京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19624号 / 医博第4131号 / 新制||医||1015(附属図書館) / 32660 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 今中 雄一, 教授 佐藤 俊哉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20220号 / 医博第4179号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 野田 亮, 教授 齊藤 博英 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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A Cochrane review conducted a meta-analysis looking only at FOBT for colorectal cancer screening. This review, based on published and unpublished data from 5 controlled trials, demonstrated that 3-card home FOBT conferred a reduction in colorectal cancer mortality of 16% (relative risk [RR]=0.84; 95% confidence interval [CI], 0.77-0.92) and a number needed to screen of 1173 (95% CI, 741-2807) to prevent 1 death from colon cancer over a 10-year period. If adjusted for adherence to screening, the reduction in mortality increased to 23% (RR=0.77; 95% CI, 0.57-0.89). In addition, long-term follow up of one of the RCTs in the review showed a continued reduction in colorectal cancer mortality of 34% (RR=0.66; 95% CI, 0.54-0.81) in subjects adhering to the FOBT screening protocol over a 13-year interval. Overall mortality did not differ between the screened and unscreened groups. A systematic review performed for the US Preventive Services Task Force (USPSTF) incorporated more recent data on colorectal cancer screening including colonoscopy. This review reached similar conclusions as above. This review also looked at office FOBT performed after digital rectal exam. It is important to note that a single office FOBT has a lower sensitivity than 3-card home FOBT and its effectiveness for reducing colorectal cancer mortality was unknown at the time of the systematic review. A subsequent 2005 Veterans Affairs prospective cohort study found that the sensitivity for detecting advanced neoplasia was only 4.9% for digital FOBT, and negative results did not decrease the likelihood of advanced neoplasia. The USPSTF review did not find any screening trials of colonoscopy but analyzed data from the National Polyp Study and a case-control study to draw its conclusions. The review reported an odds ratio for colorectal cancer mortality for patients who had colonoscopy to be 0.43 (95% CI, 0.30-63). The USPSTF review also looked at the sensitivity and adverse effects of FOBT compared to colonoscopy. One-time 3-card home FOBT had a sensitivity of 30% to 40% for detecting cancer. The sensitivity of one-time colonoscopy was difficult to determine since it was the criterion standard examination, but it was estimated to be greater than 90%, with a risk of perforation of 1/2000. The USPSTF review found both screening strategies cost-effective (<$30,000 per additional life-year gained) compared to no screening. FOBT had a cost per life-year saved of $5691 to $17,805 compared with $9038 to $22,012 for colonoscopy performed every 10 years.

The expression of calcium sensing receptor (CaSR) in the human colonic crypt epithelium is linked to cellular differentiation while its lack of expression is associated with undifferentiated and invasive colon carcinoma. Recent studies show that CaSR suppresses the malignant phenotype through a variety of pathways that inhibits growth and promotes differentiation. CaSR also promotes cytotoxic response to fluorouracil. These studies, taken together, have led me to formulate the following working hypotheses: (i), CaSR is a robust inducer of differentiation by virtue of its ability to activate and integrate diverse growth and differentiation control signals; (ii), loss of CaSR expression enable cellular escape from CaSR control and (iii), loss of CaSR expression is an underlying mechanism of malignant transformation, progression and drug resistance in colon cancer. Previous studies showed that there are endogenous small subpopulations that do not express CaSR in colon carcinoma cell lines. These cells are highly drug resistant. Indeed, immunocytochemical analyses of CaSR showed that the expression of CaSR in both the CBS and HCT116 colon carcinoma cell lines are heterogeneous. Human colon carcinoma cell lines contain small subpopulations (10-20%) that do not express CaSR (termed CaSR null cells). In order to further test my hypotheses, the isolation and characterization of CaSR null cells are required. Here, I report on the isolation, propagation, maintenance and characterization of CaSR null cells from the CBS and HCT116 human colon carcinoma cell lines. CaSR null cells grew as three-dimensional non-adherent spherical clusters with increased propensity for anchorage independent growth, cellular proliferation and invasion of matrigels. CaSR null cells