Tumor suppressors miR-143 and miR-145 and predicted target proteins API5, ERK5, K-RAS, and IRS-1 are differentially expressed in proximal and distal colon.

The colon differs regionally in local luminal environment, excretory function, and gene expression. Polycistronic microRNA (miR)-143 and miR-145 are downregulated early in colon cancer. We asked if these microRNAs (miRNAs) might be differentially expressed in the proximal vs. the distal colon, contributing to regional differences in protein expression. Primary transcripts and mature miR-143 and miR-145 were quantified by real-time PCR, putative targets were measured by Western blotting, and DNA methylation was assessed by sequencing bisulfite-treated DNA in proximal and distal normal colonic mucosa as well as colon cancers. Putative targets of these miRNAs were assessed following transfection with miR-143 or miR-145. Mean expression of mature miR-143 and miR-145 was 2.0-fold (P < 0.001) and 1.8-fold (P = 0.03) higher, respectively, in proximal than distal colon. DNA methylation or primary transcript expression of these miRNAs did not differ by location. In agreement with increased expression of miR-143 and miR-145 in proximal colon, predicted targets of these miRNAs, apoptosis inhibitor 5 (API5), ERK5, K-RAS, and insulin receptor substrate 1 (IRS-1), which are cell cycle and survival regulators, were expressed at a lower level in proximal than distal colon. Transfection of HCA-7 colon cancer cells with miR-145 downregulated IRS-1, and transfection of HT-29 colon cancer cells with miR-143 decreased K-RAS and ERK5 expression. In conclusion, miR-143 and miR-145 and the predicted target proteins API5, ERK5, K-RAS, and IRS-1 display regional differences in expression in the colon. We speculate that differences in these tumor suppressors might contribute to regional differences in normal colonic gene expression and modulate site-specific differences in malignant predisposition.

Sprouty4 is epigenetically upregulated in human colorectal cancer.

Sprouty4 (SPRY4) has been frequently reported as a tumor suppressor and is therefore downregulated in various cancers. For the first time, we report that SPRY4 is epigenetically upregulated in colorectal cancer (CRC). In this study, we explored DNA methylation and hydroxymethylation levels of SPRY4 in CRC cells and patient samples and correlated these findings with mRNA and protein expression levels. Three loci within the promoter region of SPRY4 were evaluated for 5mC levels in CRC using the combined bisulfite restriction analysis. In addition, hydroxymethylation levels within SPRY4 were measured in CRC patients. Lastly, DNA methylation and mRNA expression data were extracted from CRC patients in multiple high-throughput data repositories like Gene Expression Omnibus and The Cancer Genome Atlas. Combined in vitro and in silico analysis of promoter methylation levels of SPRY4 clearly demonstrates that the distal promoter region undergoes hypomethylation in CRC patients and is associated with increased expression. Moreover, a decrease in gene body hydroxymethylation and an increase in gene body methylation within the coding region of SPRY4 were found in CRC patients and correlated with increased expression. SPRY4 is epigenetically upregulated in CRC by promoter hypomethylation and hypermethylation within the gene body that warrants future investigation of atypical roles of this established tumor suppressor.

SPRY4 gene expression is increased in colorectal cancerThe SPRY4 promoter loses DNA methylation, and the gene body gains DNA methylation in colorectal cancerThe gene body of SPRY4 loses DNA hydroxymethylation.

Both stromal cell and colonocyte epidermal growth factor receptors control HCT116 colon cancer cell growth in tumor xenografts.

