextran sulfate sodium (AOM/DSS) model of inflammation-associated colon cancer mimics many features of IBD-associated colon cancer (5). Animals receiving AOM/DSS develop colitis followed by colon cancer. Colonocytes, initiated by the mutagen AOM, are expanded by epithelial regeneration that follows DSS-induced colonic epithelial damage. In prior AOM/DSS studies using hypomorphic Egfr^Wa2 mice, we showed that EGFR was required for Western diets to promote tumorigenesis, whereas others have shown that EGFR inhibitors reduce stem cells in experimental colon cancer (6, 7). We also demonstrated that vitamin D suppresses dysplasia in this model (8), whereas VDR deletion increased DSS colitis (9). These studies suggest that decreased VDR signals exaggerate colonic pro-inflammatory cytokines (10). In addition, we recently demonstrated that colonic epithelial VDR maintains intestinal mucosal barrier integrity to prevent microbial inflammation (11). Thus, these data indicate that EGFR and VDR exert opposing effects on colonic inflammation and tumorigenesis. Furthermore, studies in cell culture have identified an important opposing VDR-EGFR cross-talk in colon cancer cells (12-14). Investigations to dissect mechanisms of this cross-talk in vivo in colonic tumorigenesis, however, have not been reported.

The renin-angiotensin system (RAS) regulates systemic vascular tone and sodium balance (15). RAS is also mitogenic and angiogenic and contributes to neoplastic growth in breast, ovary, lung, prostate and pancreatic cancer (16). Several RAS components, including renin, angiotensin converting enzyme (ACE) and angiotensin II (Ang II), are locally up-regulated in tumors. These components are also expressed in human colonic mucosa (17). Furthermore, epidemiological studies suggest that inhibitors of the RAS reduce colonic tumorigenesis (18). In prior analyses, we demonstrated that vitamin D signals suppress renin transcription and that this limits macrophage-associated inflammation (19-21). The macrophage is implicated in DSS inflammation (22). In the current study, we therefore asked if vitamin Dand the VDR regulate colonic RAS signals modulated by Western diet or inflammation-associated colon cancer. We used Vdr^+/+ and Vdr^−/− mice and vitamin D supplementation to dissect VDR regulation of RAS signals. Since RAS can activate EGFR, we also examined VDR regulation of EGFR in colonic tumorigenesis. To examine the potential translational relevance of our findings, we measured renin expression in sporadic human colonic tumors.

Fibroblasts and macrophages are important stromal cells that drive cancer cell proliferation (23). As Ang II is a mitogen and can transactivate EGFR in non-colonic cells (24), we asked if the RAS signaling could activate EGFR and stimulate proliferation of colonic cancer cells and fibroblasts. Furthermore, as the RAS can induce inflammation (25), we examined the effects of Ang II on tumor necrosis factor-α (TNFα) in macrophages.

Finally, as studies in vitro suggest that EGFR can also regulate VDR (12, 13), we investigated potential EGFR regulation of VDR using archived tumors induced by AOM/DSS in Egfr^wildtype and Egfr^{Waved2/Waved2} mice. The Waved2 Egfr mutation abrogates nearly 90% of receptor kinase activity in vitro (26). Furthermore, EGFR can up-regulate Snail1 in vitro, and this transcription factor was shown to suppress VDR in colon cancer cells (27). We, therefore, also investigated EGFR regulation of Snail1 in AOM/DSS-induced tumors. Taken together, our findings uncover a functional VDR regulated RAS pathway in vivo that controls EGFR signals in colonic carcinogenesis.

MATERIALS AND METHODS

Materials

A defined Western style diet containing 20% fat was used for the experiments in Vdr ^−/− and Vdr^+/+ mice. This diet, which included 2% calcium and 20% lactose to prevent hypocalcemia in Vdr null mice, was modified from a previously described defined diet (6, 19). Azoxymethane was obtained from Midwest Research, the NCI Chemical Carcinogen Reference Standard Repository (Kansas City, MO). Tarceva was obtained fro OSI Pharmaceuticals. Antibodies for immunostaining and Western blotting and molecular reagents for real time PCR are provided in the Supplemental data section.

