Tumor Suppressor In Colon Research Articles

ELEVATED ADENOMATOUS POLYPOSIS COLI IN GOBLET CELLS IS ASSOCIATED WITH INFLAMMATION IN MOUSE AND HUMAN COLON

Adenomatous Polyposis Coli (APC) functions as an essential colon tumor suppressor. Roles for APC in other disease states such as Inflammatory Bowel Disease (IBD) remain less defined. In the process of characterizing Apc protein in gastrointestinal tissues from human patients with active IBD, we found a subset of goblet cells with elevated Apc staining intensity. While normal colon tissue was only sparsely populated with these “APChigh” goblet cells, tissue from patients with Ulcerative Colitis and Crohn’s Disease contained entire regions where almost every goblet cell was APChigh. In the descending colon, the APChigh goblet cells were prominent in patients with active Ulcerative Colitis but were mostly lacking in normal samples. In mice, APChigh cells were observed only in the small intestines and proximal/medial colons. Upon induction of colitis with Dextran Sodium Sulfate (DSS), the APChigh goblet cells remained in the proximal and medial colon, but also appeared in the distal colon. To better understand the link between APC expression, goblet cells, and inflammation, cultured human colon cells derived from normal tissue were depleted of APC and RNAseq analysis was performed. APC depletion resulted in reduced expression of mRNAs encoding IL1 signaling pathway components IL1b and IL1R, known regulators of Muc2 expression. Muc2 is the main constituent of goblet cell mucus, which is secreted to form a protective barrier for the epithelial cells lining the large intestine. We found that treating cancer cells lacking wild-type APC with IL1b resulted in increased IL1R and MUC2 expression. Induction of full-length APC in these cells led to a similar phenotype. Combining IL1β treatment with APC induction led to an increase of MUC2 expression greater than expected for additive affects, suggesting that APC sensitized these cells to IL1 signaling. These results illuminate a novel role for APC in goblet cells under conditions of inflammation. We postulate that the higher amount of APC in a subset of goblet cells might sensitize them to IL-1 signaling. During times of inflammation, such as active Ulcerative Colitis, goblet cells with higher APC levels would be more sensitive to IL-1-induced MUC2 expression than other goblet cells, perhaps protecting the intestinal lining from further barrier breaches.

ELEVATED ADENOMATOUS POLYPOSIS COLI IN GOBLET CELLS IS ASSOCIATED WITH INFLAMMATION IN MOUSE AND HUMAN COLON

Abstract Adenomatous Polyposis Coli (APC) functions as an essential colon tumor suppressor. Roles for APC in other disease states such as Inflammatory Bowel Disease (IBD) remain less defined. In the process of characterizing Apc protein in gastrointestinal tissues from human patients with active IBD, we found a subset of goblet cells with elevated Apc staining intensity. While normal colon tissue was only sparsely populated with these “APChigh” goblet cells, tissue from patients with Ulcerative Colitis and Crohn’s Disease contained entire regions where almost every goblet cell was APChigh. In the descending colon, the APChigh goblet cells were prominent in patients with active Ulcerative Colitis but were mostly lacking in normal samples. In mice, APChigh cells were observed only in the small intestines and proximal/medial colons. Upon induction of colitis with Dextran Sodium Sulfate (DSS), the APChigh goblet cells remained in the proximal and medial colon, but also appeared in the distal colon. To better understand the link between APC expression, goblet cells, and inflammation, cultured human colon cells derived from normal tissue were depleted of APC and RNAseq analysis was performed. APC depletion resulted in reduced expression of mRNAs encoding IL1 signaling pathway components IL1b and IL1R, known regulators of Muc2 expression. Muc2 is the main constituent of goblet cell mucus, which is secreted to form a protective barrier for the epithelial cells lining the large intestine. We found that treating cancer cells lacking wild-type APC with IL1b resulted in increased IL1R and MUC2 expression. Induction of full-length APC in these cells led to a similar phenotype. Combining IL1β treatment with APC induction led to an increase of MUC2 expression greater than expected for additive affects, suggesting that APC sensitized these cells to IL1 signaling. These results illuminate a novel role for APC in goblet cells under conditions of inflammation. We postulate that the higher amount of APC in a subset of goblet cells might sensitize them to IL-1 signaling. During times of inflammation, such as active Ulcerative Colitis, goblet cells with higher APC levels would be more sensitive to IL-1-induced MUC2 expression than other goblet cells, perhaps protecting the intestinal lining from further barrier breaches.