Colon cancer growth requires growth-promoting interactions between malignant colonocytes and stromal cells. Epidermal growth factor receptors (EGFR) are expressed on colonocytes and many stromal cells. Furthermore, EGFR is required for efficient tumorigenesis in experimental colon cancer models. To dissect the cell-specific role of EGFR, we manipulated receptor function on stromal cells and cancer cells. To assess the role of stromal EGFR, HCT116 human colon cancer cells were implanted into immunodeficient mice expressing dominant negative (DN) Egfr(Velvet/+) or Egfr(+/+). To assess the role of cancer cell EGFR, HCT116 transfectants expressing inducible DN-Egfr were implanted into immunodeficient mice. To dissect EGFR signals in vitro, we examined colon cancer cells in monoculture or in cocultures with fibroblasts for EGFR transactivation and prostaglandin synthase 2 (PTGS2) induction. EGFR signals were determined by blotting, immunostaining and real-time PCR. Tumor xenografts in Egfr(Velvet/+) mice were significantly smaller than tumors in Egfr(+/+) mice, with decreased proliferation (Ki67) and increased apoptosis (cleaved caspase-3) in cancer cells and decreased stromal blood vessels. Mouse stromal transforming growth factor alpha (TGFA), amphiregulin (AREG), PTGS2 and Il1b and interleukin-1 receptor 1 (Il1r1) transcripts and cancer cell beta catenin (CTNNB1) and cyclin D1 (CCND1) were significantly lower in tumors obtained from Egfr(Velvet/+) mice. DN-EGFR HCT116 transfectants also formed significantly smaller tumors with reduced mouse Areg, Ptgs2, Il1b and Il1r1 transcripts. Coculture increased Caco-2 phospho-active ERBB (pERBB2), whereas DN-EGFR in Caco-2 cells suppressed fibroblast PTGS2 and prostaglandin E2 (PGE2). In monoculture, interleukin 1 beta (IL1B) transactivated EGFR in HCT116 cells. Stromal cell and colonocyte EGFRs are required for robust EGFR signals and efficient tumor growth, which involve EGFR-interleukin-1 crosstalk.

Inactivation of the vitamin D receptor in APC(min/+) mice reveals a critical role for the vitamin D receptor in intestinal tumor growth.

Emerging evidence supports an inhibitory role for vitamin D in colorectal carcinogenesis; however, the mechanism remains unclear. The adenomatous polyposis coli (APC)/ß-catenin pathway plays a critical role in colorectal carcinogenesis. The purpose of our study is to explore the interactions of vitamin D and APC/ß-catenin pathways in intestinal tumor development. APC(min/+) mice with genetic inactivation of the vitamin D receptor (VDR) were generated through breeding. Intestinal tumorigenesis was compared between APC(min/+) and APC(min/+) VDR(-/-) mice at different ages. No differences were seen in the number of small intestinal and colonic tumors between APC(min/+) and APC(min/+) VDR(-/-) mice aged 3, 4, 6 and 7 months. The size of the tumors, however, was significantly increased in APC(min/+) VDR(-/-) mice in all age groups. Immunostaining showed significant increases in ß-catenin, cyclin D1, phosphorylated Stat-3 and MSH-2 levels and decreases in Stat-1 in APC(min/+) VDR(-/-) tumors compared to APC(min/+) tumors. These observations suggest that VDR signaling inhibits tumor growth rather than tumor initiation in the intestine. Thus, the increased tumor burden in APC(min/+) VDR(-/-) mice is likely due to the loss of the growth-inhibiting effect of VDR. This study provides strong evidence for the in vivo relevance of the interaction demonstrated in vitro between the vitamin D and ß-catenin signaling pathways in intestinal tumorigenesis.

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

BACKGROUND: Western 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. METHODS: Mice 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. RESULTS: Ginseng 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. CONCLUSIONS: Ginseng 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.

Epigenetic DNA Modifications Upregulate SPRY2 in Human Colorectal Cancers.

Conventional wisdom is that Sprouty2 (SPRY2), a suppressor of Receptor Tyrosine Kinase (RTK) signaling, functions as a tumor suppressor and is downregulated in many solid tumors. We reported, for the first time, that increased expression of SPRY2 augments cancer phenotype and Epithelial-Mesenchymal-Transition (EMT) in colorectal cancer (CRC). In this report, we assessed epigenetic DNA modifications that regulate SPRY2 expression in CRC. A total of 4 loci within SPRY2 were evaluated for 5mC using Combined Bisulfite Restriction Analysis (COBRA). Previously sequenced 5hmC nano-hmC seal data within SPRY2 promoter and gene body were evaluated in CRC. Combined bioinformatics analyses of SPRY2 CRC transcripts by RNA-seq/microarray and 450K methyl-array data archived in The Cancer Genome Atlas (TCGA) and GEO database were performed. SPRY2 protein in CRC tumors and cells was measured by Western blotting. Increased SPRY2 mRNA was observed across several CRC datasets and increased protein expression was observed among CRC patient samples. For the first time, SPRY2 hypomethylation was identified in adenocarcinomas in the promoter and gene body. We also revealed, for the first time, increases of 5hmC deposition in the promoter region of SPRY2 in CRC. SPRY2 promoter hypomethylation and increased 5hmC may play an influential role in upregulating SPRY2 in CRC.