Methods

Experimental animal protocol for Vdr^−/− and Vdr^+/+ mice

We used 20 Vdr^+/+ and 20 Vdr^−/− mice (28), backcrossed 10 generations to CD-1 background, to dissect the role of VDR in colonic tumorigenesis. Mice were 6-10 wks of age and included a comparable number of males and females in each genotype. For each genotype, 15 mice were treated with AOM (7.5 mg/kg) and 5 mice received saline (AOM vehicle) at days 1 and 14. After the 2nd AOM treatment mice received 1.5% DSS in the drinking water for 5 days, while saline-treated mice received water only. The DSS concentration was chosen since in preliminary studies 2% DSS caused 80% mortality in Vdr^−/− mice. Following 5 days DSS mice received tap water for 2 wks. The mice received 3 cycles of DSS and colitis disease activity index was assessed for each cycle (29). Twenty-four wks after the initial AOM injection, mice were anesthetized and treated with 30% H₂O₂ and vanadate solution as described and sacrificed 20 min later (30). Tumors were measured with a micrometer, harvested and fixed in 10% buffered formalin. Separate tumor aliquots were flash frozen in liquid nitrogen for RNA or protein. Tumors were classified according to histological grade by an GI pathologist (JH) (31). Distal colonic mucosa, cleared of any tumors, was scrape-isolated and aliquots frozen for protein and RNA. The remaining colons were fixed flat in 10% formalin for immunostaining. The Institutional Animal Care and Use Committee (IACUC) at the University of Chicago approved all animal studies.

Experimental animal protocol for Vdr wild type CF-1 mice:

CF-1 female mice age 4-6 wks were given AOM (7.5 mg/kg body wt) or saline (AOM vehicle) followed by one cycle of DSS (AOM treated) or water (saline treated) for 5 days. Mice were then fed a Western diet (20% fat, n=10) alone, or WD supplemented with cholecalciferol (500 μg/kg diet, n=10) The WD and cholecalciferol dose were previously shown to promote or inhibit AOM/DSS-induced colonic tumorigenesis, respectively (6, 32). Twelve wks after WD initiation mice were sacrificed and mucosa from left colon was harvested and RNA extracted.

Archived colonic tissue

Mouse tissue

For some experiments we used colonic tissue banked from a previous study (6). The prior study investigated the role of EGFR in Western diet-promoted colon cancer in the AOM/DSS model using Egfr^wildtype and Egfr^Waved2 mice (6). The Wa2 mutation abrogates >80% receptor kinase activity in vitro (26).

Human tissue

For studies involving sporadic human colon cancers, we obtained fresh flash frozen tumors and adjacent normal-appearing mucosa dissected free from underlying muscle from the Human Tissue Resource Center at the University of Chicago under an approved IRB protocol 10-209-A.

Cell culture and proliferation

Low passage CCD-18Co colonic fibroblasts, and HT29, HCT116 and DLD1 human colon cancer cells and RAW 264.7 murine macrophage cells were obtained from ATCC. These cell lines were authenticated by ATCC using short tandem repeat DNA fingerprinting. Cells were cultured at 37 °C in a humidified atmosphere of 5%CO₂-95% air under conditions recommended by ATCC. Cells were treated with Ang II or vehicle, or pre-treated with losartan, gefitinib or Tarceva at the indicated concentrations. For RNAi experiments cells were pretreated for 24 hrs with 20 nM Egfr siRNA or a scrambled control. Cell proliferation was measured by WST-1 assay as suggested by the manufacturer (see Supplemental Methods).

Real-time PCR

RNA was extracted from snap frozen tissue using Qiagen miRNeasy Mini Kit that captures total RNA including miRNA. Samples were homogenized with a Polytron and loaded onto an RNA-binding spin column, washed, digested with DNase I and collected in 30 μl elution buffer. RNA samples were examined by Agilent chip for RNA purity and quantified by Ribogreen. Real time PCR was performed as previously described (6) [see Supplemental Methods].

Immunohistochemistry

Tumors and normal colon were immunostained as previously described (6) [see Supplemental Methods for details]. For semi-quantitative analysis of immunostaining we used a Leica DM2500 microscope equipped with a CCD camera (Q Imaging Retiga EXI Fast1394) and captured images with Image Pro Plus (V6.3) software. DAB staining was analyzed using Fiji (ImageJ V1.48k) and the H DAB deconvolution plug-in (33, 34). Color-specific thresholds were adjusted to distinguish brown (DAB positive) and blue (DAB negative) cells and to calculate the ratio of positively stained cells. At least 5fields per tumor and 3 tumors per group were scanned for quantitation. For nuclear β-catenin, Snail1 and VDR we used ImmunoRatio web-based software (35).

Western Blotting

Colonic mucosal lysates and lysates from tumors of comparable stage were used for Western blotting. Proteins were extracted in SDS-containing Laemmli buffer, quantified by RC-DC protein assay and subjected to Western blotting as previously described (6) [see Supplemental Methods].

ELISA

Ang II was assayed by EIA in lysates prepared from colonic mucosa from left colon as suggested by the manufacturer. TNF-α was assayed in RAW264.7 conditioned media by ELISA following the manufacturer’s directions. Amphiregulin was measured in conditioned media from HT29 cells by ELISA following the manufacturer’s directions.