CADM1 suppresses c-Src activation by binding with Cbp on membrane lipid rafts and intervenes colon carcinogenesis

Cell adhesion molecules act as tumor suppressors primarily by cell attachment activity, but additional mechanisms modifying signal transduction are suggested in some cases. Cell adhesion molecule 1 (CADM1), a membrane-spanning immunoglobulin superfamily, mediates intercellular adhesion by trans-homophilic interaction and acts as a tumor suppressor. Here, we investigated CADM1-associated proteins comprehensively using proteomic analysis of immune-precipitates of CADM1 by mass spectrometry and identified a transmembrane adaptor protein, Csk-binding protein (Cbp), known to suppress Src-mediated transformation, as a binding partner of CADM1. CADM1 localizes to detergent-resistant membrane fractions and co-immunoprecipitated with Cbp and c-Src. Suppression of CADM1 expression using siRNA reduces the amount of co-immunoprecipitated c-Src with Cbp and activates c-Src in colon cancer cells expressing both CADM1 and Cbp. On the other hand, co-replacement of CADM1 and Cbp in colon cancer cells lacking CADM1 and Cbp expression suppresses c-Src activation, wound healing and tumorigenicity in nude mice. Furthermore, expression of Cbp and CADM1 was lost in 55% and 83% of human colon cancer, respectively, preferentially in tumors with larger size and/or lymph node metastasis. CADM1 would act as a colon tumor suppressor by intervening oncogenic c-Src signaling through binding with Cbp besides its authentic cell adhesion activity.

The interaction of Hemin and Sestrin2 modulates oxidative stress and colon tumor growth

Several large epidemiological and animal studies demonstrate a direct correlation between dietary heme iron intake and/or systemic iron levels and cancer risk in several cancers including colorectal cancer (CRC). However, the precise mechanisms for how heme iron contributes to CRC and how cancer cells respond to heme iron-induced stress are still unclear. Previously we have shown that one of the stress-inducible proteins, Sestrin2 (SESN2), is a novel tumor suppressor in colon by limiting endoplasmic reticulum stress and mammalian target of rapamycin complex 1 (mTORC1) signaling and tumor growth. But the relationship between heme iron and SESN2, especially in the context of colon carcinogenesis, was not investigated previously. Here, we found that hemin dose-dependently increased SESN2 expression in an oxidative stress and nuclear factor (erythroid-derived 2)-like 2 (NFE2L2, NRF2)-dependent manner. Since SESN2 overexpression reduced hemin-induced oxidative stress, SESN2 could be an important target of NRF2 exerting antioxidant function. Indeed, expression of several oxidative stress responsive proteins such as NRF2 and its target genes was reduced by SESN2. Although we formerly reported that SESN2 expression was reduced after p53 mutation in colon tumors, mouse colon tumors, which have intact p53 and NRF2, induced SESN2 expression in response to iron stimulus. Although SESN2 overexpression decreased murine colon tumor cell growth both in vitro and in vivo, it rendered colon cancer cells more resistant to hemin-induced apoptosis and therefore promoted tumor growth during hemin treatment. Taken together, although SESN2 generally suppresses tumorigenesis, it can produce tumor-promoting role in iron-rich environment by suppressing oxidative stress-associated cancer cell death.

Abstract 478: Regulation of MUC13 by microRNA in colorectal cancer

Abstract Colorectal cancer (CRC) represents a serious health problem with high incidence and mortality worldwide. The main reason of high mortality is the late diagnosis. For the Duke stage I, the average 5-years survival rate is 90%. In comparison to patients diagnosed at IV stage, the 5-years survival rate decreases to 3%. Mucinous CRC is generally defined as having greater than 50 % of the tumor area with a mucinous differentiation by histological examination. The increased incidence occurs in proximal colon and among younger patients, as compared to nonmucinous adenocarcinoma. Mucinous CRCs have been found to have a higher Duke stage at diagnosis, and, consequently a poorer prognosis. Mucines are surface or secreted glycoproteins with high molecular weight. Dysregulation of their expression may lead to malignant transformation. MUC13 overexpression influences colon cancer tumorigenesis and metastasis via multiple oncogenic proteins. Recently, we have observed that rs1532602 polymorphism in microRNA (miRNA) binding sites in MUC13 gene was associated with significantly decreased risk of CRC. The aim of the present study is to investigate regulation in vitro by miRNAs as a potential mechanism for increased MUC13 expression in CRC. Two different miRNAs were selected in this investigation. MiRNA-4647 is predicted to bind to the rs1532602 in MUC13. MiRNA-137 functions as a tumour suppressor in colon. Additionally, we investigated the impact of miRNAs mentioned above on regulation of MUC13 in a cohort of 113 patients with sporadic CRC. The present work provides important insights into role of miRNAs which bind to MUC13. Definition of functions, mechanisms of MUC13 regulation and its impact on CRC may contribute to better prediction and individual treatment of CRC. Acknowledgement: Grant Agency of the Ministry of Health of the Czech Republic (AZV MZ 15-26535A) and Grant Agency of the Czech Republic GACR 17-16857S. Citation Format: Linda Bartu, Alena Opattova, Pavel Vodicka, Veronika Vymetalkova. Regulation of MUC13 by microRNA in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 478.

