Urszula Dougherty

Intestinal epithelial vitamin D receptor signaling inhibits experimental colitis

The inhibitory effects of vitamin D on colitis have been previously documented. Global vitamin D receptor (VDR) deletion exaggerates colitis, but the relative anticolitic contribution of epithelial and nonepithelial VDR signaling is unknown. Here, we showed that colonic epithelial VDR expression was substantially reduced in patients with Crohns disease or ulcerative colitis. Moreover, targeted expression of human VDR (hVDR) in intestinal epithelial cells (IECs) protected mice from developing colitis. In experimental colitis models induced by 2,4,6-trinitrobenzenesulfonic acid, dextran sulfate sodium, or CD4(+)CD45RB(hi) T cell transfer, transgenic mice expressing hVDR in IECs were highly resistant to colitis, as manifested by marked reductions in clinical colitis scores, colonic histological damage, and colonic inflammation compared with WT mice. Reconstitution of Vdr-deficient IECs with the hVDR transgene completely rescued Vdr-null mice from severe colitis and death, even though the mice still maintained a hyperresponsive Vdr-deficient immune system. Mechanistically, VDR signaling attenuated PUMA induction in IECs by blocking NF-κB activation, leading to a reduction in IEC apoptosis. Together, these results demonstrate that gut epithelial VDR signaling inhibits colitis by protecting the mucosal epithelial barrier, and this anticolitic activity is independent of nonepithelial immune VDR actions.

miR‐143 and miR‐145 are downregulated in ulcerative colitis: Putative regulators of inflammation and protooncogenes

Background: miR‐143 and miR‐145 are believed to function as colon cancer tumor suppressors, as they inhibit colon cancer cell growth and are downregulated in sporadic colonic tumors. We speculated that miR‐143 and miR‐145 might also be downregulated and contribute to malignant transformation of colonic epithelium in longstanding ulcerative colitis (UC). Methods: Biopsies were obtained 20 cm proximal to the anus from individuals with quiescent UC and from normal controls. RNA and proteins were extracted and measured. miR‐143 and miR‐145 were quantified by real‐time polymerase chain reaction (PCR) and miR‐145 was also assessed by in situ hybridization. Putative targets of these miRNAs, K‐RAS, API5, MEK‐2 (miR‐143), and IRS‐1 (miR‐145) were determined by western blotting. To assess the effects of miR‐143 and miR‐145 on these predicted targets, HCT116 and HCA‐7 colorectal cancer cells were transfected with miR‐143 and miR‐145 and expression levels of these proteins were measured. Results: In UC, miR‐143 and miR‐145 were significantly downregulated 8.3‐fold (3.4–20.1) (P < 0.0001) and 4.3‐fold (2.3–7.8) (P < 0.0001), respectively, compared to normal colon. In contrast, IRS‐1, K‐RAS, API5, and MEK‐2 were upregulated in UC, consistent with their assignments as targets of these miRNAs. Furthermore, transfected miR‐143 and miR‐145 significantly downregulated these proteins in HCT116 or HCA‐7 cells. Conclusions: Compared to normal colonic mucosa, in chronic UC miR‐143 and miR‐145 were significantly downregulated and their predicted targets, IRS‐1, K‐RAS, API5, and MEK‐2 were upregulated. We postulate that loss of these tumor suppressor miRNAs predispose to chronic inflammation and neoplastic progression in IBD. (Inflamm Bowel Dis 2011;)

EGFR Signals Downregulate Tumor Suppressors miR-143 and miR-145 in Western Diet–Promoted Murine Colon Cancer: Role of G1 Regulators

Epidermal growth factor receptors (EGFR) contribute to colonic tumorigenesis in experimental models of colon cancer. We previously showed that EGFR was also required for colonic tumor promotion by Western diet. The goal of this study was to identify EGFR-regulated microRNAs that contribute to diet-promoted colonic tumorigenesis. Murine colonic tumors from Egfrwt and hypomorphic Egfrwa2 mice were screened using micro RNA (miRNA) arrays and miR-143 and miR-145 changes confirmed by Northern, real-time PCR, and in situ analysis. Rodent and human sporadic and ulcerative colitis (UC)-associated colon cancers were examined for miR-143 and miR-145. Effects of EGFR on miR-143 and miR-145 expression were assessed in murine and human colonic cells and their putative targets examined in vitro and in vivo. miR-143 and miR-145 were readily detected in normal colonocytes and comparable in Egfrwt and Egfrwa2 mice. These miRNAs were downregulated in azoxymethane and inflammation-associated colonic tumors from Egfrwt mice but upregulated in Egfrwa2 tumors. They were also reduced in human sporadic and UC colon cancers. EGFR signals suppressed miR-143 and miR-145 in human and murine colonic cells. Transfected miR-143 and miR-145 inhibited HCT116 cell growth in vitro and in vivo and downregulated G1 regulators, K-Ras, MYC, CCND2, cdk6, and E2F3, putative or established targets of these miRNAs. miRNA targets Ras and MYC were increased in colonic tumors from Egfrwt but not Egfrwa2 mice fed a Western diet. EGFR suppresses miR-143 and miR-145 in murine models of colon cancer. Furthermore, Western diet unmasks the tumor suppressor roles of these EGFR-regulated miRNAs. Mol Cancer Res; 9(7); 960–75. ©2011 AACR.