Statistical Methods

Tumor incidence was defined as the percentage of mice with at least one tumor and compared between genotypes using Fisher exact test. Western blotting densitometry and ELISA data were summarized as mean±SD, and compared by unpaired Student’s t-test. Reverse transcriptase reactions were run in duplicate and assayed in triplicate and Ct values averaged. Untransformed Ct values were compared between groups Error! Bookmark not defined.. Relative abundance, expressed as 2(ΔΔCt), was calculated by exponentiating differences in Ct between mucosa from AOM/DSS-treated mice and mucosa from vehicle-treated mice with values normalized to β-actin mRNA as a reference gene. For all statistical analyses, p values <0.05 were considered statistically significant.

RESULTS

VDR suppresses inflammation and tumor development

To examine the role of the VDR in colitis-associated colon cancer we compared tumorigenesis in Vdr^+/+ and Vdr^−/− mice. Fig. 1 summarizes the protocol (Fig 1A) and clinical colitis score for the 3^rd DSS cycle (Fig 1B). Clinical disease activity scores were low in Vdr^+/+ mice, reflecting the low concentration of DSS chosen to prevent high mortality in Vdr^−/− mice (80% mortality with 2% DSS). In agreement with prior studies, Vdr deletion increased colonic inflammation induced by DSS (9). All mice in the Vdr^−/− group developed tumors (adenomas or cancers), compared to only 47% in Vdr^+/+ group (n=15 mice/genotype, p=0.001) [Fig 1C]. While tumor burdens were modest secondary to low DSS concentrations and calcium supplementation, Vdr-dependent differences in tumor incidence were significant, consistent with differences in inflammation (Fig 1B) (37). VDR loss appeared to increase tumor progression, with cancers in 27% Vdr^/− mice, compared to only 7% in Vdr^+/+ mice (Fig 1C, p=0.1). Tumors in Vdr^−/− group were also significantly larger (Fig 1D).

Fig 1. VDR suppresses AOM/DSS inflammation and tumorigenesis.

A. Study design. VDR^+/+ and VDR^−/− mice were treated with AOM and three cycles of DSS. B. Colitis index in 3rd DSS cycle (*p<0.05, compared to VDR^+/+). C. Tumor incidence and stage. VDR deletion increased tumor incidence (n=15 AOM/DSS treated mice/genotype) and appeared to increase tumor progression to cancer (27% vs. 7%, p=0.1). There were 11 adenomas and 4 cancers in the VDR^−/− group (n=15 total) and 6 adenomas and one cancer in the VDR^+/+ group (n=15 total). D. Tumor size. Mean±SD (*p<0.05, compared to VDR^+/+).

VDR negatively regulates EGFR signals

We next asked whether VDR modulates EGFR signals, since EGFR and VDR have opposing effects on tumorigenesis in this model. As shown in Fig 2A and quantified in Fig 2B, VDR deletion significantly increased activation of EGFR and ErbB2, and stimulated effectors AKT, ERK and STAT3. While βcatenin plays a critical role in colonic tumorigenesis, in prior studies we showed that EGFR controls βcatenin in AOM/DSS tumors in vivo, consistent with findings in colon cancer cells in vitro (6, 38). In agreement with these studies, we found that VDR deletion, which increases EGFR signals, also significantly enhanced nuclear βcatenin in malignant colonocytes, 49.2±11.3% in tumors from Vdr^/− vs. 28.8±7.1% in tumors from Vdr^+/+ mice (Fig 2C, p<0.05, n=4 adenomas/genotype). Not surprisingly, βcatenin targets, Myc and cyclin D1 (6, 39, 40) were also increased in Vdr^−/− tumors (Figs 2A-B) consistent with reports that vitamin D signals suppress Myc and cyclin D1 in colon cancer cells (41, 42).

Fig 2. VDR deletion stimulates EGFR signals and increases nuclear β-catenin accumulation in tumors.

A. EGFR signals. Tumor lysates from VDR+/+ and VDR−/−mice were probed for the indicated proteins. B. Quantitative densitometry (*p<0.05; †p<0.005 ‡p<0.0005, compared to VDR+/+ tumors, n = 4 tumors/genotype). C. Nuclear β-catenin. Tumors were stained for β-catenin and nuclear β-catenin quantified. Shown are representative tumors (*p<0.05 compared to VDR+/+, n=3 tumors/genotype).

