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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23811号 / 医博第4857号 / 新制||医||1058(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 小林 恭, 教授 万代 昌紀, 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

Black men have the highest burden of prostate cancer (PCa) compared to all other races. Early detection of PCa is controversial, thus preference based PCa testing is recommended. PCa testing intention can be used as a proxy for testing preferences. Intention is known to predict behavior; however there is a gap between testing intention and testing behavior. The aims of this study were to examine the PCa testing intention-prostate specific antigen (PSA) testing gap and identify social cognitive variables that moderate the gap. Two hundred and sixteen black men participated in this longitudinal study. Results indicated PCa testing intention was a positive but moderate predictor of three PSA testing outcomes, p<.05. Men who tested in accordance with their PCa testing intention (positive or negative) ranged from 52% to 58%. Men who intended to test but did not, were the group most responsible for the PCa intention-PSA testing gap. History of PCa testing had an independent main effect on medical claim of a PSA test between time one interview and one year after time one interview, p<.05. A significant knowledge of PCa testing controversy by PCa testing intention interaction effect on medical claim of a PSA test between time one and time two interview was found, p<.05. Men who do not know about the testing controversy are more likely to have a positive intention and fulfill their testing intention. Conversely, men who are aware and appreciate the controversy surrounding testing are more ambivalent about testing. Social cognitive variables were associated with PCa testing intention-PSA testing outcomes. These variables should be considered when designing interventions to help black men to manage their risk for PCa in a manner that is consistent with their testing preferences. / Public Health

The PPP2R2A gene encodes the B55α regulatory subunit of PP2A. Here we report that PPP2R2A is hemizygously lost in ~42% of prostate adenocarcinomas, correlating with reduced expression, poorer prognosis, and an increased incidence of hemizygous loss (>75%) in metastatic disease. Of note, PPP2R2A homozygous loss is less common (5%) and not increased at later tumor stages. Reduced expression of B55α is also seen in prostate tumor tissue and cell lines. Consistent with the possibility that complete loss of PPP2R2A is detrimental in prostate tumors, PPP2R2A deletion in cells with reduced but present B55α reduces cell proliferation by slowing progression through multiple phases in the cell cycle. Remarkably, B55α-low cells also appear addicted to lower B55α expression, as even moderate increases in B55α expression are toxic. Reconstitution of B55α expression in prostate cancer (PCa) cell lines with low B55α expression reduces proliferation, inhibits transformation and blocks xenograft tumorigenicity. Mechanistically, we show B55α reconstitution reduces phosphorylation of proteins essential for centrosomal maintenance, and induces centrosome collapse and chromosome segregation failure; a first reported link between B55α/PP2A and the vertebrate centrosome. These effects are dependent on a prolonged metaphase to anaphase checkpoint and are lethal to PCa cells addicted to low levels of B55α. Thus, we propose the reduction in B55α levels associated with hemizygous loss is necessary for centrosomal integrity in PCa cells, leading to selective lethality of B55α reconstitution. Such a vulnerability could be targeted therapeutically in the large pool of patients with hemizygous PPP2R2A deletions, using pharmacologic approaches that enhance PP2A/B55α activity. With that aim and considering the limitations of conventional 2D cell culture in mimicking the tumor environment and predicting drug responses in animal models and humans, we also established 3D organoid cultures of PCa cells and immortalized human prostate epithelial cells (hPrEC) in Matrigel. This allowed us to explore cell to extracellular matrix (ECM) interactions. PC3 cells initially form round organoids in Matrigel, followed by an invasive switch to where cell protrusions invade the surrounding ECM. Strikingly, B55α reconstitution, dramatically suppressed rupture of the basement lamina and ECM invasion, while proliferation appeared not affected. Tracking organoid growth at defined time points or using live imaging, shows that protrusions in PC3 organoids are very dynamic and resemble invadopodia. Interestingly, reconstitution of B55α in PC3 organoids just prior the invasive switch results in reduction of invasive leads and those protrusions that appear to initiate keep forming and collapsing, with most organoids remain round. Our previous phosphoproteomics data in 2D culture suggests that cell-to-ECM signaling is likely altered with B55α reconstitution, identifying potential B55α/PP2A substrates among key mediators of integrin signaling. In sum, reconstitution of B55α suppresses invasion in PC3 organoids, possibly by regulating potential B55α substrates in focal adhesion signaling, such as Paxillin and/or Talin. Alternatively, centrosomal defects due to dephosphorylation of B55α substrates (e.g. HAUS6, NEDD1) may cause microtubule defects that preclude invasion. Further studies are required to identify the mechanism. Moreover, because our studies presented above are based on prostate cancer cell lines with undefined genetic alterations, we have immortalized primary human PrEC by a novel approach to generate a cell model to study cooperation of PPP2R2A loss with step-wise introduction of specific oncogenes and/or tumor suppressor gene alterations in transformation, tumorigenicity and invasion. Our newly develop method combines expression of hTERT, which appears insufficient for immortalization of hPrEC with CDKN2A knockout, which we predicted will prevent stress-induced replicative senescence. We have obtained two independent immortalized clones (hPrEC-T-ΔN2A) using this method and confirmed their identity using PCR and western blot analyses. Although cytogenetic analysis showed these two clones are of mixed population with minor alterations in karyotype, 4 out of 11 cells examined in clone 1 appear completely normal. We also find that the clones exit the cell cycle upon contact inhibition and induce p53 expression when treated with flavopiridol, further supporting that hPrEC-T-ΔN2A clones exhibit the features of normal cells. Characterization in 3D culture reveals that the clones are likely of basal epithelial origin. Finally, soft agar and clonogenic assays show hPrEC-T-ΔN2A clones are highly proliferative but not transformed. We are using these cell models to dissect the role of PPP2R2A depletion in step-wise transformation of immortalized PrEC and hope to develop a defined 3D organoid system to study invasion, which could also be suitable for drug screens. Altogether our work has very significantly advanced our understanding of B55α in suppressing transformation in prostate cancer cells and has developed novel tools for further mechanistic characterization of PPP2R2A haploinsufficiency and the development of potential pharmacologic therapeutic agents. / Biomedical Sciences

Background: Prostate cancer remains the second most common cause of cancer-related death in men in the United States (Siegel et al. 2017). Observational studies of patients with prostate cancer have found associations between diet and prostate cancer severity, but the evidence is inconsistent or inconclusive. The purpose of this thesis is to implement a validated international healthy diet score and evaluate whether or not it is associated with prostate cancer severity. Objective: The objectives of this thesis were: Chapter 1: examine whether an association exists between diet quality, using the validated Healthy Diet Score, and the severity of prostate cancer, and Chapter 2: examine the agreement between two methods of dietary data collection (an abridged FFQ and a longer previously validated FFQ) with respect to macronutrients and main food groups. Methods: We used observational data from the Randomized Intervention for Cardiovascular and Lifestyle Risk Factors in Prostate Cancer Patients (RADICAL PC), a multi-centre Canadian prospective cohort study into which men with a new diagnosis of prostate cancer or who were being treated with androgen deprivation therapy were enrolled. To complete objective 1 (Chapter 1) of this dissertation, a cross-sectional analysis was completed using baseline data collected in the RADICAL PC study. In order to evaluate the association of diet with prostate cancer severity, the relationship between the Healthy Diet Score and prostate cancer severity (stage and grade) was assessed. The second objective (Chapter 2) is a comparability sub-study comparing an abridged FFQ with a long, validated FFQ in a subgroup of participant (N=130) enrolled in the RADICAL PC study. Results: Chapter 1: In the cross-sectional analysis of baseline data collected in RADICAL PC, a higher diet score was not significantly associated with prostate cancer severity. An association between age and the high-risk prostate cancer category was found to be statistically significant (OR: 1.04, 95%CI 1.02-1.05, p<0.00). Chapter 2: There