Upregulation of polycistronic microRNA-143 and microRNA-145 in colonocytes suppresses colitis and inflammation-associated colon cancer.

Because ADAM17 promotes colonic tumorigenesis, we investigated potential miRNAs regulating ADAM17; and examined effects of diet and tumorigenesis on these miRNAs. We also examined pre-miRNA processing and tumour suppressor roles of several of these miRNAs in experimental colon cancer. Using TargetScan, miR-145, miR-148a, and miR-152 were predicted to regulate ADAM17. miR-143 was also investigated as miR-143 and miR-145 are co-transcribed and associated with decreased tumour growth. HCT116 colon cancer cells (CCC) were co-transfected with predicted ADAM17-regulating miRNAs and luciferase reporters controlled by ADAM17-3'UTR. Separately, pre-miR-143 processing by colonic cells was measured. miRNAs were quantified by RT-PCR. Tumours were induced with AOM/DSS in WT and transgenic mice (Tg) expressing pre-miR-143/miR-145 under villin promoter. HCT116 transfection with miR-145, -148a or -152, but not scrambled miRNA inhibited ADAM17 expression and luciferase activity. The latter was suppressed by mutations in ADAM17-3'UTR. Lysates from colonocytes, but not CCC, processed pre-miR-143 and mixing experiments suggested CCC lacked a competency factor. Colonic miR-143, miR-145, miR-148a, and miR-152 were downregulated in tumours and more moderately by feeding mice a Western diet. Tg mice were resistant to DSS colitis and had significantly lower cancer incidence and tumour multiplicity. Tg expression blocked up-regulation of putative targets of miR-143 and miR-145, including ADAM17, K-Ras, XPO5, and SET. miR-145, miR-148a, and miR-152 directly suppress colonocyte ADAM17 and are down-regulated in colon cancer. This is the first direct demonstration of tumour suppressor roles for miR-143 and miR-145 in an in vivo model of colonic tumorigenesis.

Correction: Stuckel, et al.; Enhanced CXCR4 Expression Associates with Increased Gene Body 5-Hydroxymethylcytosine Modification but Not Decreased Promoter Methylation in Colorectal Cancer. Cancers 2020, 12, 539.

The authors would like to make a correction to their published paper [...].

Enhanced CXCR4 Expression Associates with Increased Gene Body 5-Hydroxymethylcytosine Modification but not Decreased Promoter Methylation in Colorectal Cancer.

In colorectal cancer (CRC), upregulation of the C-X-C motif chemokine receptor 4 (CXCR4) is correlated with metastasis and poor prognosis, highlighting the need to further elucidate CXCR4's regulation in CRC. For the first time, DNA methylation and 5-hydroxymethylcytosine aberrations were investigated to better understand the epigenetic regulation of CXCR4 in CRC. CXCR4 expression levels were measured using qPCR and immunoblotting in normal colon tissues, primary colon cancer tissues and CRC cell lines. Publicly available RNA-seq and methylation data from The Cancer Genome Atlas (TCGA) were extracted from tumors from CRC patients. The DNA methylation status spanning CXCR4 gene was evaluated using combined bisulfite restriction analysis (COBRA). The methylation status in the CXCR4 gene body was analyzed using previously performed nano-hmC-seal data from colon cancers and adjacent normal colonic mucosa. CXCR4 expression levels were significantly increased in tumor stromal cells and in tumor colonocytes, compared to matched cell types from adjacent normal-appearing mucosa. CXCR4 promoter methylation was detected in a minority of colorectal tumors in the TCGA. The CpG island of the CXCR4 promoter showed increased methylation in three of four CRC cell lines. CXCR4 protein expression differences were also notable between microsatellite stable (MSS) and microsatellite instable (MSI) tumor cell lines. While differential methylation was not detected in CXCR4, enrichment of 5-hydroxymethylcytosine (5hmC) in CXCR4 gene bodies in CRC was observed compared to adjacent mucosa.

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 beta-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-beta, 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.

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.