VDR negatively regulates renin-angiotensin system (RAS) in AOM/DSS colonic tumors

The renin-angiotensin system (RAS) is a potential link between VDR and EGFR signals, as vitamin D is a negative regulator of the RAS; and the RAS in turn can transactivate EGFR (19, 24). Furthermore, the RAS is mitogenic and angiogenic for many tumors and RAS components are increased in other neoplastic tissue (16). We, therefore, examined the effect of Vdr deletion on colonic RAS by staining tumors for RAS components. Renin was greater in malignant colonocytes from Vdr^−/− mice compared to Vdr^+/+ mice (Fig 3A, upper panel). AT1 receptor expression was also greater in tumors fromVdr^−/− mice, compared to Vdr^+/+ mice and was readily detectable in tumor stromal cells (Fig. 3A, middle panel). As the RAS is known to drive blood vessel development, we also examined Nestin1, a marker of angiogenesis. Nestin-1 was 2.6±0.4-fold greater in tumors from Vdr^−/−, compared to Vdr^+/+ mice (Fig. 3A, lower panel).

Fig. 3. VDR deletion increases the RAS components in tumors and adjacent colonic mucosa.

A. Immunostaining for renin, AT-1 and Nestin-1 in colonic tumors. Note the white arrows on brown staining cells. Semiquantitative analyses of DAB staining (% positve cells) are indicated in right panels (*p<0.05, compared to VDR+/+ tumors, n=3 tumors/genotype). B. RAS transcripts. mRNAs were measured by qPCR in the distal colonic mucosa and expressed as fold-VDR+/+ (n=4 mice/group; *p<0.05 compared to Vdr^+/+ vehicletreated mice, ‡p<0.005, †p<0.001, compared to Vdr^+/+ AOM/DSS-treated mice). C. RAS proteins. Indicated proteins were probed by Western blotting in lysates from the distal colonic mucosa. D. Densitometry of RAS proteins. Mean±SD (*p<0.05, †p<0.005 compared to AOM/DSS-treated Vdr^+/+ mice, n=4 mice/group). E. Ang II measured by ELISA in distal colonic mucosa. (*p<0.05, compared to genotype-matched vehicle-treated mice, †p<0.05 compared to AOM/DSS-treated Vdr^+/+ mice; n= 3 mice/genotype/treatment condition). F. Dietary cholecalciferol suppresses renin and angiotensinogen levels in colonic mucosa. CF-1 mice, treated with saline or AOM/DSS, were fed Western diet, or WD supplemented with cholecalciferol. Angiotensinogen and renin were measured in distal colonic mucosa by real tine PCR (*, †p<0.05 compared to WD alone, n=5 mice/group). G. Renin and VDR in human colon cancers. Lysates prepared from colonic tumors (T), and adjacent normal appearing mucosa (N), were probed for renin, phospho-active EGFR (pEGFR) and pan EGFR and VDR as well as β-actin as a loading control. H. Densitometries of renin, VDR and pEGFR in tumors (mean±SD) were normalized to adjacent mucosa (*p<0.05; blots are representative of N=9 tumors and matched normal-appearing colonic mucosa).

VDR regulation of colonic RAS - field effect

Molecular abnormalities in colons harboring tumors are frequently widespread, with derangements in normal-appearing mucosa (43). To investigate more generalized “field effects”, we examined mRNA levels of several of the RAS components in distal colonic mucosa. In mice treated with saline alone (no AOM/DSS), angiotensin converting enzyme (ACE) transcripts were elevated in Vdr^−/− compared to Vdr^+/+ mice (Fig 3B). With AOM/DSS treatment, angiotensinogen (Agt), renin (Ren), ACE and angiotensin II receptor type 1A (Agtr1a) transcripts were up-regulated in Vdr^−/− mice compared to Vdr^+/+ mice (Fig 3B). Protein levels were also significantly higher in Vdr^−/− mice as shown in Fig 3C and quantified in Fig 3D. Levels of colonic mucosal Ang II, a major RAS effector, were significantly elevated in AOM/DSS-treated mice, compared to vehicle-treated mice matched for Vdr genotype. Increases were greater in Vdr^−/− mice (Fig 3E), consistent with greater increases in up-stream RAS components in Vdr^−/− mice. Colonic mucosal VEGF protein levels were also elevated in AOM/DSS-treated Vdr−/− mice compared to Vdr+/+ mice (Fig C-D). The latter results are consistent with differences in tumor nestin-1 levels by Vdr genotype (Fig 3A) and with prior reports in other tissue of positive VEGF regulation by Ang II and negative regulation by VDR (44, 45). To assess the effects of supplemental vitamin D on colonic mucosal RAS we measured transcripts of renin and angiotensinogen in colonic mucosa prepared from AOM/DSS- or saline-treated Vdr^+/+ CF-1 mice fed Western diet or Western diet supplemented with cholecalciferol. As shown in Fig 3F, cholecalciferol significantly decreased expression of these genes in both control mice (no AOM/DSS) and AOM/DSS-treated mice. Thus, VDR gain of function inhibits RAS signaling, whereas VDR loss of function enhances colonic RAS signaling. With only 5 mice in the AOM/DSS alone group and 5 mice in the AOM/DSS + VD3 group, the study was not powered for tumor prevention. We noted, however, that there were 4 tumors in WD alone group vs. 1 in the VD3 treated group (p=0.1). To assess the translational relevance of these observations we examined renin expression in human colon cancers. EGFR (pEGFR) activation and renin levels were increased in human colon cancers, emphasizing the potential relevance of up-regulated RAS in sporadic colonic tumorigenesis (Fig 3G,H). VDR levels were variable and not different in human tumors, suggesting that supplemental vitamin D by binding VDR might suppress tumor-associated RAS that we speculate promotes colonic tumorigenesis.

