Bile acids have been studied for many years for their role in either promoting (Deoxycholic Acid) or suppressing (Ursodeoxycholic Acid) colon tumor development in animal models. However, the molecular mechanisms of both DCA's and UDCA's biological effects in colon tumorigenesis is still unclear. The cholesterol-like composition of bile acids and evidence of deregulating signal transduction pathways, such as the p42/44 MAP kinase cascade, led us to identify the plasma membrane as a target for bile acid-mediated effects. Specifically, plasma membrane microdomains such as lipid rafts and caveolae are particularly capable of altering mitogenic signaling due to have their role as platforms to concentrate receptors and assemble signal transduction machinery. In this study I tested the hypothesis that the growth suppressive effects of UDCA are mediated by stimulating membrane microdomains to activate protein degradation machinery to facilitate the down-regulation of receptor tyrosine kinase activity. We found that UDCA suppresses EGF-induced ERK activation, promotes interactions between EGFR and Caveolin-1 membrane fractions, whereas DCA causes redistribution. EGFR proteins that are localized to membrane fractions in the UDCA treated cells are extensively ubiquitinylated and we present evidence that this yields recruitment of the ubiquitin ligase c-Cbl to membrane fractions. UDCA increases the rate of EGFR degradation, whereas DCA sustains its' stability. I present evidence that UDCA's growth inhibitory effects on colon cancer cells may be mediated by recruitment of protein degradation machinery to membrane domains that are enriched with signaling receptors, a mechanism which has not been previously described. Importantly, I demonstrate for the first time a novel mechanism by which UDCA promotes growth inhibition, through increasing the rates of degradation of EGFR, thereby down-regulating mitogenic signaling in the cell. These experiments show exciting insights into the mechanism of bile acids and represent potential mechanisms for other chemopreventive agents.

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We have hypothesized that dietary modulation of intestinal miRNA expression may contribute to the chemoprotective effects of nutritional bioactives (fish oil and pectin). Using a rat colon carcinogen model, we determined miRNAs-let-7d, miR-15b, miR-107, miR-191 and miR-324-5p were modulated by fish oil + pectin. We also demonstrated that BACE1 and PTEN are targets of miR-107 and miR-21, respectively. To further elucidate the biological effects of diet and carcinogen on miRNAs, we integrated global miRNAs, total and polysomal gene expression datasets obtained from the above mentioned study and used four computational approaches. We demonstrated that polysomal profiling is tightly related to microRNA changes when compared with total mRNA profiling. In addition, diet and carcinogen exposure modulated a number of microRNAs and complementary gene expression analyses showed that oncogenic PTK2B, PDE4B, and TCF4 were suppressed by the chemoprotective diet at both the mRNA and protein levels. To determine the function of select diet and colon carcinogen modulated miRNAs and to validate their targets, we carried out a series of loss and gain of function experiments along with luciferase reporter assays. We verified that PDE4B and TCF4 are direct targets of miR-26b and miR-203, respectively. PTK2B was determined to be an indirect target of miR-19b. In addition, microRNA physiological function was assessed by examining effects on apoptosis and cell proliferation. To better understand how the colonic stem cell population responds to environmental factors such as diet and carcinogen, we investigated the chemoprotective effects of dietary agents on miRNAs in colonic stem cells obtained from Lgr5-EGFP-IRES-creERT2 knock in mice injected with AOM. We demonstrated that based on relative expression of miR-125a-5p, miR-190b and miR-191 in stem cells vs. daughter cells and differentiated cells, these miRNAs may be stem cell specific miRNAs. We also identified miR-21 to be significantly reduced in stem cells compared to differentiated cells and selectively modulated by these dietary agents in stem cells. In summary, our results indicate for the first time that fish oil plus pectin protect against colon tumorigenesis in part by modulating a subset of miRNAs and their target genes (mRNAs) implicated in the regulation of the colon stem cell niche and tumor development.

Final publication is available at http://cancerres.aacrjournals.org/cgi/pmidlookup?view=long&pmid=26018088 / Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(医科学) / 甲第19635号 / 医科博第73号 / 32671 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 妹尾 浩, 教授 渡邊 直樹, 教授 椛島 健治 / 学位規則第4条第1項該当