Integrative genomic approaches to dissect clinically-significant relationships between the VDR cistrome and gene expression in primary colon cancer

Recently, we undertook a pan-cancer analyses of the nuclear hormone receptor (NR) superfamily in The Cancer Genome Atlas (TCGA), and revealed that the vitamin D receptor (NR1I1/VDR) was commonly and significantly down-regulated specifically in colon adenocarcinoma cohort (COAD). To examine the consequence of down-regulated VDR expression we re-analyzed VDR chromatin immunoprecipitation sequencing (ChIP-Seq) data from LS180 colon cancer cells (GSE31939). This analysis identified 1809 loci that displayed significant (p.adj<0.01) differential binding of the VDR in response 1,25(OH)2D3 treatment; 947 peaks annotated to 672 genes. We examined expression patterns in the COAD cohort of 286 tumors compared to 41 normal samples and revealed that VDR bound genes were significantly positively correlated to VDR expression compared to the background transcriptome, suggesting direct regulation by VDR. Gene set enrichment analyses revealed significant enrichment for genes known to be regulated by a number of other transcription factors including SMADs and JUN. Filtering VDR associated genes for those that were commonly and significantly altered in COAD revealed a cohort of 27 differentially expressed genes. The expression patterns of these genes clustered tumors and significantly associated with disease free survival. For instance, males with low expression of Lectin, Galactoside Binding Soluble 4 (LGALS4, encodes the colon tumor suppressor, Galactin 4) had significantly shorted disease free survival. These analyses suggest that reduced expression of VDR in colon cancer (but neither loss nor mutation) changes the actions of the VDR by both dampening the expression of tumor suppressors (e.g. LGALS4) whilst either stabilizing or not down-regulating expression of oncogenes (e.g. Carbonic Anhydrase 9 (CA9)). These integrative genomic approaches are relatively generic and applicable to the study of any transcription factor.

Critical function of the necroptosis adaptor RIPK3 in protecting from intestinal tumorigenesis.

Necroptosis is a programmed form of non-apoptotic cell death that requires the kinase activity of the receptor interacting protein kinase 3 (RIPK3). Although in vitro data suggests that cancer cells lacking expression of RIPK3 are invasive, the physiological role of RIPK3 in a disease-relevant setting remains unknown. Here we provide evidence that RIPK3 has a critical role in suppressing colorectal cancer (CRC). RIPK3-deficient mice were highly susceptible to colitis-associated CRC and exhibited greater production of pro-inflammatory mediators and tumor promoting factors. Tumorigenesis in RIPK3-deficiency resulted from uncontrolled activation of NF-κB, STAT3, AKT and Wnt-β-catenin signaling pathways that enhanced the ability of intestinal epithelial cells (IECs) to aberrantly proliferate in the face of the sustained inflammatory microenvironment and promote CRC. We found that RIPK3 expression is reduced in tumors from patients with inflammatory bowel diseases, and further confirmed that expression of RIPK3 is downregulated in human CRC and correlated with cancer progression. Thus, our results reveal that the necroptosis adaptor RIPK3 has key anti-inflammatory and anti-tumoral functions in the intestine, and define RIPK3 as a novel colon tumor suppressor.

A genetic polymorphism (rs17251221) in the calcium-sensing receptor is associated with ovarian cancer susceptibility.

Calcium-sensing receptor (CaSR) is a G-protein‑ coupled receptor that senses blood calcium. In vivo, CaSR is required for normal epidermal differentiation by mediating calcium signaling. CaSR was confirmed to be a tumor suppressor in colon and breast cancer. The single-nucleotide polymorphism (SNP) rs17251221, located on the intron, is a genetic variation of the CaSR gene. We analyzed rs17251221 in ovarian cancer using an allelic discrimination assay. Cycling probes were used for genotyping 290 ovarian cancer patients and 312 age-matched cancer-free females. rs17251221 and clinicopathological characteristics of ovarian cancer were analyzed statistically. The AG and GG genotypes were confirmed to appear in fewer cancer cases than in controls and the genotype distribution between cases and controls was statistically significant. The AG+GG genotype was correlated with low ovarian cancer risk, while rs17251221 was not associated with clinicopathological variables including age at diagnosis, tumor size, histologic type, pathological subtype, lymph node metastasis, CA-125 expression, clinical stage, or degree of differentiation. The rs17251221 polymorphism genotype was not correlated with survival in ovarian cancer. These results suggest that the G allele of the CaSR rs17251221 polymorphism is protective against ovarian cancer and the homozygous GG genotype may be a protective genotype as well. The rs17251221 may play an important role in the development of ovarian cancer and could be used as a biomarker for predicting ovarian cancer.