Losartan and Vitamin D Inhibit Colonic Tumor Development in a Conditional Apc-Deleted Mouse Model of Sporadic Colon Cancer.

Colorectal cancer is a leading cause of cancer deaths. The renin-angiotensin system (RAS) is upregulated in colorectal cancer, and epidemiologic studies suggest RAS inhibitors reduce cancer risk. Because vitamin D (VD) receptor negatively regulates renin, we examined anticancer efficacy of VD and losartan (L), an angiotensin receptor blocker. Control Apc+/LoxP mice and tumor-forming Apc+/LoxP Cdx2P-Cre mice were randomized to unsupplemented Western diet (UN), or diets supplemented with VD, L, or VD+L, the latter to assess additive or synergistic effects. At 6 months, mice were killed. Plasma Ca2+, 25(OH)D3, 1α, 25(OH)2D3, renin, and angiotensin II (Ang II) were quantified. Colonic transcripts were assessed by qPCR and proteins by immunostaining and blotting. Cancer incidence and tumor burden were significantly lower in Cre+ VD and Cre+ L, but not in the Cre+ VD+L group. In Apc+/LoxP mice, VD increased plasma 1,25(OH)2D3 and colonic VDR. In Apc+/LoxP-Cdx2P-Cre mice, plasma renin and Ang II, and colonic tumor AT1, AT2, and Cyp27B1 were increased and VDR downregulated. L increased, whereas VD decreased plasma renin and Ang II in Cre+ mice. VD or L inhibited tumor development, while exerting differential effects on plasma VD metabolites and RAS components. We speculate that AT1 is critical for tumor development, whereas RAS suppression plays a key role in VD chemoprevention. When combined with L, VD no longer increases active VD and colonic VDR in Cre- mice nor suppresses renin and Ang II in Cre+ mice, likely contributing to lack of chemopreventive efficacy of the combination.

Lithocholic acid down-regulation of NF-kappaB activity through vitamin D receptor in colonic cancer cells.

Lithocholic acid (LCA), a secondary bile acid, is a vitamin D receptor (VDR) ligand. 1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the hormonal form of vitamin D, is involved in the anti-inflammatory action through VDR. Therefore, we hypothesize that LCA acts like 1,25(OH)(2)D(3) to drive anti-inflammatory signals. In present study, we used human colonic cancer cells to assess the role of LCA in regulation of the pro-inflammatory NF-kappaB pathway. We found that LCA treatment increased VDR levels, mimicking the effect of 1,25(OH)(2)D(3). LCA pretreatment inhibited the IL-1beta-induced IkappaBalpha degradation and decreased the NF-kappaB p65 phosphorylation. We also measured the production of IL-8, a well-known NF-kappaB target gene, as a read-out of the biological effect of LCA expression on NF-kappaB pathway. LCA significantly decreased IL-8 secretion induced by IL-1beta. These LCA-induced effects were very similar to those of 1,25(OH)(2)D(3.) Thus, LCA recapitulated the effects of 1,25(OH)(2)D(3) on IL-1beta stimulated cells. Mouse embryonic fibroblast (MEF) cells lacking VDR have intrinsically high NF-kappaB activity. LCA pretreatment was not able to prevent TNFalpha-induced IkappaBalpha degradation in MEF VDR (-/-), whereas LCA stabilized IkappaBalpha in MEF VDR (+/-) cells. Collectively, our data indicated that LCA activated the VDR to block inflammatory signals in colon cells.

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.

Protein Kinase-zeta inhibits collagen I-dependent and anchorage-independent growth and enhances apoptosis of human Caco-2 cells.