EGFR mediates angiotensin II-induced colon cancer cell and colonic fibroblast proliferation

We used cell culture to dissect Ang II-induced responses in malignant and non-malignant colonic cells. Colon cancer cells, colonic fibroblasts and macrophage cells express AT1 receptors (Fig 4A). Ang II stimulated proliferation of HT29, HCT116 and DLD1 colon cancer cells and colonic fibroblasts (Fig 4B). Losartan, a specific AT1 inhibiter, blocked Ang II induced mitogenic effects (Fig 4B). We infer that Ang II mitogenic effects are mediated by EGFR since gefitinib blocked Ang II-induced proliferation (Fig 4C). Similar results were obtained with Tarceva (Supplemental Fig S1). Receptor knock down with EGFR siRNA also blocked Ang II-induced proliferation (Fig 4C). Basal proliferation was also controlled by EGFR since treatment with gefitinib, Tarceva or EGFR siRNA alone also reduced HT29 cell proliferation (Fig 4C and Supplemental Fig S1). Ang II was shown previously to activate EGFR in non-colonic cells (24). In this study we showed that Ang II activated EGFR signals in HT29 cells (Fig. 4D-E). In data not shown, Ang II also transactivated EGFR in HCT116 and DLD1 cells.

Fig. 4. Ang II activates EGFR and stimulates colon cancer cell proliferation by an EGFR-dependent mechanism.

A. AT1 expression. Lysates from indcated cells were probed for AT1 and βactin as loading control. B. Ang II induces colon cancer cell and colonic fibroblast proliferation. Cells were treated with 50 nM Ang II alone or pre-treated with 1 μM losartan and cell proliferation assessed. (*p<0.005, compared to vehicle-treated cells). C. Gefitinib and EGFR siRNA block Ang II-stimulated HT29 cell proliferation. Cells were pre-treated with 1 μM gefitinib (G), vehicle (control), EGFR siRNA (20 nM) or scrambled oligonucleotide, followed by treatment with 50 ng/ml Ang II or 10 ng/ml EGF for 48 hrs. Inset Western blot of EGFR in HT29 cells treated with 20 nM scrambled (scr) or 20 nM EGFR siRNA for 48 hrs. (*,†p<0.05, compared to vehicle-treated control cells; ‡p<0.05 compared to Ang II alone; ◇p<0.05 compared to EGF alone). D & E. Ang II transactivates EGFR. HT29 cells were treated with indicated Ang II concentrations for 2.5-10 min. Cell lysates were probed for indicated proteins including β-actin as loading control (D). Note that pErbB2 runs as a broad band above a non-specific band (NS). Time- and dose-response of indicated phospho-active proteins to Ang II treatment. (E). Cell culture results were replicated in independent platings.

RAS signals (Ang II) induce inflammation in macrophage cells

The macrophage is implicated in DSS inflammation (22). We observed that macrophages were more abundant in tumors from Vdr^−/− mice (Fig. 5A-B). As shown in Fig. 5C, colonic TNF-α was increased in vehicle-treated Vdr^−/− animals, compared to Vdr^+/+ mice. Following AOM/DSS treatment, colonic mucosal IL1β, IL6 and TNF-α were up-regulated, with significantly greater increases in Vdr^−/− mice (Fig. 5C). Macrophage RAW264.7 cells express AT1 receptors (Fig. 4A). To directly examine the effect of the RAS on macrophage function we treated RAW264.7 cells with Ang II. As expected, Ang II significantly increased TNF-α secretion and the AT1 inhibitor losartan blocked this increase (Fig 5D).

Fig. 5. VDR deletion increases macrophage infiltration and inflammation in vivo; Ang II induces macrophage TNF-α in vitro. A Macrophage staining.