Abstract 2037: The butyrate transporter SLC5A8 is a tumor suppressor in colon linked to dietary fiber content

Abstract Dietary fiber has long been known to protect against colonic inflammation and colon cancer. Short-chain fatty acids (SCFA) such as butyrate generated in colonic lumen by bacterial fermentation of dietary fiber mediate most of these protective effects, but the underlying molecular mechanisms are poorly understood. We examined the role of the plasma membrane transporter Slc5a8 in the beneficial effects of these bacterial metabolites against colitis and colon cancer. Slc5a8 is expressed on the luminal membrane of colonic epithelial cells and also in mucosal immune cells including the antigen-presenting dendritic cells. The transporter mediates the entry of SCFAs into colonic epithelium from the lumen and also into dendritic cells in the lamina propria. We interrogated the role of the transporter in colon by comparing the function of dendritic cells and progression of experimentally induced colitis and colon cancer between wild type mice and Slc5a8-null mice. Since the amount of SCFAs generated in colonic lumen depends on the fiber content in the diet, we used two different diets, one with optimal fiber content (FC diet) and the other with no fiber (FF diet). These studies have shown that Slc5a8 is obligatory for butyrate-dependent inhibition of histone deacetylases in colonic epithelium and dendritic cells and also for the maintenance of the intestinal barrier function; but this obligatory need for the transporter is evident only when the mice are fed FF diet. The transporter is dispensable with FC diet. Compared to wild type mice, Slc5a8-null mice exhibit increased susceptibility to dextran sulfate sodium-induced colitis and azoxymethane/dextran sulfate sodium-induced colon cancer, again only with FF diet and not with FC diet. Butyrate induces the immunosuppressive enzymes indoleamine dioxygenase 1 (IDO1) and aldehyde dehydrogenase 1A2 (Aldh1A2) in dendritic cells in an Slc5a8-dependent manner. This is corroborated by the decreased expression of these two enzymes in the colon of germ-free mice compared to conventional mice. IDO1 and Aldh1A2 play a critical role in dendritic cells to maintain an immunosuppressive phenotype. Butyrate, which induces the expression of both enzymes, promotes the ability of dendritic cells to convert naive T cells into FoxP3-positive immunosuppressive Tregs and also their ability to suppress interferon-gamma-secreting pro-inflammatory T cells. As such, Slc5a8-null mice have decreased Tregs and increased IFN-gamma-positive T cells in colon. We conclude that Slc5a8 may be dispensable in colon under dietary conditions associated with high fiber content when the luminal concentrations of SCFAs are high, but the transporter is indispensable for protection against colonic inflammation and colon cancer with a diet containing suboptimal fiber which results in decreased concentrations of SCFAs in the lumen. Thus, Slc5a8 is a conditional tumor suppressor in colon linked to dietary fiber content. Citation Format: Vadivel Ganapathy, Ashish Gurav, Nagendra Singh. The butyrate transporter SLC5A8 is a tumor suppressor in colon linked to dietary fiber content. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2037. doi:10.1158/1538-7445.AM2015-2037

Abstract 2815: Sulindac reversal of 15-PGDH mediated resistance to colon tumor chemoprevention with NSAIDs

Abstract Background: Non-steroidal anti-inflammatory drugs (NSAIDs) protect against colorectal cancer development by inhibiting cyclooxygenase (COX) enzymes responsible for the production of tumor growth promoting prostaglandins. 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a colon tumor suppressor that degrades prostaglandins and antagonizes COX-mediated oncogenesis. Deletion of 15-PGDH in mice results in an increased susceptibility to azoxymethane (AOM)-induced colon tumor formation and resistance to the chemopreventive effects of the COX-2 selective inhibitor, celecoxib. Similar resistance to colon adenoma prevention with celecoxib has been shown in humans with low colonic 15-PGDH levels. In this study, we expanded the panel of NSAIDs tested in a mouse colon tumorigenesis model to include sulindac and aspirin to determine if these non-selective COX-1 and COX-2 inhibitors would be a better treatment for the prevention of colon adenomas in mice genetically lacking 15-PGDH. Materials and Methods: 15-PGDH wild-type and knockout mice on a FVB/NJ background were administered azoxymethane (AOM) by i.p. injection once per week for 6 weeks at 10mg/kg dose. Concurrent with the first AOM injection, all mice were fed for the length of the study with either a control (AIN-76A) diet or diet supplemented with celecoxib (1250 ppm), sulindac (167 ppm), or aspirin (300 ppm). Mice were euthanized 14 weeks after the last AOM injection, and the colons were removed and examined under a dissecting microscope to identify all tumors and inflammatory lesions. The effect of NSAID treatment on COX inhibition was determined by measuring colonic PGE2 levels. Results: We demonstrate a significant difference in tumor formation between aspirin and sulindac treated 15-PGDH knockout mice compared to celecoxib treatment. Sulindac significantly prevented colon tumor formation while aspirin had no protective effect, despite a comparable reduction in colonic mucosal PGE2 levels by both sulindac and aspirin. Additionally, we find that treatment of celecoxib or sulindac for an extended period of time results in a significant increase in colonic inflammation in both wild-type and knockout mice, with lymphoid aggregates and large proximal colon inflammatory lesions appearing, and that the incidence of inflammatory lesions correlate with NSAID efficacy. Conclusions: These findings suggest sulindac may be the most active NSAID for colon chemoprevention in populations with low 15-PGDH, but raise the caution that this agent may also give rise to inflammatory lesions in the colon. Citation Format: Stephen P. Fink, Dawn M. Dawson, Yongyou Zhang, Adam Kresak, Earl G. Lawrence, Peiying Yang, Yanwen Chen, Jill S. Barnholtz-Sloan, Joseph E. Willis, Levy Kopelovich, Sanford D. Markowitz. Sulindac reversal of 15-PGDH mediated resistance to colon tumor chemoprevention with NSAIDs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2815. doi:10.1158/1538-7445.AM2015-2815

Slc5a8, a Na+-coupled high-affinity transporter for short-chain fatty acids, is a conditional tumour suppressor in colon that protects against colitis and colon cancer under low-fibre dietary conditions.