Epidermal growth factor receptor signaling is up-regulated in human colonic aberrant crypt foci.

Aberrant crypt foci (ACF) are collections of abnormal colonic crypts with heterogeneous molecular and pathologic characteristics. Large and dysplastic ACF are putative precursors of colon cancer with neoplastic risk related to increased proliferation. In this study, we examined the role of epidermal growth factor receptor (EGFR) signaling in regulating ACF proliferation. Using magnification chromoendoscopy, we collected large ACF with endoscopic features of dysplasia and separately biopsied adjacent mucosa. Transcript levels were measured by real-time PCR, proteins were assessed by Western blotting, and levels were expressed as fold changes of adjacent mucosa. K-ras and B-Raf mutations were assessed by PCR and Ras activation by the ratio Ras-GTP / (Ras-GTP + Ras-GDP). At the RNA level, 38% of ACF were hyperproliferative, with proliferating cell nuclear antigen (PCNA) mRNA >/=2-fold of adjacent mucosa. Hyperproliferative ACF had significantly increased mRNA levels of EGFR (6.0 +/- 1.7-fold), transforming growth factor-alpha (14.4 +/- 5.0-fold), heparin-binding EGF-like growth factor (4.5 +/- 1.4-fold), cyclin D1 (4.6 +/- 0.7-fold), and cyclooxygenase-2 (COX-2; 9.3 +/- 4.2-fold; P < 0.05). At the protein level, 46% of ACF were hyperproliferative (PCNA, 3.2 +/- 1.2-fold). In hyperproliferative ACF, 44% possessed significant increases in four EGFR signaling components: EGFR (9.5 +/- 1.3-fold), phosphoactive ErbB2 (2.6 +/- 0.4-fold), phosphoactive extracellular signal-regulated kinase (3.7 +/- 1.1-fold), and cyclin D1 (3.4 +/- 0.8-fold; P < 0.05). Ras was activated in 46% of ACF (3.2 +/- 0.4-fold; P < 0.05), but K-ras mutations were present in only 7% of ACF. In contrast to COX-2 mRNA, the protein was not increased in hyperproliferative ACF. In summary, we have shown that ACF with up-regulated PCNA possess increased EGFR signaling components that likely contribute to the enhanced proliferative state of dysplastic-appearing ACF.

Epidermal Growth Factor Receptor Signaling Is Required for Microadenoma Formation in the Mouse Azoxymethane Model of Colonic Carcinogenesis

Colonic carcinogenesis involves the progressive dysregulation of homeostatic mechanisms that control growth. The epidermal growth factor (EGF) receptor (EGFR) regulates colonocyte growth and differentiation and is overexpressed in many human colon cancers. A requirement for EGFR in colonic premalignancy, however, has not been shown. In the current study, we used a specific EGFR antagonist, gefitinib, to investigate this role of the receptor in azoxymethane colonic premalignancy. The azoxymethane model shares many clinical, histologic, and molecular features of human colon cancer. Mice received azoxymethane i.p. (5 mg/kg/wk) or saline for 6 weeks. Animals were also gavaged with gefitinib (10 mg/kg body weight) or vehicle (DMSO) thrice weekly for 18 weeks, a dose schedule that inhibited normal receptor activation by exogenous EGF. Compared with control colonocytes [bromodeoxyuridine (BrdUrd), 2.2+/-1.2%], azoxymethane significantly increased proliferation (BrdUrd, 12.6+/-2.8%), whereas gefitinib inhibited this hyperproliferation (BrdUrd, 6.2+/-4.0%; <0.005). Azoxymethane significantly induced pro-transforming growth factor-alpha (6.4+/-1.3-fold) and increased phospho-(active) EGFR (5.9+/-1.1-fold), phospho-(active) ErbB2 (2.3+/-0.2-fold), and phospho-(active) extracellular signal-regulated kinase (3.3+/-0.4-fold) in premalignant colonocytes. Gefitinib inhibited activations of these kinases by >75% (P<0.05). Gefitinib also significantly reduced the number of large aberrant crypt foci and decreased the incidence of colonic microadenomas from 75% to 33% (P<0.05). Gefitinib concomitantly decreased cell cycle-regulating cyclin D1 and prostanoid biosynthetic enzyme cyclooxygenase-2 in microadenomas, suggesting that these regulators are key targets of EGFR in colonic carcinogenesis. These results show for the first time that EGFR signaling is required for early stages of colonic carcinogenesis. Our findings suggest, moreover, that inhibitors of EGFR might be useful in chemopreventive strategies in individuals at increased risk for colonic malignancies.

