The native polyamines putrescine, spermidine, and spermine are small positively-charged molecules that can interact with negatively-charged macromolecules like DNA, RNA and proteins. These interactions aid in nucleic acid and protein conformational stability, thereby, making polyamines essential building blocks for cells. Polyamines are involved in various cellular functions including gene regulation, protein synthesis, and cell proliferation. Rapidly-proliferating cells, such as cancer cells, utilize high polyamine levels for cell growth. Targeting the polyamine addiction of cancers is a validated anti-cancer strategy. To achieve the maximal reduction of polyamine levels, polyamine blocking therapy (PBT) employs several compounds in combination. First, one can inhibit polyamine biosynthesis using difluoromethylornithine (DFMO). DFMO inhibits ornithine decarboxylase (ODC); a rate limiting enzyme required for the generation of putrescine. Unfortunately, inhibition of polyamine biosynthesis is often insufficient because cells escape DFMO pressure by upregulating polyamine import. To address this escape pathway, a polyamine transport inhibitor (PTI) is included with DFMO to reduce cell growth via PBT. There are two types of PTIs: polyamine-based and non-polyamine-based PTIs. Chapter 1 provides an overview of the field. In Chapter 2, we developed a novel polyamine-based PTI which is smaller and less toxic than the known PTIs and was equally potent when used in PBT. In Chapter 3, we identified the first non-polyamine-based, PTI (GW5074) that successfully reduced human cancer cell growth when dosed with DFMO. In Chapter 4, we discovered new DFMO adjunct agents which inhibit the far upstream element binding protein 1 (FUBP1). This type of PBT significantly reduced cancer cell growth and was directed at polyamine biosynthesis via a two-fold mechanism involving direct inhibition of polyamine biosynthesis (DFMO) and downregulation of upstream transcription factors like c-myc. In summary, we have described three categories of compounds that can be used as adjunctive agents for DFMO in anti-cancer therapies.

Statistics on breast cancer tell a sobering tale. According to the American Cancer Society (1978), breast cancer is found in some 90,000 women in America each year: approximately one out of thirteen women. The majority of breast cancers are discovered by women themselves. It is currently the leading cause of death among women aged 40 to 44, and kills some 43,000 women annually. The risk of breast cancer increases with age; about 75% of all breast carcinoma occurs in women aged 50 or above.

Neuroblastoma is an aggressive pediatric cancer that is poorly responsive to traditional cancer therapies. Oncolytic viral (OV) vectors such as Zika virus (ZIKV) and Parainfluenza virus type 5 (P/V virus) are promising neuroblastoma therapeutics, but the role of innate immune responses in the effectiveness of OV killing is not well understood. Previous studies showed the neuroblastoma cell line SK-N-AS (expressing low CD24) had low permissivity for ZIKV infection, and this restriction was relieved by ectopic CD24 expression (CD24-high cells). Compared to permissive CD24-high cells, the non-permissive CD24-low cells had elevated basal levels of IRF-1, NF?B and phosphorylated STAT1; these cells also showed higher levels of interferon (IFN)-induced antiviral genes and activity against IFN- sensitive viruses (e.g., VSV). Media-transfer experiments showed that the inherent antiviral state of CD24-low cells was not dependent on a secreted factor. Transcriptomics analysis revealed that CD24 expression suppressed basal expression of antiviral genes. These data support the proposal that CD24 alters basal and induced antiviral states and may represent a novel biomarker for susceptibility of cells to OV infection. We extended these studies to test the hypothesis that epigenetic modulators could alter the antiviral state of neuroblastoma cells to enhance OV killing. Pre-treatment with the DNA methyltransferase inhibitor 5-Azacytidine enhanced P/V virus-mediated death of SK-N-AS cells in a dose- and MOI-dependent manner. While killing of SK-N-AS cells by the P/V virus alone was minimally caspase-dependent, combined treatment with 5-Azacytidine and P/V virus shifted cell killing to a largely caspase-dependent mechanism. 5-Azacytidine pre-treatment of SK-N-AS cells dampened P/V virus growth, which correlated with higher expression of antiviral mediators such as interferon-beta and OAS2. These studies demonstrate that antiviral responses mediated by a host factor (CD24) and by epigenetic modulators (5-Azacytidine) can be exploited to enhance tumor cell killing by RNA OVs.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24507号 / 医博第4949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 村川 泰裕, 教授 金子 新, 教授 永井 純正 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Metastasis and recurrent tumors caused by drug resistance are the main reasons for breast-cancer associated deaths. The mechanisms are still not fully understood. In this study, we address novel mechanisms underlying which localized cancer metastasis turns wildly aggressive to multiple organs during anticancer treatment. We show that Krüppel-like factor 8 (KLF8) expression can be over-induced by chemotherapeutic drugs. Data from large-cohorts of patients shows that after chemotherapy there is a close correlation between the aberrant high levels of KLF8 and C-X-C chemokine receptor type 4 (CXCR4) with drug resistance, metastasis, and poor prognosis. To mimic the drug-induced upregulation, KLF8 or CXCR4 was overexpressed in the lung-specific metastatic cell line LM2. We unexpectedly found that KLF8 or CXCR4 overexpressing cells metastasized extensively to multiple organs, particularly the C-X-C motif chemokine ligand 12 (CXCL12)-rich organs. We found that KLF8 or CXCR4 overexpression enhanced angiogenesis involving increased expression and secretion of vascular endothelial growth factor (VEGF) protein. We also found that KLF8 or CXCR4 overexpression strongly enhanced the formation of filopodia-like protrusions (FLPs) and proliferation. In addition, we opened a new avenue for developing an effective anticancer therapy against Herceptin-resistance, which is a major problem during the treatment of human epidermal growth factor receptor 2 (HER2) positive breast cancer patients. Hypoxia-inducible factor 1 subunit alpha (HIF-1α) plays a critical role in drug-resistance. However, no active drug that can selectively target HIF-1α is available. Cerium oxide nanoparticles (CNPs) have recently emerged as anticancer agents with minimal side effects on normal tissues. We proved that CNPs showed a strong ability to sensitize Herceptin-resistant breast cancer cells to the cytotoxicity of Herceptin. Herceptin combined with CNPs was shown to effectively eliminate Herceptin-resistant breast cancer cells via HIF-1α and VEGF signaling suppression. We suggest great potential for their combination to overcome drug resistance, metastasis, and tumor relapse.