Tumors from Vdr^+/+ (left panel) and Vdr^−/− mice (right panel) were stained with anti-CD68 antibodies. B. Macrophage quantification (*p<0.05 compared to Vdr^+/+ mice, n=3 tumors/group). C. Proinflammatory cytokine levels. Colonic mucosal TNFα is increased in vehicle-treated (control, no AOM/DSS) Vdr^−/− compared to Vdr^+/+ mice. IL1β, IL6 and TNFα are further increased in colonic mucosa from AOM/DSS-treated mice, with greater increases in Vdr^−/− mice compared to Vdr^+/+ mice (‡p<0.001 compared to vehicle-treated Vdr^+/+; *<p<0.05, ††p<0.005, compared to vehicle-treated Vdr^+/+ mice; †,**p<0.0001, compared to AOM/DSS-treated Vdr^+/+; ‡‡p<0.0001, compared to AOM/DSS-treated Vdr^+/+; n=4 control mucosa/genotype or 4 tumors/genotype). D. Ang II induces TNF-α in macrophage cells. RAW264.7 cells were pre-treated with 1 μM losartan or vehicle for 2 hrs and then treated with indicated concentrations of Ang II or vehicle and TNF-α assayed by ELISA (*p<0.05 compared to untreated cells). Cell culture results were replicated in 3 independent platings.

EGFR signals suppress VDR in AOM/DSS colonic tumors

While we demonstrated that VDR sufficiency inhibits EGFR signals in the AOM/DSS model (Fig 2), we next asked the converse: does EGFR control VDR in this model? To address this question we examined tumors from Egfr^+/+ and hypomorphic Egfr^Wa2/Wa2 mice. Nuclear VDR levels in malignant colonocytes were reduced in Egfr^wildtype mice, whereas nuclear VDR levels were maintained in malignant colonocytes from Egfr^Wa2/Wa2 mice, with positive nuclei in 17.1±3.0% vs. 30.2±11.5% respectively (Fig 6A). Nuclear VDR staining in normal colonic epithelial cells from Egfr^+/+ and Egfr^Wa2 mice treated with saline (no AOM/DSS) were comparable with 30.9±9.0% vs. 31.9±13.6% positive nuclei (Fig 6A). Western blotting confirmed VDR levels were significantly decreased in tumors from Egfr^wildtype mice, compared to Egfr^Wa2/Wa2 mice (Figs 6B). Thus, EGFR signals reduced VDR expression and VDR signals (nuclear VDR) in colonic tumors.

Fig. 6. EGFR suppresses VDR and up-regulates Snail1 in AOM/DSS-induced tumors.

A. VDR immunostaining. VDR expression in colonic mucosa and tumors from Egfr^wildtype and Egfr^Wa2/Wa2 mice. Shown are representative tumors. Note the decreased VDR staining in tumors from Egfr^wildtype mouse compared to Egfr^Wa2/Wa2 mouse, 17.1±3.0% vs. 30.2±11.5%*, respectively (*p<0.05 compared to VDR in tumors from Egfr^wildtype mouse; n=3 tumors/genotype). B. VDR Western blotting left panel:representative blot of lysates from control mucosa and colonic tumors probed for VDR and β-actin as loading control. Right panel VDR densitometry (*p<0.05 compared to VDR in normal mucosa from Egfr^wildtype mice; †p<0.05 compared to VDR in tumors from Egfr^wildtype mice, n=3 tumors/genotype). C. Snail1 immunostaining Tumors from Egfr^wildtype and Egfr^Wa2/Wa2 mice were stained for Snail1. There were 42.8±5.4% nuclei positive for Snail1 in tumors from Egfr^wildtype mice, compared to 23.5±2.1%* Snail1 positive nuclei in tumors from Egfr^Waved-2 mice (*p<0.05, n=3 tumors/genotype). D. Snail1 Western blotting left panel: representative blot of lysates from control mucosa and tumors probed for Snail1. Right panel: Snail1 densitometry (*p<0.05, compared to Snail1 in normal mucosa from Egfr^wildtype mice; †p<0.05, compared to Snail1 in tumors from Egfr^wildtype mice). E. Proposed model for EGFR-VDR cross-talk. Under physiological conditions VDR signals inhibit the RAS signals (see Fig. 3F). With tumorigenesis, EGFR is activated and suppresses VDR (see Fig. 2A,B and Fig. 6A,B). Down-regulation of VDR increases renin secretion from colon cancer cells, which in turn up-regulates Ang II in the colonic mucosa (see Fig. 3A-E). Ang II binds AT1 receptors to transactivate EGFR, thereby stimulating colon cancer cell proliferation and activating fibroblasts and macrophages in tumor stroma (Figs. 4,5).