Epidermal Growth Factor Receptor Controls Flat Dysplastic Aberrant Crypt Foci Development and Colon Cancer Progression in the Rat Azoxymethane Model

Purpose: Colonic carcinogenesis deranges growth-regulating epidermal growth factor receptors (EGFR). We previously showed that EGFR signals were up-regulated in human aberrant crypt foci (ACF), putative colon cancer precursors. The azoxymethane model of colon cancer recapitulates many aspects of human colonic tumors. Recent studies indicate that flat dysplastic ACF with increased β-catenin are tumor precursors in this model. We asked, therefore, if EGFR signals are required for flat dysplastic ACF development and cancer progression. Experimental Design: Rats received azoxymethane or saline, and standard chow or chow supplemented with gefitinib, an EGFR inhibitor, for 44 weeks. EGFR signals were quantified in normal colon, flat ACF, and tumors by computerized analysis of immunostains and Western blots. K-ras mutations were assessed by PCR and mRNA for egfr ligands by quantitative real-time PCR. Results: EGFR inhibition with gefitinib decreased the incidence of flat dysplastic ACF from 66% to 36% and tumors from 71% to 22% (P < 0.05). This inhibitor also reduced the overexpressions of cyclin D1 and Cox-2 in flat ACF. Furthermore, in flat ACF, EGFR blockade decreased the up-regulation of c-Jun, FosB, phosphorylated active signal transducers and activators of transcription 3, and CCAAT/enhancer binding protein-β, potential regulators of cyclin D1 and Cox-2. In colonic tumors, EGFR blockade significantly decreased angiogenesis, proliferation, and progression while also increasing apoptosis (P < 0.05). Gefitinib also inhibited the activations of extracellular signal–regulated kinase, Src, and AKT pathways in tumors. Conclusions: We have shown for the first time that EGFR promotes the development of flat dysplastic ACF and the progression of malignant colonic tumors. Furthermore, we have mechanistically identified several transcription factors and their targets as EGFR effectors in colonic carcinogenesis.

Sprouty-2 Controls c-Met Expression and Metastatic Potential of Colon Cancer Cells: Sprouty/c-Met Upregulation in Human Colonic Adenocarcinomas

Sprouty negatively regulates receptor tyrosine kinase signals by inhibiting Ras/extracellular signal-regulated kinase (ERK) pathways. Sprouty is downregulated in breast, prostate and liver cancers and appears to function as a tumor suppressor. The role of sprouty in colonic neoplasia, however, has not been investigated. Sprouty-2 protein and mRNA transcripts were significantly upregulated in human colonic adenocarcinomas. Strikingly, the c-Met receptor was also upregulated in tumors with increased sprouty-2. To delineate a potential causal relationship between sprouty-2 and c-Met, K-ras mutant HCT-116 colon cancer cells were transduced with purified TAT-sprouty-2 protein or stably transfected with full-length human sprouty-2 gene. Sprouty-2 upregulation significantly increased cell proliferation by accelerating cell cycle transition. Sprouty-2 transfectants showed strong upregulation of c-Met protein and mRNA transcripts and hepatocyte growth factor-stimulated ERK and Akt phosphorylation and enhanced cell migration and invasion. In contrast, knockdown of c-Met by small interfering RNA (siRNA) significantly decreased cell proliferation, migration and invasion in sprouty-2 transfectants. Further, knockdown of sprouty-2 by siRNA in parental HT-29 and LS-174T colon cancer cells also decreased cell invasion. Sprouty-2 transfectants formed significantly larger tumor xenografts and showed increased proliferation and angiogenesis and suppressed apoptosis. Sprouty-2 tumors metastasized to the liver from cecal orthotopic implants, suggesting that sprouty-2 might also enhance metastatic signals. Thus, in colon cancer sprouty functions as an oncogene and its effects are mediated in part by c-Met upregulation.