Mammalian colon harbours trillions of bacteria under physiological conditions; this symbiosis is made possible because of a tolerized response from the mucosal immune system. The mechanisms underlying this tolerogenic phenomenon remain poorly understood. In the present study we show that Slc5a8 (solute carrier gene family 5a, member 8), a Na(+)-coupled high-affinity transporter in colon for the bacterial fermentation product butyrate, plays a critical role in this process. Among various immune cells in colon, dendritic cells (DCs) are unique not only in their accessibility to luminal contents but also in their ability to induce tolerogenic phenotype in T-cells. We found that DCs exposed to butyrate express the immunosuppressive enzymes indoleamine 2,3-dioxygenase 1 (IDO1) and aldehyde dehydrogenase 1A2 (Aldh1A2), promote conversion of naive T-cells into immunosuppressive forkhead box P3(+) (FoxP3(+)) Tregs (regulatory T-cells) and suppress conversion of naive T-cells into pro-inflammatory interferon (IFN)-γ-producing cells. Slc5a8-null DCs do not induce IDO1 and Aldh1A2 and do not generate Tregs or suppress IFN-γ-producing T-cells in response to butyrate. We also provide invivo evidence for an obligatory role for Slc5a8in suppression of IFN-γ-producing T-cells. Furthermore, Slc5a8 protects against colitis and colon cancer under conditions of low-fibre intake but not when dietary fibre intake is optimal. This agrees with the high-affinity nature of the transporter to mediate butyrate entry into cells. We conclude that Slc5a8 is an obligatory link between dietary fibre and mucosal immune system via the bacterial metabolite butyrate and that this transporter is a conditional tumour suppressor in colon linked to dietary fibre content.

Interplay between chromatin-modifying enzymes controls colon cancer progression through Wnt signaling

Cancer progression is associated with epigenetic alterations, such as changes in DNA methylation, histone modifications or variants incorporation. The p400 ATPase, which can incorporate the H2A.Z variant, and the Tip60 histone acetyltransferase are interacting chromatin-modifying proteins crucial for the control of cell proliferation. We demonstrate here that Tip60 acts as a tumor suppressor in colon, since mice heterozygous for Tip60 are more susceptible to chemically induced preneoplastic lesions and adenomas. Strikingly, heterozygosity for p400 reverses the Tip60-dependent formation of preneoplastic lesions, uncovering for the first time pro-oncogenic functions for p400. By genome-wide analysis and using a specific inhibitor in vivo, we demonstrated that these effects are dependent on Wnt signaling which is antagonistically impacted by p400 and Tip60: p400 directly favors the expression of a subset of Wnt-target genes and regulators, whereas Tip60 prevents β-catenin acetylation and activation. Taken together, our data underline the physiopathological importance of interplays between chromatin-modifying enzymes in the control of cancer-related signaling pathways.

SPDEF Functions as a Colorectal Tumor Suppressor by Inhibiting β-Catenin Activity

Expression of the SAM pointed domain containing ETS transcription factor (SPDEF or prostate-derived ETS factor) is regulated by Atoh1 and is required for the differentiation of goblet and Paneth cells. SPDEF has been reported to suppress the development of breast, prostate, and colon tumors. We analyzed levels of SPDEF in colorectal tumor samples from patients and its tumor-suppressive functions in mouse models of colorectal cancer (CRC). We analyzed levels of SPDEF messenger RNA and protein in more than 500 human CRC samples and more than 80 nontumor controls. Spdef(-/-)and wild-type mice (controls) were either bred with Apc(Min/+) mice, or given azoxymethane (AOM) and dextran sodium sulfate (DSS), or 1,2-dimethylhydrazine and DSS, to induce colorectal tumors. Expression of Spdef also was induced transiently by administration of tetracycline to Spdef(dox-intestine) mice with established tumors, induced by the combination of AOM and DSS or by breeding with Apc(Min/+) mice. Colon tissues were collected and analyzed for tumor number, size, grade, and for cell proliferation and apoptosis. We also analyzed the effects of SPDEF expression in HCT116 and SW480 human CRC cells. In colorectal tumors from patients, loss of SPDEF was observed in approximately 85% of tumors and correlated with progression from normal tissue, to adenoma, to adenocarcinoma. Spdef(-/-); Apc(Min/+) mice developed approximately 3-fold more colon tumors than Spdef(+/+); Apc(Min/+) mice. Likewise, Spdef(-/-) mice developed approximately 3-fold more colon tumors than Spdef(+/+) mice after administration of AOM and DSS. After administration of 1,2-dimethylhydrazine and DSS, invasive carcinomas were observed exclusively in Spdef(-/-) mice. Conversely, expression of SPDEF was sufficient to promote cell-cycle exit in cells of established adenomas from Spdef(dox-intestine); Apc(Min/+) mice and in Spdef(dox-intestine) mice after administration of AOM + DSS. SPDEF inhibited the expression of β-catenin-target genes in mouse colon tumors, and interacted with β-catenin to block its transcriptional activity in CRC cell lines, resulting in lower levels of cyclin D1 and c-MYC. SPDEF is a colon tumor suppressor and a candidate therapeutic target for colon adenomas and adenocarcinoma.