EGFR signals in AOM/DSS colonic tumors induce Snail1, a negative regulator of VDR expression

To investigate potential EGFR-dependent mechanisms that might suppress VDR in colonic tumors, we examined the transcription factor Snail1. Other investigators have shown that EGFR can up-regulate Snail1 and that Snail1 in turn can suppress VDR (27, 46). As shown in Fig 6C-D, Snail1 was increased in tumors from Egfr^wildtype mice compared to Egfr^Wa2/Wa2 mice. EGF signals also increased Snail1 in HT29 colon cancer cells (Supplemental Fig S2). Thus, EGFR induction of Snail1 is a potential mechanism by which EGFR suppresses VDR in colonic tumorigenesis (Fig 6E).

DISCUSSION

Prior studies showed that EGFR promotes colonic tumor development, whereas vitamin D inhibits tumorigenesis in models of colon cancer (6, 8, 32, 47-49). To examine how VDR alters colonic tumorigenesis and EGFR signals, we treated Vdr^−/− and Vdr^+/+ mice with AOM/DSS. VDR signals suppressed colonic tumorigenesis, whereas VDR deletion increased tumor development, enhanced EGFR and βcatenin signals and up-regulated the colonic RAS. The effects of VDR deletion on nuclear βcatenin levels are in agreement with prior investigations by our laboratory and others (47, 50). Since βcatenin plays a critical role in colonic tumorigenesis, increased nuclear βcatenin is likely a key factor in enhanced tumorigenesis that occurs in VDR null mice. The effects of VDR deletion on renin in the colon are consistent with prior reports that vitamin D is a negative transcriptional regulator of renin (20). The potential translational relevance of these studies is emphasized by our finding that renin is up-regulated in human colon cancers. Mechanistically, Ang II transactivated EGFR and stimulated colon cancer cell proliferation by an AT1-mediated EGFR-dependent mechanism. In preliminary studies Ang II caused a 25% increase in amphiregulin (AREG) secretion in HT29 cells (p<0.05). RAS signals also activated fibroblasts and macrophages, key cellular components of tumor stroma (23). Thus, in colitis-associated colon cancer the RAS and EGFR pathways are up-regulated and their signals are negatively controlled by VDR (Figure 6E). Taken together, these findings highlight a potentially important VDR-dependent mechanism that suppresses EGFR and RAS signaling and likely contributes to chemoprevention by vitamin D.

RAS components, including renin, ACE, Ang II and AT1, have been detected in many tissues, including colon (17) and implicated in the development of breast, ovary, lung and prostate cancer (16). Antihypertensive agents that block the RAS signals may inhibit colonic or pancreatic tumorigenesis in humans (18, 51). In the current report we showed that colonic RAS components were up-regulated in AOM/DSS-treated Vdr^/− mice. These changes reflected generalized field effects that we predict promote growth of mutated colonocytes. Interestingly, renin up-regulation was detected in transforming colonocytes, whereas AT1 receptors were increased in tumor stroma. These findings uncover potentially important paracrine mechanisms in the microenvironment that drive stromal cell-cancer cell cross-talk. Presumably, release of angiotensinogen, renin and ACE into the extracellular space in colonic mucosa would increase Ang II to stimulate malignant colonocytes and stromal cells. These local RAS paracrine networks are still little understood (52). In contrast to Vdr deletion, dietary supplementation with cholecalciferol suppressed colonic mucosal renin and angiotensinogen in both control and AOM/DSS treated Vdr^+/+ mice fed a WD. This dose of cholecalciferol was previously shown to inhibit AOM/DSStumorigenesis (32). In agreement with these results, in prior studies we showed that Iα,25 dihydroxyvitamin D3 inhibited increases in inflammation-induced angiotensinogen in other tissues (53)

AOM/DSS colonic tumorigenesis is promoted by inflammation and TNF-α plays a pathogenic role (54). We showed that Ang II increased TNF-α secretion from the macrophage by an AT1-mediated mechanism. The macrophage is a major source of TNF-α in tumor stroma and contributes to inflammation (22). We also showed that VDR suppresses accumulation of tumor-associated macrophages and reduces pro-inflammatory cytokine release as both were increased in Vdr^−/− mice. In prior studies we showed that vitamin hormone inhibited TNF-α release from macrophage (10). We speculate that suppression of macrophage recruitment and activation are likely essential for the antineoplastic effects of VDR in this model.