were highly resistant to fluorouracil and expressed abundant amount of thymidylate synthase and survivin. Molecular profiling showed a high level of expression of the previously reported cancer stem cell markers CD133, CD44 and Nanog in CaSR null cells. A significant increase in the expression of epithelial-mesenchymal transitional (EMT) molecules and transcription factors was also observed. These include N-cadherin, β-catenin, vimentin, fibronectin, Snail1, Snail2, Twist and FOXC2. The expression of the tumor suppressive E-cadherin and miR145, on the other hand, was greatly reduced while the expression of oncogenic micro RNAs: miR21, miR135a and miR135b was significantly up-regulated. CaSR null cells possess a myriad of cellular and molecular features that drive and sustain the malignant phenotype. I conclude that CaSR null constitutes a highly malignant and drug resistant phenotype of colon cancer. I discovered that CaSR null cells, cultured in defined human embryonic stem cell culture medium, can be induced to differentiate and acquire CaSR expression when the medium of the null cells was changed to conventional cell culture medium containing fetal bovine serum. I hypothesize that expression of CaSR can alter the phenotype of the CaSR null cells. The objectives of this study were then three folds: (i), determine if induction of CaSR expression could circumvent the molecular phenotype of the CaSR null cells; (ii), determine if CaSR was required in altering the null phenotype and (iii), determine the underlying mechanism of CaSR induction. I hypothesize that if CaSR is a strong promoter of differentiation, then without CaSR, the constraint exerted by CaSR will not be functional and pathways normally inhibited by CaSR will be activated. I found that induction of CaSR expression led to a more indolent phenotype which includes the acquisition of epithelial morphology, down-regulated expression of cancer stem cell markers, down-regulated expression of thymidylate synthase and survivin and increased sensitivity to fluorouracil. Molecular profiling also revealed that the induction of CaSR expression was linked to a down-regulated expression of EMT molecules, EMT associated transcription factors and oncogenic miRNAs with a concurrent up-regulated expression of tumor-suppressive molecules. With the exception of the cancer stem cell markers, the reversal of molecular features, upon the induction of CaSR expression, was directly linked to the expression and function of CaSR because blocking CaSR induction by shRNA circumvented such reversal. I further report that demethylation of the CaSR gene promoter underlie CaSR induction. I conclude that induction of CaSR expression in CaSR null cells resulted in a more indolent phenotype concurrent with a variety of molecular changes and that these changes (with the exception of stem cell markers) are dependent on the expression and function of CaSR. I further conclude that methylation of the CaSR gene promoter is an underlying mechanism of maintaining the CaSR null phenotype while promoter demethylation is an underlying mechanism responsible for CaSR induction. CaSR null is a phenotype of the rapidly proliferating, undifferentiated crypt stem cells at the base of colonic crypts. Differentiation of crypt stem cells toward the apex of a crypt (in the direction of the lumen), on the other hand, is tightly linked to CaSR expression. What induces CaSR expression as the crypt stem cells migrate up the crypts is unknown. I hypothesize that as the colonic crypt stem cells migrate up the crypt, they become increasingly exposed to the colonic fluid in the lumen and components in the colonic fluid can trigger the induction of CaSR expression. Both Ca2+ and vitamin D are good candidates because either Ca2+ or vitamin D can stimulate CaSR expression in the parental CBS and HCT116 human colon carcinoma cells. Certainly, Ca2+ and vitamin D are not the only components involved in regulating CaSR expression. A variety of minerals in the colonic fluid may also serve as good candidates in the induction of CaSR. Of interest is Aquamin, a calcium-rich mineralized extract from the red marine algae, Lithothamnion calcareum, which has been shown to induce differentiation in colon carcinoma cells and possess chemopreventive properties against colon polyp formation in mice fed a high fat diet. CaSR null cells cultured in defined human embryonic stem cell culture medium were used to test this hypothesis because they offer an in vitro model in determining the triggers and the underlying mechanisms of CaSR induction that may resemble that of the colonic crypt stem cells in vivo. I found that all three agonists (Ca2+, vitamin D and Aquamin) induced CaSR mRNA and