Abstract 26: Deletion of Slc5a8 in mice promotes metabolic syndrome, colonic inflammation, and colon cancer: A phenomenon dependent on dietary fiber content

Abstract SLC5A8 is a candidate tumor suppressor that is silenced in colon cancer. It is a transporter that mediates concentrative entry of short-chain fatty acids (SCFAs: acetate, propionate, and butyrate) into colon cells. SCFAs are produced at high concentrations in colon by bacterial fermentation of dietary fiber (acetate, ∼60 mM; propionate, ∼20 mM; butyrate, ∼10 mM). Among SCFAs, propionate and butyrate are inhibitors of histone deacetylases (HDACs). These HDAC inhibitors induce differentiation in normal colon cells but cause apoptosis in colon cancer cells. They also suppress colonic inflammation. We postulate that the transporter is silenced in colon cancer to prevent the entry of these HDAC inhibitors into tumor cells. We have demonstrated the tumor-suppressive function of SLC5A8 in vitro in colon cancer cells and hence hypothesized that deletion of Slc5a8 in mice would promote inflammation and cancer in colon. Surprisingly however, there was no difference between wild type (WT) mice and Slc5a8-null (KO) mice in progression of colonic inflammation and colon cancer in experimental model systems when mice were fed optimal dietary fiber. SLC5A8 transports butyrate and propionate with a Michaelis constant of ∼0.05 mM; under in vivo conditions where butyrate and propionate are present at &amp;gt;10 mM in colon, the transporter plays only a minor role in the entry of these compounds into colon cells. At high concentrations, they diffuse into cells bypassing the transporter. As such we postulated that Slc5a8-null mice would have enhanced colonic inflammation and colon cancer only under low-fiber dietary conditions where butyrate/propionate concentrations in colon are low enough that the transporter is obligatory for their entry into colon cells. To test this hypothesis, we maintained WT and KO mice with a low-fiber diet starting at 4-weeks of age. We found that, under these conditions, the KO mice gained more weight than WT mice, became obese with increased abdominal fat, and exhibited hyperglycemia. When dextran sulfate sodium was administered in drinking water (2% for 1 week), KO mice died from severe colonic inflammation whereas WT mice were able to withstand the treatment and did not die. We then examined the incidence of colon cancer in these mice by crossing them with ApcMin/+ mouse. Under optimal dietary fiber conditions, there was no difference between Slc5a8+/+/ApcMin/+ mice and Slc5a8-/-/ApcMin/+ mice in the incidence of colon cancer. But, under low-fiber dietary conditions, the incidence of colon cancer was much higher in Slc5a8-/-/ApcMin/+ mice than in Slc5a8+/+/ApcMin/+ mice. These studies show that Slc5a8 indeed functions as a tumor suppressor in colon in vivo, but only if dietary fiber content is low. In addition, under these dietary conditions, Slc5a8 also protects against metabolic syndrome and colonic inflammation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 26. doi:1538-7445.AM2012-26

Abstract A44: Suppression of insulin-induced fatty acid synthase gene expression and colon cancer cell proliferation by members of the Krüppel-like family of transcription factors