Cancer remains one of the most common causes of mortality around the world and continues to evade standardization of diagnostic and therapeutic guidelines. As a multifaceted and heterogenous disease, development of standard screening options and monitoring cancer progression in patients is a challenge. In this study, we hypothesized that this challenge could be addressed by a biomarker that plays an essential and central role in oncogenesis across multiple cancer types. Previous studies from our labs and others have shown that Chaperonin Containing TCP-1 (CCT) is present in multiple cancers and plays an important role in oncogenesis as a protein folding complex of many oncogenes. Increased CCT levels in tissues correlate with increased cancer stage and severity, while inversely correlating with patient outcomes. For this project, we investigated the potential of the second subunit, CCT2, as a biomarker for adult cancers and pediatric cancers including breast cancer, lung cancer, and neuroblastoma. While most cancers have high levels of CCT, we used bioinformatics and histological studies to build a foundation for which cancers would benefit the most from our studies and were available for testing the feasibility of CCT2 as a biomarker. Since CCT2 has been studied in adult cancers previously, we first used breast cancer cells spiked in blood to adjust CCT2 as a biomarker for liquid biopsy. Once we determined a range of use for CCT2 in liquid biopsy, we tested it in lung cancer patients. Both showed increased recovery and promising circulating cells that were cytokeratin negative and CCT2 positive. To determine if CCT2 would also work in cancers with very different origins, we looked at pediatric cancers. We used neuroblastoma as our test model to determine if the CCT complex had a similar significance and role in pediatric cancers as in adult cancers. We completed overexpression and knockdown studies which showed results that were congruent with what CCT's role in adult cancer has been. We therefore tested sensitivity to CCT drug inhibition and sensitivity in liquid biopsy detection and determined that CCT is vital for tumorigenesis in neuroblastoma. These results show that CCT has the unique potential as a novel biomarker for cancer detection and that the second subunit could be used as a readout for the presence of the entire complex.

Pancreatic ductal adenocarcinoma (PDAC) is highly chemo-resistant and has a five year survival rate of < 8%. Risk factors of pancreatic cancer, such as chronic pancreatitis, help to elicit a pro-tumor immune response, and highly fibrotic environment that promotes tumorigenesis. To study how chronic pancreatitis promotes cancer initiation, traditional KRasG12D mice and double mutant Akt1Myr/KrasG12D mice were used to model microenvironment changes. Akt1Myr/KrasG12D mice were more susceptible to chronic tissue damage, accelerated tumor development and metastatic disease. These mice exhibited histological changes consistent with immune cell privilege, where M2 macrophages and non-cytotoxic eosinophils were co-localized with fibrotic regions. IL-5 expression was up regulated in pancreatic cells undergoing acinar to ductal metaplasia and then diminished in advanced lesions. Tumor cells treated with IL-5 exhibit increased migration and activation through STAT5 signaling. Collectively, the results suggest that eosinophils, which are responsive to IL-5, are key mediators in the pancreatic environment subjected to chronic inflammation and injury. Current therapeutics fall short in increasing patient survival. There remains an urgent need for innovative treatments and thus we tested difluoromethylornithine (DFMO) in combination with a novel polyamine transport inhibitor, Trimer44NMe, against Gemcitabine-resistant PDAC cells. Prior clinical failures when targeting polyamine biosynthesis with DFMO monotherapy may be due to tumor escape via an undefined polyamine transport system. In pancreatic tumor cells DFMO alone and with Trimer44NMe significantly reduced PDAC cell viability by inducing apoptosis or cell cycle arrest. In vivo orthotopic PDAC growth with DFMO treatment resulted in decreased c-Myc expression, a readout of polyamine pathway dysfunction. Moreover, dual inhibition significantly prolonged survival of tumor-bearing mice, and increased M1 macrophage infiltration and reduced FoxP3 expression. Collectively, these studies demonstrate that targeting polyamine pathways in PDAC is a promising immunomodulating therapy that increases survival.