Colonic carcinogenesis is accompanied by abnormalities in multiple signal transduction components, including alterations in protein kinase C (PKC). The expression level of PKC-zeta, an atypical PKC isoform, increases from the crypt base to the luminal surface and parallels crypt cell differentiation in normal colon. In prior studies in the azoxymethane model of colon cancer, we showed that PKC-zeta was down-regulated in rat colonic tumors. In this study, we showed that PKC-zeta is expressed predominantly in colonic epithelial and not stromal cells, and loss of PKC-zeta occurs as early as the adenoma stage in human colonic carcinogenesis. To assess the regulation of growth and differentiation by PKC-zeta, we altered this isoform in human Caco-2 colon cancer cells using stable constitutive or inducible expression vectors, specific peptide inhibitors or small interfering RNA. In ecdysone-regulated transfectants grown on collagen I, ponasterone A significantly induced PKC-zeta expression to 135% of empty vector cells, but did not alter nontargeted PKC isoforms. This up-regulation was accompanied by a 2-fold increase in basal and 4-fold increase in insulin-stimulated PKC-zeta biochemical activity. Furthermore, PKC-zeta up-regulation caused >50% inhibition of cell proliferation on collagen I (P < 0.05). Increased PKC-zeta also significantly enhanced Caco-2 cell differentiation, nearly doubling alkaline phosphatase activity, while inducing a 3-fold increase in the rate of apoptosis (P < 0.05). In contrast, knockdown of this isoform by small interfering RNA or kinase inhibition by myristoylated pseudosubstrate significantly and dose-dependently increased Caco-2 cell growth on collagen I. In transformation assays, constitutively up-regulated wild-type PKC-zeta significantly inhibited Caco-2 cell growth in soft agar, whereas a kinase-dead mutant caused a 3-fold increase in soft agar growth (P < 0.05). Taken together, these studies indicate that PKC-zeta inhibits colon cancer cell growth and enhances differentiation and apoptosis, while inhibiting the transformed phenotype of these cells. The observed down-regulation of this growth-suppressing PKC isoform in colonic carcinogenesis would be predicted to contribute to tumorigenesis.

miR-193a-3p is a Key Tumor Suppressor in Ulcerative Colitis-Associated Colon Cancer and Promotes Carcinogenesis through Upregulation of IL17RD.

Purpose: Patients with ulcerative colitis are at increased risk for colorectal cancer, although mechanisms underlying neoplastic transformation are poorly understood. We sought to evaluate the role of microRNAs in neoplasia development in this high-risk population.Experimental Design: Tissue from 12 controls, 9 ulcerative colitis patients without neoplasia, and 11 ulcerative colitis patients with neoplasia was analyzed. miRNA array analysis was performed and select miRNAs assayed by real-time PCR on the discovery cohort and a validation cohort. DNA methylation of miR-193a was assessed. Following transfection of miR-193a-3p, proliferation, IL17RD expression, and luciferase activity of the 3'UTR of IL17RD were measured. Tumor growth in xenografts as well as EGFR signaling were assessed in HCT116 cells expressing IL17RD with either a mutant 3' untranslated region (UTR) or wild-type (WT) 3'UTR.Results: miR-31, miR-34a, miR-106b, and miR-193a-3p were significantly dysregulated in ulcerative colitis-neoplasia and adjacent tissue. Significant down-regulation of miR-193a-3p was also seen in an independent cohort of ulcerative colitis cancers. Changes in methylation of miR-193a or expression of pri-miR-193a were not observed in ulcerative colitis cancer. Transfection of miR-193a-3p resulted in decreased proliferation, and identified IL17RD as a direct target of miR-193a-3p. IL17RD expression was increased in ulcerative colitis cancers, and miR-193a-3p treatment decreased growth and EGFR signaling of HCT116 cells in xenografts expressing both IL17RD with WT 3'UTR compared with cells expressing IL17RD with mutant 3'UTR.Conclusions: miR-193a-3p is downregulated in ulcerative colitis neoplasia, and its loss promotes carcinogenesis through upregulation of IL17RD. These findings provide novel insight into inflammation-driven colorectal cancer and could suggest new therapeutic targets in this high-risk population. Clin Cancer Res; 23(17); 5281-91. ©2017 AACR.

ADAM17 is a Tumor Promoter and Therapeutic Target in Western Diet-associated Colon Cancer.

PURPOSE: Epidermal growth factor receptors (EGFR) are required for tumor promotion by Western diet. The metalloprotease, ADAM17 activates EGFR by releasing pro-EGFR ligands. ADAM17 is regulated by G-protein-coupled receptors, including CXCR4. Here we investigated CXCR4-ADAM17 crosstalk and examined the role of ADAM17 in tumorigenesis. EXPERIMENTAL DESIGN: We used CXCR4 inhibitor, AMD3100 and ADAM17 inhibitor, BMS566394 to assess CXCR4-ADAM17 crosstalk in colon cancer cells. We compared the expression of CXCR4 ligand, CXCL2, and ADAM17 in mice fed Western diet versus standard diet. Separately, mice were treated with marimastat, a broad-spectrum ADAM17 inhibitor, or AMD3100 to assess EGFR activation by ADAM17 and CXCR4. Using Apc-mutant Min mice, we investigated the effects of ADAM17/10 inhibitor INCB3619 on tumorigenesis. To assess the effects of colonocyte ADAM17, mice with ADAM17 conditional deletion were treated with azoxymethane (AOM). ADAM17 expression was also compared in colonocytes from primary human colon cancers and adjacent mucosa. RESULTS: CXCL12 treatment activated colon cancer cell EGFR signals, and CXCR4 or ADAM17 blockade reduced this activation. In vivo, Western diet increased CXCL12 in stromal cells and TGFα in colonocytes. Marimastat or AMD3100 caused >50% reduction in EGFR signals (P < 0.05). In Min mice, INCB3619 reduced EGFR signals in adenomas and inhibited intestinal tumor multiplicity (P < 0.05). In the AOM model, colonocyte ADAM17 deletion reduced EGFR signals and colonic tumor development (P < 0.05). Finally, ADAM17 was upregulated >2.5-fold in human malignant colonocytes. CONCLUSIONS: ADAM17 is a Western diet-inducible enzyme activated by CXCL12-CXCR4 signaling, suggesting the pathway: Western diet→CXCL12→CXCR4→ADAM17→TGFα→EGFR. ADAM17 might serve as a druggable target in chemoprevention strategies. Clin Cancer Res; 23(2); 549-61. ©2016 AACR.

The renin-angiotensin system mediates EGF receptor-vitamin d receptor cross-talk in colitis-associated colon cancer.

PURPOSE: We previously showed that EGF receptor (EGFR) promotes tumorigenesis in the azoxymethane/dextran sulfate sodium (AOM/DSS) model, whereas vitamin D suppresses tumorigenesis. EGFR-vitamin D receptor (VDR) interactions, however, are incompletely understood. Vitamin D 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 Western blotting, 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(wild-type) mice. Angiotensin II (Ang II)-induced EGFR activation was studied in cell culture. RESULTS: Vdr deletion significantly increased tumorigenesis, activated EGFR and ß-catenin 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 with tumors from Egfr(Waved2) mice, tumors from Egfr(wild-type) mice showed upregulated Snail1, a suppressor of VDR, and downregulated VDR. CONCLUSIONS: VDR suppresses the colonic RAS cascade, limits EGFR signals, and inhibits colitis-associated tumorigenesis, whereas EGFR increases Snail1 and downregulates VDR in colonic tumors. Taken together, these results uncover a RAS-dependent mechanism mediating EGFR and VDR cross-talk in colon cancer.

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.

Upregulation of glycogen synthase kinase 3ß in human colorectal adenocarcinomas correlates with accumulation of CTNNB1.

INTRODUCTION: Mutations of the adenomatous polyposis coli (APC) tumor suppressor gene or the CTNNB1 protooncogene have been implicated in the initiation of most human colorectal epithelial neoplasms. Glycogen synthase kinase 3ß (GSK3B) serves a critical role in regulating their functions by phosphorylating both APC and CTNNB1 to facilitate CTNNB1 degradation. The current studies were performed to investigate whether GSK3B itself is regulated during the process of colorectal tumorigenesis. PATIENTS AND METHODS: We examined the expression of GSK3B and CTNNB1 in tissue samples from 24 human colorectal adenocarcinomas by Western immunoblotting analysis, kinase activity assays and immunohistochemistry. Normal colonic mucosa from the same colectomy specimens were used as a reference for comparison. RESULTS: We demonstrated that GSK3B expression levels and kinase activities were markedly and significantly increased in colorectal adenocarcinomas in all 24 cases compared with paired adjacent normal-appearing colonic mucosa. These increases correlated with significantly increased expression of CTNNB1 in the same tumors. Similar results were obtained in several cultured human colon cancer cell lines, demonstrating GSK3B levels correlated with CTNNB1 expression. CONCLUSION: Though APC and CTNNB1 regulation by GSK3B are frequently disrupted by mutations in colon cancers, our observations suggest that increased functional GSK3B might drive other growth-promoting signals in colorectal tumorigenesis.