Abstract Obesity and the associated hyperinsulinemia are major risk factors for colorectal cancer (CRC). The Fatty Acid Synthase (FASN) gene encodes the protein responsible for de novo synthesis of fatty acids. FASN mRNA and protein are insulin-induced in the colonic epithelium, over-expressed early in CRC, and positively associated with tumor cell proliferation, cell survival and chemo-resistance. FASN and its transcriptional regulators may therefore provide mechanistic links between obesity and CRC predisposition and may be exploitable for CRC prevention. Recent attention has focused on several members of the Krüppel-like family (KLF) of transcription factors in pathogenesis of CRC. Published studies have demonstrated that Krüppel-like Factors 4 and 6 (KLF4, KLF6) are important suppressors of CRC development. Recent work from our laboratory and others has implicated KLF9 as a third potential KLF protein that may be a tumor suppressor in colon and rectum. In the present study, we evaluated the possible actions of KLF9 and KLF4 to oppose the inductions by insulin of colon cancer cell FASN expression and proliferation. Addition of insulin to cell medium to mimic hyperinsulinemia (5nM-25nM, compared to vehicle) led to an increase in proliferation (13–25%, P&amp;lt;0.05, evaluated by MTT assay) of the human colorectal cancer cell line HT-29. An increased insulin level also led to induction of FASN mRNA abundance (P=0.018). Gene expression and cell proliferation were examined after transfection of HT-29 cells with siRNAs targeting KLF4, KLF9, and KLF13 (the latter is highly similar in primary sequence to KLF9). Knockdown of KLF9 caused induction of FASN at both the mRNA (P&amp;lt;0.001) and protein (P=0.01) levels. FASN mRNA levels also increased with siRNA knockdown of KLF13 (P=0.001) and tended (P=0.075) to increase with KLF4 knockdown. Hyperinsulinemia elicited a greater increase in proliferation of cells transfected with KLF9 siRNA than of cells transfected with control (scrambled) siRNA. While transfection of KLF9 expression vector produced a trend of reduced proliferation (P=0.082; compared to empty vector), hyperinsulinemia rescued cells from this effect. A hyperinsulinemic environment coupled with siRNA knockdown of KLF9 augmented FASN mRNA expression, compared to KLF9 knockdown alone. Increased insulin concentrations caused a marked induction of KLF4 but not KLF9 or KLF13, mRNA expression. As KLF4 has been shown to inhibit proliferation of colorectal cancer cells, it is possible that induction of KLF4 by insulin may constitute a counter-regulatory mechanism to control their proliferation. In conclusion, our findings implicate KLF family members KLF9, KLF13 and KLF4 in transcriptional repression of proto-oncogenic FASN in CRC cells. They also highlight the potential cancer-suppressive roles of KLF proteins in normo- and hyperinsulinemic conditions, which clearly warrant further investigation. Supported by NIH RO1 CA136493 and grants from the Arkansas Biosciences Institute and Sturgis Foundation. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A44.

A novel facet to consider for the effects of butyrate on its target cells. Focus on “The short-chain fatty acid butyrate is a substrate of breast cancer resistance protein”

short-chain fatty acids (SCFAs), acetate, propionate, and butyrate are produced by bacterial fermentation of dietary fiber in the colonic lumen. In the normal human large intestine, SCFA concentrations range from 70 to 100 mM; these SCFAs account for ∼80% of the anions present in the lumen ([5][1

Deregulated expression of sprouty2 and microRNA-21 in human colon cancer: Correlation with the clinical stage of the disease

Sprouty protein is a novel feedback regulator involved in downstream inactivation of several growth factor receptor pathways. Sprouty2 (Spry2) protein was shown to be downregulated in human cancers. High levels of microRNA-21 (miRNA-21) expression have been associated with poor survival and poor response to adjuvant chemotherapy in cancer patients. But the effect of Spry2 in human colon cancer remained unknown. Paired tumor and normal mucosa samples from patients were examined for their expression of Spry2 mRNA and miRNA-21 by real-time quantitative RT-PCR analysis. Our results show that Spry2 was downregulated in human colon cancer, and its expression levels were lower in advanced-stage tumors than in early-stage tumors. There was a negative correlation between the expression levels of Spry2 and miRNA-21. Furthermore, overexpression of Spry2 suppressed the growth and migration of colon cancer cells with a concomitant increase in PTEN expression and reduction of Akt and MAPK phosphorylation. Spry2 inhibited the growth and tumorigenesis of colon cancer cells in vivo. Conclusively, we show for the first time that Spry2 expression is downregulated and miRNA-21 is upregulated in the clinical samples of colon cancer, which correlates with clinical stage of disease. Thus, Spry2 functions as a tumor suppressor in colon cancer.See commentary: Sprouty keeps bowel kinases regular in colon cancer, while miR-21 targets Sprouty

Role of RUNX3 in Bone Morphogenetic Protein Signaling in Colorectal Cancer

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta (TGF-beta) superfamily, are multifunctional cytokines regulating a broad spectrum of biological functions. Recent studies show the presence of BMP receptor 1a mutations in juvenile polyposis and frequent Smad4 mutations in colon cancer, suggesting that aberrations in BMP signaling play an important role in intestinal cancer pathogenesis. However, the exact molecular mechanisms remain poorly understood. The Runt domain transcription factor RUNX3 is an integral component of signaling pathways mediated by TGF-beta and BMPs. RUNX3 is a gastric and colon tumor suppressor, functioning downstream of TGF-beta. Recently, we showed the tumor-suppressive effects of RUNX3 by its ability to attenuate beta-catenin/T-cell factors (TCFs) transactivation in intestinal tumorigenesis. Here, we explore the molecular basis of the tumor-suppressive function of the BMP pathway through RUNX3 in colorectal carcinogenesis. BMP exerted a growth-suppressive effect in HT-29, a human colorectal cancer cell line. c-Myc oncogene was found to be downregulated by BMP and/or RUNX3. We show that upregulation of RUNX3 by BMP reduces c-Myc expression. Evidence is presented suggesting that RUNX3 downregulates c-Myc expression by two parallel pathways-directly at the transcriptional level and through attenuation of beta-catenin/TCFs, downstream of BMPs in colorectal cancer cells.