was good agreement between the abridged FFQ and long FFQ for carbohydrates, proteins, whole wheat, refined grains, fish, dairy, potatoes, fruits, nuts, and soft drinks (Spearman rank correlation >0.5). Food groups including fried foods, processed meats, vegetables and total fats (nutrients) were found to have moderate correlation (Spearman rank correlation between 0.3-0.5). There was low correlation for legumes, sugars and oils. Bland-Altman plots showed good absolute agreements between the two methods, and reliability test using Spearman’s correlation showed moderate to good correlation (0.45 to 0.75 among most food groups. Conclusion: There was no clear association between a healthier diet and prostate cancer severity in men with newly diagnosed prostate cancer. There was adequate agreement between the abridged SFFQ and the long FFQ of the expected food groups, and thus the SFFQ can be considered an appropriate tool to use for measuring diet among prostate cancer patients for some food groups and nutrients. / Thesis / Master of Health Sciences (MSc)

Prostate cancer is the most commonly diagnosed disease and second most commonly caused death among men in America. Although much controversy surrounds the current methods of detection, PSA test and biopsy, no new methods have been approved as an effective method of detection. Biomarkers and non-invasive means of detection are being investigated everyday in hopes of discovering new information that could be of use in the prostate cancer field. One such non-invasive technology is the use of an electronic nose. The electronic nose technology has been utilized in the agricultural, food, biomedical, and environmental. The objective of this current study is to determine the effectiveness of the electronic nose to discriminate between prostate cancer cells (DU-145 and PC-3) and non-tumor forming cells from the urinary tract (SVHUC). Specific factors that will be investigated are incubation period and cell population. For all three cell lines, two cell populations of 75,000 and 150,000 cells were cultured and tested after 2, 8, 12, and 24 hours using a conducting polymer based hand-held electronic nose. Multivariate analysis was performed on the data and determined that the greatest discrimination between incubation periods was between 2 hours of incubation and the remaining periods of 8, 12, and 24 hour periods. This presents the idea that by 8 hours, ample volatiles were produced to be detected by the electronic nose. Additionally, when compared to one another, all three cell lines showed distinct differences. The cell lines most closely related were PC-3 and DU-145, the prostate cancer cell lines. However some variation was seen between these cell lines, which may be attributed to the presence of PSA in PC-3 cells or other factors affecting prostate cancer patients. Finally, PCA plots clearly illustrated that after 2 hours of incubation, sufficient volatiles were produced to allow the electronic nose to clearly discriminate the three cell lines from one another, demonstrating the importance of incubation period on successful discrimination. Based on the findings that the electronic nose was effective at discriminating the three cell lines, testing was completed to determine if cell population or cell maturity had the greatest effect on discrimination. The cell lines were cultured and tested immediately using an initial cell population of the highest cell population observed after a 72 hour incubation period. The results concluded that when the cell lines were tested immediately after culturing, the Cyranose was able to detect the individual cell lines in culture while also determining a range of detection for each cell line. The range of detection for DU-145 was found to be 26,200 to 262,000 cells based on interclass m-distances of 6.829-9.170 for cell populations lower than 26,200. A range of detection of 51,400 to 514,000 cells was concluded for PC-3 cells based on interclass m-distances of 5.690-7.400 for cell populations lower than 51,400. Finally, the results showed a range of detection of 19,000 to 190,000 cells for SVHUC based on interclass m-distances of 5.520-9.076 for cell populations lower than 19,000. However, when attempting to discriminate the three cell lines against one another immediately after culture, the electronic nose was unable to make clear distinctions between the three cell lines. When testing cancerous and non-cancerous cells, incubation period of the cells should be the only factor considered. It is evident that the cells need time to metabolize and produce volatiles so that the electronic nose can clearly distinguish these cells from one another in culture. / Master of Science