protein expression and inhibited cellular proliferation in the parental cells which express a heterogeneous mixture of cells with different level of CaSR expression. These agonists also induced CaSR mRNA and protein expression and inhibited cellular proliferation in the homogeneous isolated CaSR null cells. In both cases, Aquamin was found to be most potent in this regard. Induction of CaSR expression by these agonists in the CaSR null cells resulted in demethylation of the CaSR gene promoter with a concurrent increase in CaSR promoter reporter activity. Induction of CaSR expression resulted in a down-regulated expression of tumor inducers and up-regulated expression of tumor suppressors in the CaSR null cells. Again, Aquamin was found to be most potent in this regard. Taken together, I conclude that nutrients are good candidate in the induction of CaSR and differentiation in colonic epithelia cells. Similar to CaSR, transforming growth factor β (TGFβ) is also a robust promoter of differentiation in the colonic epithelium. The expression profile of both CaSR and TGFβ in the colonic epithelium is tightly linked to differentiation. Both CaSR and TGFβ expression progressively increases as the undifferentiated crypt stem cells migrate and differentiate toward the apex of a crypt in the direction of the lumen. Similar to the loss of CaSR in cancer cells, loss of TGFβ responsiveness has long been considered an underlying mechanism of early colon carcinogenesis. I hypothesize that there is functional linkage between CaSR and TGFβ function. Human colonic epithelial CBS cells originally developed from a differentiated human colon tumor, retain CaSR expression and function, TGFβ responsiveness and TGFβ receptor expression. Thus, these cells offer an opportunity to determine the functional linkage (if any) between CaSR and TGFβ. I found that knocking down CaSR expression in the CBS cells abrogated TGFβ-mediated cellular responses and attenuated the expression of TGFβ receptors. Ca2+ or vitamin D treatment induced CaSR expression with a concurrent up-regulation of TGFβ receptor expression. Ca2+ or vitamin D, however, did not induce CaSR in CaSR knocked down cells and without CaSR, there was no up-regulation of TGFβ receptor. I conclude that TGFβ receptor expression and TGFβ mediated responses requires CaSR expression and function. In summary, my research has revealed the important role of CaSR in controlling differentiation. CaSR also function as a robust tumor suppressor. My study clearly discerns the multifarious molecular signaling cascades involved in CaSR function and that methylation and demethylation regulates CaSR expression. My work has also established the importance of CaSR in the chemoprevention of colon cancer. My thoughts in regard to future studies and the potential role that CaSR could play in the management of colon cancer are given in the perspective section of this dissertation.

Background: Mortality from colorectal cancer (CRC) can be reduced drastically by early detection and early treatment. However, uptake of CRC screening is relatively low, about 50% for those whom the test is highly recommended. Objectives: We examined the influence of and racial differences in depression, insomnia, alcohol use, and tobacco use on CRC screening uptake in the US. Patients and Methods: Analysis of the 2012 National Health Information Survey data was conducted. Both weighted univariate and multiple logistic regression analyses were performed in SAS to estimate the odds ratios (ORs) and their 95% confidence intervals (CIs). A total of 21511 participants were included in the analysis. Results: Prevalence of CRC screening in the participants was 19%. Adjusting for all factors, insomnia (OR = 1.18, 95%CI = 1.06 - 1.32), moderate alcohol drinking (OR = 1.16, 95%CI = 1.01 - 1.30), past smoking (OR = 1.17, 95%CI = 1.04 - 1.32), depression (OR = 1.37, 95%CI = 1.18 - 1.58), African American (AA) race, and cancer history were positively associated with CRC screening. Females and Single were inversely associated with CRC screening prevalence. In stratified analysis by races (White and AA), depression was associated with CRC screening in both races. Marital status, smoking, cancer history and insomnia were associated with CRC screening in Whites only; while alcohol use was associated with CRC screening in AAs only. Conclusions: We have found significant associations between lifestyle factors (alcohol consumption and smoking) and mental health problems (depression and insomnia) and CRC screening uptake. To improve overall CRC screening uptake in the US, it is important to consider racial differences in predictors and tailor appropriate interventions to each racial/ethnic group.