973 15-HYDROXYPROSTAGLANDIN DEHYDROGENASE IS A THERAPEUTIC TARGET FOR BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research II1 Apr 2010973 15-HYDROXYPROSTAGLANDIN DEHYDROGENASE IS A THERAPEUTIC TARGET FOR BLADDER CANCER Monica Liebert, Stephanie Tseng-Rogenski, Lakshmi Kunju, Alon Weizer, David Wood, and Cheryl Lee Monica LiebertMonica Liebert More articles by this author , Stephanie Tseng-RogenskiStephanie Tseng-Rogenski More articles by this author , Lakshmi KunjuLakshmi Kunju More articles by this author , Alon WeizerAlon Weizer More articles by this author , David WoodDavid Wood More articles by this author , and Cheryl LeeCheryl Lee More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1920AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES 15-hydroxyprostaglandin dehydrogenase (PGDH) is an enzyme responsible for inactivation of powerful tumor promoting molecule prostaglandin E2 (PGE2). Recent studies have shown that PGDH is a tumor suppressor in colon, lung, gastric and breast cancers. The objective of this study was to determine if restoration of PGDH expression provides a therapeutic effect for bladder cancer. METHODS PGDH immunostaining was performed in 128 paraffin-embedded tissue samples. PGDH expression in 8 human bladder cancer cell lines was determined using Western blotting. Restoration of PGDH expression was accomplished using a ″Tet On″ lentiviral system (Invitrogen). Cells were first infected with a lentivirus carrying the tetracycline repressor gene. After selection with Blasticidin, infected cells were treated with a second lentivirus containing the genetic sequence for PGDH or a control reverse sequence (HDGP). Infected cells were selected by treatment in culture with Blasticidin and Zeocin. PGDH expression was induced in cells by treatment with tetracycline. Statistics were evaluated using SigmaStat v3.5. RESULTS By immunostaining, 18/19 normal urothelial samples showed strong staining (2-3+ staining, 95%). Staining was reduced in many lower stage (Ta) samples (only 22/37 showed strong staining, 59%) and lost in most invasive bladder cancer samples (6/72 samples strongly positive, 8%; p<.0001, χ2). Western blotting confirmed PGDH expression in only 2/8 bladder cancer cell lines. Restoration of PGDH expression in the PGDH-negative UM-UC3 human bladder cancer cell line slowed cell growth, and eventually the cells died with fragmentation of the cell nuclei. Previous studies assume that PGDH tumor suppressor activity is due to lower local PGE2 levels. However, attempted ″rescue″ of PGDH-expressing UM-UC-3 cells with exogenous PGE2 treatment failed, suggesting that restoration of PGDH expression has additional novel anti-tumor effects. CONCLUSIONS PGDH expression is lost early in bladder cancer progression. Restoration of PGDH expression in the UM-UC3 cell line resulted in cell death. This effect appears to be independent of the reduced level of PGE2 in the environment, a previously unappreciated and novel activity. These data indicate that PGDH restoration has therapeutic potential for bladder cancer. Ann Arbor, MI© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e378 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Monica Liebert More articles by this author Stephanie Tseng-Rogenski More articles by this author Lakshmi Kunju More articles by this author Alon Weizer More articles by this author David Wood More articles by this author Cheryl Lee More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

GPR109A Is a G-protein–Coupled Receptor for the Bacterial Fermentation Product Butyrate and Functions as a Tumor Suppressor in Colon

Short-chain fatty acids, generated in colon by bacterial fermentation of dietary fiber, protect against colorectal cancer and inflammatory bowel disease. Among these bacterial metabolites, butyrate is biologically most relevant. GPR109A is a G-protein-coupled receptor for nicotinate but recognizes butyrate with low affinity. Millimolar concentrations of butyrate are needed to activate the receptor. Although concentrations of butyrate in colonic lumen are sufficient to activate the receptor maximally, there have been no reports on the expression/function of GPR109A in this tissue. Here we show that GPR109A is expressed in the lumen-facing apical membrane of colonic and intestinal epithelial cells and that the receptor recognizes butyrate as a ligand. The expression of GPR109A is silenced in colon cancer in humans, in a mouse model of intestinal/colon cancer, and in colon cancer cell lines. The tumor-associated silencing of GPR109A involves DNA methylation directly or indirectly. Reexpression of GPR109A in colon cancer cells induces apoptosis, but only in the presence of its ligands butyrate and nicotinate. Butyrate is an inhibitor of histone deacetylases, but apoptosis induced by activation of GPR109A with its ligands in colon cancer cells does not involve inhibition of histone deacetylation. The primary changes in this apoptotic process include down-regulation of Bcl-2, Bcl-xL, and cyclin D1 and up-regulation of death receptor pathway. In addition, GPR109A/butyrate suppresses nuclear factor-kappaB activation in normal and cancer colon cell lines as well as in normal mouse colon. These studies show that GPR109A mediates the tumor-suppressive effects of the bacterial fermentation product butyrate in colon.