京都大学 / 新制・論文博士 / 博士(医学) / 乙第13427号 / 論医博第2231号 / 新制||医||1053(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 松田 道行, 教授 渡邊 直樹, 教授 川口 義弥 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Glioblastoma (GBM) is a deadly, malignant brain tumor with a poor long-term prognosis. The current median survival is approximately fifteen to seventeen months with the standard of care therapy which includes surgery, radiation, and chemotherapy. An important factor contributing to recurrence of GBM is high resistance of GBM cancer stem cells (CSCs), for which a systematically delivered single drug approach will be unlikely to produce a viable cure. Therefore, multi-drug therapies are needed. Currently, only temozolomide (TMZ), which is a DNA alkylator, affects overall survival in GBM patients. CSCs regenerate rapidly and over-express a methyl transferase which overrides the DNA-alkylating mechanism of TMZ, leading to drug resistance. Idasanutlin (RG7388, R05503781) is a potent, selective MDM2 antagonist that additively kills GBM CSCs when combined with diagnostics in a truly theranostic manner for enhancing personalized medicine against GBM. The goal of this thesis was to develop a multi-drug therapy using mutli-functional nanoparticles (NPs) that preferentially target the GBM CSC subpopulation and provide in vivo preclinical imaging capability. Polymer-micellar NPs composed of poly(styrene-<i>b</i>-ethylene oxide) (PS-<i>b</i>-PEO) and poly(lactic-<i>co</i>-glycolic) acid (PLGA) were developed investigating both single and double emulsion fabrication techniques as well as combinatinos of TMZ and RG7388. The NPs were covalently bound to a 15 base-pair CD133 aptamer in order to target a specific epitope on the CD133 antigen expressed on the surface of GBM CSC subpopulation. For theranostic functionality, the NPs were also labelled with a positron emission tomography (PET) radiotracer, zirconium-89 (⁸⁹Zr). The NPs maintained a small size of less than 100 nm, a relatively neutral charge and exhibited the ability to produce a cytotoxic effect on CSCs. There was a slight increase in killing with the aptamer-bound NPs compared to those without a targeting agent. This work has provided a potentially therapeutic option for GBM specific for CSC targeting and future in vivo biodistribution

The enhanced expression of histone deacetylases (HDACs) in a variety of malignancies drew attention to investigate a new category of anti-cancer drugs that are based on the inhibition of those enzymes. Valproic acid (VPA) is a well-known antiepileptic drug that exhibits antitumor activities through inhibition of HDACs class I and IIa. Cancer stem cells (CSCs) have been recognized to drive the tumor growth and progression hence; attention has been given to target this small subpopulation of CSCs rather than the whole bulk tumor cells. CD133 is considered to be a CSC marker in several tumors and its transcription is strongly influenced by epigenetic changes that will be altered upon administration of histone deacetylase inhibitors (HDACi) in cancer treatment. Therefore, we evaluated the epigenetic and cytotoxic effects of treatment with 1 mM VPA in combination with other chemotherapeutics and its influence on the expression of CD133 in human neuroblastoma (NB) cell lines. Our results revealed that addition of VPA to DNA-damaging chemotherapeutics induced a synergistic anti-tumor effect that was associated with caspase-3 dependent induction of apoptosis in UKF-NB-4 cells. This synergism was related to the increase of the acetylation status of histones H3 and H4 and was only produced either by...

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.

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第24136号 / 医博第4876号 / 新制||医||1060(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 藤田 恭之, 教授 波多野 悦朗, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Colorectal cancer (CRC) is one of the most prevalent cancers worldwide and is recognized as the second leading cause of cancer-related deaths in North America. CRC follows a hierarchal tumor organization, the root being a small population of self-renewing and highly tumorigenic colon cancer stem cells (CSC). There is an epigenetic signature that exists within these colon CSCs contributing to their maintenance and dynamic plasticity. A key hallmark of this colon CSC epigenetic signature is the Histone-3 Lysine-9 di-methylation (H3K9me2) histone mark which is overrepresented in many types of cancer. Pharmacological modulation of this epigenetic signature (i.e through H3K9me2 modulation) serves as a novel way to selectively target and eliminate human CSCs while sparing normal progenitor cells. Direct inhibitors of key methyltransferases such as G9a, have been identified to have high specificity, however none of these inhibitors have shown success during early stages of clinical trials, leaving us to question the clinical relevance. My research has shown that the overexpression of histone methyltransferase G9a in colon cancer serves as a risk factor for patients and is associated with shorter-relapse free survival. G9a activity has been shown to be essential for the maintenance of embryonic-like transcriptional signature which promotes self-renewal, tumorigenicity and an undifferentiated state. This work provides insights into the role of G9a as a driver of a cancer stem cell phenotype. To combat the toxicity and issues associated with targeting the catalytic activity of G9a, I utilized a phenotypic screening pipeline consisting of thousands of clinically-approved compounds, and identified CSC-bioactive epigenetic inhibitors showing promise in CSC-like models. RNA-seq profiling, dose response treatments and molecular techniques were used to confirm the selectivity of these candidates to colon-CSC like cells with minimal impact on normal progenitors. The lead candidate compound, vanoxerine (VXN) restricts organoid-initiating capacity of patient derived colon CSCs in serial 3D organoid formation assays that I developed throughout my research project. Using two murine syngeneic models resembling microsatellite instability and stability in CRC, I found this compound to be successful in decreasing primary tumor volumes compared to vehicle control mice, through epigenetic reprogramming and infiltration of immune cells. Drug treated tumors that were harvested, dissociated and re-injected into secondary mice showed diminished tumor initiating capacity compared to vehicle controls. Furthermore, the target of vanoxerine, SLC6A3, was investigated and the expression pattern characterized revealed a new potential biomarker for colon cancer stem cells. This SLC6A3-G9a axis discovered for the first time in colon cancer and serves as a novel and important pathway to block H3K9me2 deposition in CRC, rewiring the CSC epigenome and suppressing neoplastic self-renewal and tumor-initiating functions. Together, the identified repurposed compound selectively targets and modulates the epigenetic signature of CSCs which diminishes the tumor initiating function of these cells. This hints at an interesting interaction between CRC stemness and tumor immunology, a promising future therapeutic avenue.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24499号 / 医博第4941号 / 新制||医||1064(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 伊藤 貴浩, 教授 中島 貴子, 教授 藤田 恭之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM