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DNA-damaging chemotherapeutics are a consistently employed for the treatment of cancer, and are regularly part of first-line combination therapeutic regimens. However, these regimens often display limited activity in cancers including of the lung and breast. Novel therapeutic strategies are urgently required. Understanding the molecular mechanisms regulating DNA-damaging chemotherapeutics activity will lead to such novel strategies. Activating transcription factor 3 (ATF3) is a stress inducible gene that plays a significant role in regulating cellular stress including DNA damage. This thesis investigates the role of ATF3 in mediating the cytotoxic effects of two commonly employed DNA-damaging chemotherapeutics, cisplatin and doxorubicin. This study also identifies other independent ATF3 inducing agents in potential novel combination therapeutic strategies. In this study, cell line models of cisplatin-resistance were generated independently from two non-small cell lung cancer (NSCLC) cell lines, Calu6 and H23. Full transcriptome RNA-sequencing analysis identified ATF3 as the most highly dysregulated apoptosis regulatory gene in the cisplatin-resistant compared to their respective parental cell lines following treatment with cisplatin. Further characterization identified cisplatin induced activation of JNK as the key regulator of ATF3 induction in these cell lines. Restoring JNK activity resulted in induced ATF3 expression and re-sensitization of the resistant cell lines to cisplatin treatment. FDA-approved 1200 compound library screens were employed to identify agents that can enhance cisplatin cytotoxicity as well as whose cytotoxicity was dependent on ATF3 expression. Vorinostat, an HDAC inhibitor, was identified in both screens and importantly displayed synergistic cytotoxicity in combination with cisplatin. In addition, ATF3 was also induced with treatments of the DNA damaging agent doxorubicin in these NSCLC cell lines including in the cisplatin resistant models. This work suggested a different mechanism of ATF3 induction by doxorubicin and the potential role of ATF3 in mediating doxorubicin cytotoxicity was further investigated in breast cancer cells. Doxorubicin robustly increased ATF3 expression in human breast cancer cell lines and tumor tissue ex-vivo. Loss of ATF3 in MEFs resulted in reduced sensitivity to doxorubicin treatment compared to wild-type MEFs. Employing the same library screen as above, compounds with ATF3 dependent mechanisms that could enhance doxorubicin cytotoxicity were identified. Vorinostat, as well as, the nucleoside analogues trifluridine and 6-mercaptopurine induced ATF3 expression and enhanced doxorubicin cytotoxicity. This study demonstrated that ATF3 plays an important role in mediating the cytotoxic effect of the DNA-damaging chemotherapeutics cisplatin and doxorubicin. It also provides rationale for ATF3 as a potential therapeutic target, and for the incorporation of ATF3 inducing agents in novel combination therapeutic strategies.

Ovarian cancer (OVCA) is the most lethal gynecological cancer with a 5-year survival rate less than 50%. Despite new therapeutic strategies, such as targeted therapies and immune checkpoint blockers (ICBs), tumor recurrence and drug resistance remain key obstacles in achieving long term therapeutic success. Therefore, there is an urgent need to understand the cellular and molecular mechanisms of immune dysregulation in chemoresistant ovarian cancer in order to harness the host’s immune system to improve cancer survival. Early diagnosis and residual disease are key determinants of favorable survival in OVCA; however, CA125 which is the conventional marker is not reliable and has modest diagnostic accuracy. There is therefore an urgent need to discover reliable biomarkers to optimize individualized treatment and diagnostic recommendations. Plasma gelsolin (pGSN; an actin binding protein) is the secreted isoform of the gelsolin (GSN) gene implicated in inflammatory disorders, colon cancer and prostate cancer. Increased expression of total GSN is associated with poor survival of patients with gynecological cancers. As to whether this is due to pGSN is yet to be investigated. Increased expression of pGSN is significantly associated with the down-regulation of immune cell markers; however, the exact mechanism has not been explored. If and how pGSN is involved in the cellular and molecular mechanisms of OVCA remains to be determined. In our current research, we have demonstrated that pGSN is involved in the regulation of immune cells, early diagnosis, tumor recurrence and chemoresistance in OVCA, using standard in vitro techniques and human clinical samples (North America, Asia and public datasets). We have shown that pGSN is highly expressed and secreted in chemoresistant OVCA cells than their chemosensitive counterparts. pGSN, secreted and transported via exosomes, upregulated HIF1α–mediated pGSN expression in chemoresistant OVCA cells in an autocrine manner as well as conferred cisplatin resistance in otherwise chemosensitive OVCA cells. pGSN also induced the OVCA expression of the antioxidant and tumor growth promoter, glutathione (GSH), by activating Nuclear factor erythroid 2-related factor 2 (NRF2), a response that attenuated cisplatin (CDDP)-induced apoptosis. In human tumor tissues, increased pGSN mRNA and protein expressions were significantly associated with advanced tumor stage, suboptimal residual disease, tumor recurrence, chemoresistance and poor survival regardless of patients’ ethnic background and histologic subtypes. Increased Infiltration of CD8+ T cells was significantly associated with favorable patient survival; however, increased pGSN hindered the survival impact of these infiltrated CD8+ T cells. Further investigation revealed that pGSN induced CD8+ T cell death via caspase-3 activation, an action that resulted in decreased IFNγ levels. Increased epithelial pGSN expression was significantly associated with reduced survival benefits of infiltrated M1 macrophages, through caspase-3-dependent apoptosis as well as reduced production of TNFα and iNOS. The clinical application of circulatory pGSN as a biomarker for early detection and patients’ survival was investigated. Pre-operative circulating pGSN presented as a favorable and independent biomarker for early disease detection and residual disease prediction compared with CA125. The test accuracy of pGSN was significantly enhanced when combined with CA125 in multianalyte index assay. The findings suggest that pGSN is a potential target for chemoresistant OVCA and presents as a diagnostic marker for early stage disease and surgical outcomes, interventions that could maximize the therapeutic success of immunotherapies.