Endothelial cells and fibroblasts also express AT1 receptors and are important in tumor progression (23, 55, 56). Increases in tumor angiogenesis (detected by Nestin-1 staining) in Vdr^−/− mice are consistent with the up-regulated RAS in these mice as RAS is a known driver of angiogenesis. Ang II also stimulates colonic fibroblast proliferation by a losartan-sensitive mechanism. Thus, VDR inhibition of the RAS likely contributes to many of VDR’s anti-proliferative, anti-angiogenic and anti-inflammatory effects. Further supporting the importance of the RAS in this model, AT1 deletion mitigates DSS colitis (57). While the RAS signals promoting proliferation could also contribute to healing DSS colitis, presumably enhanced inflammation and proliferation of transforming cells are dominant over healing DSS colitis. In addition to the current report showing VDR suppression of nuclear β-catenin, EGFR signals and colonic renin transcription, VDR signals have been shown to inhibit cell cycling and increase apoptosis in colon cancer cells (58, 59). Other potential chemopreventive mechanisms involving VDR warrant future investigation. In this regard in preliminary studies Vdr deletion increased Notch and Hedgehog signaling, two other oncogenic pathways in colon cancer.

In prior studies we demonstrated that EGFR signals play a critical role in colonic tumorigenesis (6, 48, 49). The AT1 receptor can transactivate EGFR (24). Here we established that Ang II transactivates EGFR in colon cancer cells and increases proliferation by an EGFR-dependent mechanism. Thus, by suppressing colonic renin we predict that VDR signals would inhibit EGFR activation by the RAS. We also demonstrated that EGFR signals suppressed VDR expression in tumors, confirming in vivo a novel antagonistic cross-talk between EGFR and VDR in colon cancer development. Interestingly, in the AOM rat model (49), VDR down-regulation was mitigated in tumors from animals supplemented with gefitinib (Supplemental data Fig S3). Thus, EGFR signals down-regulate VDR in a model inflammation-associated colon cancer and perhaps also in AOM-induced tumors, a model of sporadic colon cancer. Insporadic human colon cancers we found that renin and phospho-active EGFR were increased, whereas VDR expression was variable in agreement with other studies (60). In addition, we showed that EGFR signals up-regulated Snail1, a transcription factor important in tumor epithelial-to-mesenchymal transition, consistent with our prior studies (61). Other investigators showed that Snail1 was up-regulated in the AOM/DSS model (62). Since Snail1 can suppress VDR transcription (27), we speculate that Snail1 up-regulation may contribute to VDR down-regulation by EGFR in AOM/DSS tumorigenesis [Figure 6E] (27, 62). In preliminary studies we determined that EGF induced Snail1 in colon cancer cells in vitro (Supplemental Fig S2). Snail1 up-regulation by EGFR signals was not accompanied by reductions in VDR (Fig S2) in cell culture, however, suggesting that our in vitro conditions in human colon cancer cells were insufficient to mimic EGF-induced VDR down-regulation that we observed in vivo in mouse model of inflammation-associated colon cancer. This may reflect differences between human colon cancer and the mouse model. In addition to our findings of EGFR and Snail1, several other mechanisms have been proposed to inhibit VDR signaling (63).

In summary, using genetic approaches and animal models of colon cancer we have experimentally identified a novel mechanism involving RAS that may mediate the colon cancer chemopreventive effects of vitamin D. These in vivo results extend prior findings in cell culture, demonstrating an important cross-talk between VDR and EGFR in colonic tumorigenesis (12-14). Future studies to quantify the magnitude of RAS inhibition to the anti-inflammatory and chemopreventive effects of vitamin D are warranted. We speculate that the RAS may play a critical role in human IBD-associated colon cancer and that vitamin D, together with RAS inhibitors, might provide a useful chemopreventive strategy for this high-risk group.

Statement of Translational Relevance.

Colon cancer is a leading cause of cancer-related deaths. In addition to the central role played by βcatenin in colonic tumorigenesis, we previously demonstrated that EGFR signals are important in neoplastic progression. Our group and others demonstrated that vitamin D inhibits colon cancer development through anti-proliferative and anti-inflammatory activity. Because vitamin D is also a transcriptional inhibitor of renin and the renin angiotensin system (RAS) is upregulated in non-colonic cancers, we hypothesized that RAS inhibition is another mechanism of tumor suppression by vitamin D. In VDR null mice, we found that RAS was up-regulated in colitis-associated tumors and adjacent mucosa and accompanied by EGFR activation. Dietary supplementation with vitamin D3 suppressed colonic mucosal RAS. The RAS effector, angiotensin II stimulated colon cancer cell proliferation and activated fibroblasts and macrophages. Ang II also activated EGFR. Furthermore, EGFR was required for Ang II-induced mitogenesis. Thus, renin suppression likely contributes to vitamin D anti-tumor effects. Since vitamin D also exerts RAS-independent effects such as p21^Waf1 induction, our studies suggest that therapies combining vitamin D and RAS inhibitors might be an effective chemopreventive strategy for inflammation-associated colon cancer as occurs in inflammatory bowel diseases.