Ursodeoxycholic Acid Suppresses Cox-2 Expression in Colon Cancer: Roles of Ras, p38, and CCAAT/Enhancer-Binding Protein

In the azoxymethane (AOM) model of experimental rodent colon cancer, cholic acid and its colonic metabolite deoxycholic acid (DCA) strongly promote tumorigenesis. In contrast, we showed that ursodeoxycholic acid (UDCA), a low abundance bile acid, inhibited AOM tumorigenesis. Dietary UDCA also blocked the development of tumors with activated Ras and suppressed cyclooxygenase-2 (Cox-2) upregulation in AOM tumors. In this study, we compared the effect of dietary supplementation with tumor-promoting cholic acid to chemopreventive UDCA on Cox-2 expression in AOM tumors. Cholic acid enhanced Cox-2 upregulation in AOM tumors, whereas UDCA inhibited this increase and concomitantly decreased CCAAT/enhancer binding protein β (C/EBPβ), a transcriptional regulator of Cox-2. In HCA-7 colon cancer cells, DCA activated Ras and increased C/EBPβ and Cox-2 by a mechanism requiring the mitogen-activated protein kinase p38. UDCA inhibited DCA-induced p38 activation and decreased C/EBPβ and Cox-2 upregulation. Using transient transfections, UDCA inhibited Cox-2 promoter and C/EBP reporter activation by DCA. Transfection with dominant-negative 17N-Ras abolished DCA-induced p38 activation and C/EBPβ and Cox-2 upregulation. Taken together, these studies have identified a transcriptional pathway regulating Cox-2 expression involving Ras, p38, and C/EBPβ that is inhibited by UDCA. These signal transducers are novel targets of UDCAs chemopreventive actions.

American ginseng suppresses Western diet-promoted tumorigenesis in model of inflammation-associated colon cancer: role of EGFR

BackgroundWestern diets increase colon cancer risk. Epidemiological evidence and experimental studies suggest that ginseng can inhibit colon cancer development. In this study we asked if ginseng could inhibit Western diet (20% fat) promoted colonic tumorigenesis and if compound K, a microbial metabolite of ginseng could suppress colon cancer xenograft growth.MethodsMice were initiated with azoxymethane (AOM) and, two weeks later fed a Western diet (WD, 20% fat) alone, or WD supplemented with 250-ppm ginseng. After 1 wk, mice received 2.5% dextran sulfate sodium (DSS) for 5 days and were sacrificed 12 wks after AOM. Tumors were harvested and cell proliferation measured by Ki67 staining and apoptosis by TUNEL assay. Levels of EGF-related signaling molecules and apoptosis regulators were determined by Western blotting. Anti-tumor effects of intraperitoneal compound K were examined using a tumor xenograft model and compound K absorption measured following oral ginseng gavage by UPLC-mass spectrometry. Effects of dietary ginseng on microbial diversity were measured by analysis of bacterial 16S rRNA.ResultsGinseng significantly inhibited colonic inflammation and tumorigenesis and concomitantly reduced proliferation and increased apoptosis. The EGFR cascade was up-regulated in colonic tumors and ginseng significantly reduced EGFR and ErbB2 activation and Cox-2 expression. Dietary ginseng altered colonic microbial diversity, and bacterial suppression with metronidazole reduced serum compound K following ginseng gavage. Furthermore, compound K significantly inhibited tumor xenograft growth.ConclusionsGinseng inhibited colonic inflammation and tumorigenesis promoted by Western diet. We speculate that the ginseng metabolite compound K contributes to the chemopreventive effects of this agent in colonic tumorigenesis.

The Thr300Ala variant in ATG16L1 is associated with improved survival in human colorectal cancer and enhanced production of type I interferon

Objective ATG16L1 is an autophagy gene known to control host immune responses to viruses and bacteria. Recently, a non-synonymous single-nucleotide polymorphism in ATG16L1 (Thr300Ala), previously identified as a risk factor in Crohns disease (CD), was associated with more favourable clinical outcomes in thyroid cancer. Mechanisms underlying this observation have not been proposed, nor is it clear whether an association between Thr300Ala and clinical outcomes will be observed in other cancers. We hypothesised that Thr300Ala influences clinical outcome in human colorectal cancer (CRC) and controls innate antiviral pathways in colon cancer cells. Design We genotyped 460 patients with CRC and assessed for an association between ATG16L1 Thr300Ala and overall survival and clinical stage. Human CRC cell lines were targeted by homologous recombination to examine the functional consequence of loss of ATG16L1, or introduction of the Thr300Ala variant. Results We found an association between longer overall survival, reduced metastasis and the ATG16L1 Ala/Ala genotype. Tumour sections from ATG16L1 Ala/Ala patients expressed elevated type I interferons (IFN-I)-inducible, MxA, suggesting that differences in cytokine production may influence disease progression. When introduced into human CRC cells by homologous recombination, the Thr300Ala variant did not affect bulk autophagy, but increased basal production of type I IFN. Introduction of Thr300Ala resulted in increased sensitivity to the dsRNA mimic poly(I:C) through a mitochondrial antiviral signalling (MAVS)-dependent pathway. Conclusions The CD-risk allele, Thr300Ala, in ATG16L1 is associated with improved overall survival in human CRC, generating a rationale to genotype ATG16L1 Thr300Ala in patients with CRC. We found that Thr300A alters production of MAVS-dependent type I IFN in CRC cells, providing a mechanism that may influence clinical outcomes.