Despite progress being made in our understanding of triple negative breast cancer (TNBC), the overall survival and disease-free survival for TNBC patients continues to be considerably poorer than their ER/PR/HER2+ counterparts. Metastasis and chemoresistance are the pivotal issues holding back the long-term success of TNBC treatments. In addition to the bulk tumor cells, cancer stem cells (CSCs) have emerged as important targets for alleviating TNBC progression and relapse. Cisplatin, a platinum based chemotherapeutic agent, has shown promising potential for the treatment of TNBC in clinical trials; however, cisplatin treatment is associated with tumor hypoxia that in turn promotes CSC enrichment and drug resistance. My work is to develop a combinational treatment to improve the long-term therapeutic potential of cisplatin that not only targeted the bulk TNBC population but also ALDHhigh and CD44+/CD24- CSC populations. Through clinical dataset analysis, I found that patient TNBC tumors expressed high levels of epidermal growth factor receptor (EGFR) and hypoxia genes. A similar expression pattern was demonstrated in cisplatin-resistant ovarian cancer. I therefore developed a combinational therapeutic to co-inhibit EGFR and hypoxia using metformin (an AMPK activator) and gefitinib (an EGFR inhibitor), which sensitized bulk TNBC cells to cisplatin and also led to the effective inhibition of both CD44+/CD24- and ALDHhigh CSCs. I obtained similar results by using clinically relevant TNBC patient samples ex vivo. Since these drugs are already frequently used in the clinic, this study illustrates a novel, clinically translatable therapeutic approach to improve the long-term therapeutic outcome of cisplatin for TNBC treatment.

Small Cell Lung Cancer (SCLC) is a highly aggressive neuroendocrine tumor with a 5% survival rate over 5 years. Though SCLC comprises 13% of all cases of lung cancer the median survival time of 14.5 months has seen little improvement over the last four decades. Standard treatment relies on DNA damaging agents such as Cisplatin/Etoposide (EP) which induce a high response rate of 60-70%. Despite this initial response, nearly all patients will relapse rendering first-line therapies ineffective. Furthermore, SCLC has been shown to develop chemotherapy cross-resistance in which resistance to first-line chemotherapies will confer resistance to additional DNA damaging agents thereby reducing treatment efficacy and duration of response. Cross-Resistance constitutes a major clinical issue whose underlying mechanisms remain a mystery. The modest improvements in SCLC patient outcomes over the decades may be partially explained by the existing systems of study. Current methodologies of SCLC study rely on cell lines, patient samples, and Genetically Engineered Mouse Models which have little functional correlation to clinical outcomes. While few sources have proposed Patient Derived Xenograft (PDX) systems as an improved alternative, significant data remains sparse. Without a robust model system which accurately recapitulates patient outcomes, molecular pathways driving resistance cannot be uncovered. Here we present the generation of 34 SCLC PDX models which maintain both genomic and functional fidelity. Furthermore, treatment of a 30-model subset with first-line chemotherapy EP and a novel chemotherapy Olaparib/Temozolomide (OT) allowed for functional and molecular comparison between groups. Our findings demonstrate incomplete independent resistance mechanisms between EP and OT treatment with a small overlap of 31 genes involved in glycolysis and xenobiotic metabolism.

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第24794号 / 医博第4986号 / 新制||医||1066(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 寺田 智祐, 教授 武藤 学, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Background: Mannitol has been used in the past for the prevention of cisplatin-induced nephrotoxicity. Studies on its efficacy have conflicting results. An educational newsletter was designed for local oncologists on the conflicting data of mannitol use in preventing cisplatin-induced nephrotoxicity. Purpose: The purpose of this study was to determine whether a pharmacist-created newsletter intervention led to changes in the mannitol prescribing practices of local oncologists. Methods: A newsletter describing the paucity of evidence to support mannitol use to prevent cisplatin-induced nephrotoxicity was distributed via e-mail to local oncologists in October 2010. Mannitol prescribing rates were retrospectively evaluated before and after newsletter distribution. The Mann-Whitney U test was used to compare nonparametric continuous data. The chi-square test was used for nominal data. Descriptive statistics were performed for baseline demographics, and odds ratios were calculated for possible risk factors for acute kidney injury (AKI). The primary endpoint was a change in mean mannitol dose before and after the newsletter intervention. The secondary endpoint was the difference in the rate of AKI before and after the intervention. Data were collected for 67 patients with various malignancies. Results: There was a difference in the average mannitol dose before and after newsletter intervention (P = .02). The rates of AKI before and after newsletter were similar. Conclusion: A pharmacist-led newsletter intervention was associated with significantly decreased rates of mannitol usage after intervention.

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