Cervical cancer is the leading cause of cancer related deaths and the second most common cancer amongst South African women. The key cause for cervical cancer development is sexual transmission and persistent infection with high-risk Human Papillomavirus (HPV). However, it takes several years from infection to cervical cancer development, suggesting that other factors contribute to the disease. Exposure of neoplastic epithelial cells to Seminal Fluid (SF) has been shown to promote cell proliferation in culture and growth of explants in mice injected with HeLa cervical adenocarcinoma cells. Since the majority of cervical cancer cases are squamous cell carcinoma, in this study, we examined the effect of SF on cancer cell proliferation, EMT, motility and gene expression using two squamous cell carcinoma cell line model systems, SiHa and Me180. This study shows that SF significantly enhanced cell proliferation in both cell lines. Using confocal microscopy and phalloidin staining, it was further shown that SF caused morphological changes and induced stress fibre formation. SF upregulated the expression of EMT transcription factors Snail, Twist and ZEB1. EMT induction was confirmed by the increase of N-cadherin and a decrease in E-cadherin protein expression. Additionally, results showed that the induction of EMT transcription factors Snail, Twist and ZEB1 by SF occurs via EP4 receptor, ERK1/2 and COX signaling pathways. To investigate the effect of SF on migration and invasion, transwell migration assays were used. SF significantly enhanced directional cell migration and invasion of SiHa and Me180 cells. Cell invasion was associated with an increase in MMP-2 and MMP-9. SF also induced proinflammatory and angiogenic gene expression in cervical squamous carcinoma cells. SF mediated induction of inflammatory and angiogenic genes was shown to be associated with AP-1 and NFkB transcription factors. A small molecule inhibitor of nuclear import, INI-43 inhibited the nuclear localization and activity of SF activated NF-kB as well as the expression of SF induced inflammatory and angiogenic genes. Employing ectocervical tissue biopsies, SF caused the upregulation of EMT transcription factors, MMPs, inflammatory and angiogenic genes. Taken together, these results suggest that SF may play a role in promoting EMT and enhances the migratory and invasive potential of cervical squamous cell carcinoma. These findings together implicate SF as a possible factor that may promote cervical cancer progression.

According to the Global Cancer Statistics 2020, prostate cancer (PCa) is the second most commonly diagnosed male cancer and second leading cause of cancer death among men globally. Prostate cancer is known to be more aggressive among men of African origin with reasons not fully known. Previous studies have revealed PCa to be of a serious disease burden among African populations with PCa being the major cause of male cancer mortality. Prostate specific antigen (PSA) has long been introduced as a biomarker for screening in PCa diagnosis. However, serum PSA has low sensitivity for PCa diagnosis which has led to serious harm such as overdiagnosis and other complications of treatment for indolent disease. This makes it imperative to search for other novel biomarkers with high sensitivity and specificity for early diagnosis and management of prostate cancer. This study was aimed to characterize plasma and urinary cfDNA and tumour exosome cargo as diagnostic biomarker for PCa in South African populations with the goal of discovery of reliable, non-invasive, and novel biomarkers of PCa. We performed miRNA sequencing of exosomal RNA extracted from high and low Gleason's score PCa plasma samples. We performed differential expression (DE) of TCGA data and exosomal miRNA data and which we identified 185 miRNA and 65 miRNAs respectively. A comparison of the differential expressed TCGA miRNA and exosomal miRNA showed 13 miRNAs common between the two data with 7 of the 13 miRNAs expressed in the same direction. We further validated the expression of the 7 miRNAs using real time PCR in exosomal miRNA of high and low Gleason's score PCa samples and benign prostatic hyperplasia (BPH). We also performed whole exome sequencing of urinary cell free DNA and we identified 31 mutated genes. We reported for the first time an association between 27 of these genes and PCa in African populations. Four of the genes have earlier been identified as promising biomarker for prostate cancer diagnosis among African men. We also performed real time PCR quantification of cell free DNA to determine the concentration and DNA integrity of cfDNA in PCa and BPH of plasma and urine samples and which we were able to identify significantly higher plasma cfDNA level in PCa than BPH samples. We identified herein putative diagnostic biomarkers in plasma and urinary cfDNA and exosomes cargo for diagnosis of PCa in South African populations.