Aberrant activation of the canonical Wnt signaling pathway accounts for the vast majority of colorectal cancers. The Rac1 GTPase is overexpressed in colon cancer, and its splice variant, Rac1b, is preferentially expressed in colon tumours. Rac1 and Rac1b have both been previously shown to crosstalk with the canonical Wnt signaling pathway in colon cancer; however, the specific means by which this crosstalk occurs were unclear. This study examines the molecular mechanisms of Rac1/1b in the cooperation with canonical Wnt signaling in colon cancer. In a colon cancer cell line with dysregulated Wnt signaling, the constitutively active Rac1 mutant, V12Rac1, was observed to transcriptionally upregulate the expression of a gene set associated with cellular migration. Further, V12Rac1-mediated promotion of cell migration was dependent on its nuclear localization. Previous work in our lab has shown a Rac1-specific activator, Tiam1, is present in the nucleus at the promoter of Wnt target genes upon Wnt3a stimulation; and that exogenous introduction of Tiam1 increased the expression of a Wnt-responsive reporter (TopFlash). Given the importance of nuclear localization of Rac1 in the promotion of tumourigenic processes, we demonstrated that knockdown of endogenous Tiam1 reduced TopFlash expression, proving reverse specificity and strengthening the evidence of a nuclear role for Rac1. Since some functional differences exist between Rac1 and Rac1b, we also examined Rac1b for transcriptional targets following induction, and identified the RhoA effector, ROCK2, which has been previously associated with cell migration. ROCK2 demonstrated a positive correlation with Rac1b transcript expression in primary colon tumours as compared to matched normal tissue specimens. Interestingly, the observed induction in ROCK2 transcript did not translate into a detectable change in protein expression or kinase activity. Like Rac1, Rac1b also promotes cellular motility, which is dependent on nuclear localization. Cell migration can be negatively regulated by E-cadherin. Following Rac1b knockdown in HT29 cells, we show that Rac1b might contribute to motility through upregulation of the E-cadherin-repressor, Slug. Taken together, we provide greater insight into the mechanistic roles of Rac1 and Rac1b in transcriptionally regulating target genes to promote cellular processes, such as cell migration, in colon cancer with dysregulated canonical Wnt signaling.

Glucagon-like peptide-2 (GLP-2) is an intestinotrophic and intestinal anti-inflammatory hormone. Hence, I hypothesized that treatment with degradation-resistant hGly2GLP-2 increases, while blocking endogenous GLP-2 decreases colorectal cancer (CRC) in rodents. In mice, treatment with dextran sodium sulphate (DSS) and azoxymethane (AOM) induced colitis-associated CRC, which was further increased by treatment with hGly2GLP-2 and reduced by blocking endogenous GLP-2 with the antagonist hGLP-23-33. Moreover, while colonic damage score (CDS) was not altered by hGly2GLP-2 or hGLP-23-33 treatment, hGly2GLP-2 increased small intestinal growth and hGLP-23-33 reduced jejunal crypt cell proliferation. In rats fed with of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and high fat (HF) diet for aberrant crypt foci (ACF) induction, treatment with hGly2GLP-2 increased small intestinal growth and ACF occurrence. Moreover, in rats fed with PhIP-HF diet for tumour induction, early treatment with hGly2GLP-2 appears to increase the occurrence of intestinal tumours. Collectively, these findings indicate a pro-carcinogenic role for both exogenous and endogenous GLP-2.

Folate blood levels in North America have dramatically increased over the past decade owing to folic acid (FA) fortification and widespread supplement use. Furthermore, over 50% of newly diagnosed colorectal cancer (CRC) patients use vitamin supplements containing FA while receiving chemotherapy whose mechanisms of action are based on interruption of folate metabolism. This study therefore investigated whether FA supplementation can affect chemosensitivity of human colon cancer cells to 5FU, the cornerstone of CRC treatment, using a xenograft model. FA supplementation was associated with a non-dose dependent decrease in chemosensitivity, where mice receiving 8 mg FA did not respond to 5FU and had greater tumor growth with treatment, compared to 2 (control) or 25 mg FA. Results of this study pose concern given the drastically increased intake of FA, particularly among recently diagnosed CRC patients, and from mandatory fortification. Further studies are warranted to confirm our findings and to elucidate underlying mechanisms.

Folate is an essential cofactor in one-carbon transfer reactions including nucleotide biosynthesis, thereby playing an important role in DNA replication and repair. In cancer cells, folate depletion interrupts DNA synthesis, thereby causing cancer cell death. This has been the basis for chemotherapy using antifolates and 5-fluorouracil (5FU). We determined the effect of folate deficiency on the sensitivity of colon cancer cells to 5FU in a well established in vitro model of folate deficiency. Folate deficient cells had lower intracellular folate concentrations, had functional evidence of intracellular folate depletion, proliferated less, and had increased chemosensitivity to 5FU with and without Leucovorin. These data suggest that folate deficiency significantly enhances the sensitivity of colon cancer cells to 5FU based chemotherapy via changes in intracellular folate. Dietary or other strategies to deplete intracellular folate concentrations in colon cancer cells to enhance chemosensitivity to 5FU are worthy of further investigation.

Glucagon-like peptide-2 (GLP-2) is an intestinotrophic and intestinal anti-inflammatory hormone. Hence, I hypothesized that treatment with degradation-resistant hGly2GLP-2 increases, while blocking endogenous GLP-2 decreases colorectal cancer (CRC) in rodents. In mice, treatment with dextran sodium sulphate (DSS) and azoxymethane (AOM) induced colitis-associated CRC, which was further increased by treatment with hGly2GLP-2 and reduced by blocking endogenous GLP-2 with the antagonist hGLP-23-33. Moreover, while colonic damage score (CDS) was not altered by hGly2GLP-2 or hGLP-23-33 treatment, hGly2GLP-2 increased small intestinal growth and hGLP-23-33 reduced jejunal crypt cell proliferation. In rats fed with of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and high fat (HF) diet for aberrant crypt foci (ACF) induction, treatment with hGly2GLP-2 increased small intestinal growth and ACF occurrence. Moreover, in rats fed with PhIP-HF diet for tumour induction, early treatment with hGly2GLP-2 appears to increase the occurrence of intestinal tumours. Collectively, these findings indicate a pro-carcinogenic role for both exogenous and endogenous GLP-2.