Bone morphogenetic protein 2 (BMP2) is a secreted growth factor that is essential for proper embryonic development and proliferation. Our laboratory discovered a nuclear variant of BMP2 (nBMP2) which is produced when translation is initiated at an alternative start codon within the BMP2 gene. When translation occurs at the downstream start codon, the resulting protein lacks the ER signal peptide, thereby allowing cytoplasmic translation and nuclear localization. Our aim is to distinguish the role of this nuclear localized variant from secreted BMP2. Overexpression of nBMP2 in HEK293 and HT29 cell lines resulted in a higher percentage of cells in proliferative phases of the cell cycle. We determined that nBMP2 does not regulate cell cycle progression by inducing hyperphosphorylation of retinoblastoma protein (Rb), but it may regulate the cell cycle by interacting with ROC1. In order to examine the role of nBMP2 in vivo, we have generated a mouse model in which a mutation of the nuclear localization signal (NLS) disrupts nuclear localization of nBMP2. Aberrant crypts were more abundant in nBmp2NLStm azoxymethane (AOM) treated mice than in wild type mice. Furthermore, H&E staining of colonic tissue showed that mutant mice have increased levels of dysplasia and aberrant crypt foci. This work suggests that nBMP2 is involved in regulating cell cycle progression and proliferation, and therefore may play a role in tumorigenesis.

Medullary carcinoma (MC) of the colon is a rare and unique histologic subtype of colorectal cancer. It is commonly associated with deficient mismatch repair proteins and has a strong association with Lynch syndrome. Diagnosis is challenging as it does not have the usual immunohistochemical stains on pathology seen in colorectal adenocarcinoma. Here, we discuss an interesting case of MC of the colon that was metastatic on presentation and constituted a diagnostic challenge.

Over the past two decades, protein kinases have been intensively investigated as targets to treat neoplastic diseases. Many protein kinase inhibitors not only have therapeutic potential but are becoming invaluable reagents for the study of cell signaling. We aspired to use our Cyclin-Dependent Kinase 8 inhibitor, Q-12, as a probe for biomarker discovery for CDK8 inhibitor sensitive tumor types. Q-12 shows potent inhibition of cell viability and induction of apoptosis process in some triple-negative breast cancer and colorectal cancer cell lines in vitro. Western blot results indicate that the reduction of STAT1 phosphorylation could be a robust indicator of CDK8 target engagement in all three cancer cell lines used upon Q-12 treatment. Q-12 treatment of triple-negative breast cancer cell line (MDA-MB-468) decreases STAT1 phosphorylation but increases STAT3 phosphorylation. Q-12 activity in MDA-MB-468 cell is dependent on the activation of STAT3 phosphorylation. All results suggest that there may be a critical STAT1 to STAT3 ratio that may serve as a biomarker for CDK8 inhibitor sensitivity. In this precision medicine era, the discovery of biomarker is urgently needed to minimize the risks of severe side-effects by traditional chemotherapy and improve diagnosis and monitor therapy response across a wide spectrum of disease, especially heterogenous type of disease, like cancer.

Purpose: Developing new and efficient approaches for the early diagnosis of colorectal cancer (CRC) is an important issue. Circulating extracellular nanovesicles (ENVs) present a promising class of cancer markers. Cells of well-differentiated adenocarcinomas retain the molecular characteristics of colon epithelial cells, and the ENVs secreted by these cells may have colon-specific surface markers. We hypothesize that an increase in the number of ENVs carrying colon-specific markers could serve as a diagnostic criterion for colorectal cancer. Experimental design: Potential colon-specific markers were selected based on tissue-specific expression profile and cell surface membrane localization data. Plasma was collected from CRC patients (n = 48) and healthy donors (n = 50). The total population of ENVs was isolated with a two-phase polymer system. ENVs derived from colon epithelium cells were isolated using immune-beads with antibodies to colon-specific markers prior to labelling with antibodies against exosomal tetraspanins (CD63 and CD9) and quantification by flow cytometry. Results: The number of ENVs positive for single colon cancer markers was found to be significantly higher in the plasma of CRC patients compared with healthy donors. The efficacy of detection depends on the method of ENV labelling. The diagnostic efficacy was estimated by ROC analysis (the AUC varied between 0.71 and 0.79). The multiplexed isolation of colon-derived ENVs using immune-beads decorated with antibodies against five markers allowed for a further increase in the diagnostic potency of the method (AUC = 0.82). Conclusions: ENVs derived from colon epithelium may serve as markers of differentiated CRC (adenocarcinomas). The composition of ligands used for capturing colon-derived ENVs and their method of labelling are critical for the efficacy of this proposed diagnostic approach.