Yuzhuo Wang

Prostate cancer is the most commonly diagnosed cancer and the leading cause of cancer-related deaths in North American men. Androgen-deprivation therapy (ADT) is the standard treatment for patients with either locally-advanced or metastatic prostate cancer (PCa). While most PCa patients initially respond to androgen ablation, progression to recurrent castration-resistant prostate cancer (CRPC) commonly occur. Current therapies for CRPC, e.g. next-generation ADT including Enzalutamide and Abiraterone, can extend patients’ lives but are not curative as resistance to their use eventually emerges. As such, there is an urgent need to identify hitherto unrecognized but critical molecular mechanisms driving CRPC, which may in turn lead to novel treatments that can be used in combination with ADT for more effective therapy. In this doctoral dissertation, we examined the transcriptome profiling data of hormone-naïve prostate cancer (HNPC) patient-derived xenografts (PDXs) for gene expression changes at various time points after castration. Particular attention was given to expression changes that appeared early during CRPC development, indicative of genes acting as potential CRPC drivers. Eighty genes were found to be significantly upregulated at the CRPC stage, while 7 of them also showed elevated expression before CRPC development. Among the latter, Growth Factor Receptor Bound Protein 10 (GRB10), was the most significantly and consistently upregulated gene. Moreover, we found that GRB10 expression was elevated in clinical CRPC compared to HNPC in several clinical cohorts. Functionally, we found that GRB10 knockdown markedly reduced prostate cancer cell proliferation and AKT activity. Further investigation suggests that GRB10 is transcriptionally regulated by androgen receptor through an androgen responsive element located in GRB10’s intron. Mechanistically, through unbiased immunoprecipitation-mass spectrometry (IP-MS), we demonstrated that GRB10 could directly bind to and reduce the expression of protein phosphatase 2A (PP2A), a well-established tumor suppressor. These data suggest that GRB10 plays an important role in CRPC development and progression. This research demonstrates the functional roles of GRB10 in CRPC development and defines its molecular mechanisms driving ADT resistance. This study improves our understanding of the mechanisms underlying prostate cancer progression, paving the road for developing therapeutic agents that would improve the efficacy of current CRPC treatments.

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Metastatic prostate cancer (mPCa) is currently incurable. Docetaxel-based chemotherapy, used as first-line treatment for advanced PCa, is marginally effective. As PCa is a heterogeneous disease, use of therapeutics targeting multiple pathways may improve its treatment outcome. Aneustat is first-of-a-class of multivalent immuno-oncology drug candidates; a Phase-I trial has shown it is well-tolerated by patients and has immunomodulatory activity. The main goal of this PhD project is to determine whether Aneustat can be used to improve docetaxel-based therapy of advanced PCa. In vitro, Aneustat markedly inhibited human metastatic C4-2 PCa cell proliferation/migration in a dose-dependent manner and, combined with docetaxel, showed synergistic growth inhibition. In vivo, a combination of Aneustat and docetaxel synergistically enhanced anticancer activity in a clinically relevant, patient-derived xenograft (PDX) metastatic PCa model without inducing major host toxicity (inhibition of tumor growth, lung micro-metastasis, kidney invasion). Gene expression analysis of microarray data obtained from xenografts, using Ingenuity Pathway Analysis (IPA) and Oncomine software, indicated that Aneustat+docetaxel, as distinct from the single drugs, targeted multiple pathways and cancer-driving genes. Aneustat alone significantly inhibited growth of human LNCaP cells/xenografts; glucose consumption, lactic acid secretion and glycolysis-related gene expressions of LNCaP cells were markedly reduced, indicating it inhibited aerobic glycolysis. Treatment of LNCaP xenografts and first-generation PCa PDX with Aneustat led to marked changes in host immune cell levels (mouse/human), i.e. a higher ratio of CD8⁺T/Treg cells, higher Natural Killer (NK) cell numbers, lower Treg cell and MDSC numbers – changes favoring the host anticancer immune response. This study shows that combined use of Aneustat and docetaxel can lead to marked, synergistically increased anticancer activity, both in vitro and in vivo. As indicated by IPA and Oncomine analyses, this is due to the combination-induced expansion of the targeting of pathways and cancer-driving genes. Furthermore, as found with first-generation PDX PCa model, Aneustat has immunomodulatory properties, likely stemming from its inhibition of aerobic glycolysis, that may lead to stimulation of the anticancer immune response in immunocompetent hosts. Since a clinically relevant PDX metastatic PCa model was used in this study, treatment with Aneustat+docetaxel is likely valuable for clinical management of advanced PCa.

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Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer in North American males and a leading cause of cancer deaths. The lack of effective treatment options for advanced PCa such as AR-positive castration-resistant PCa (CRPC-AD) and the highly aggressive AR-negative CRPC, e.g. neuroendocrine PCa (CRPC-NE) presents a critical, unmet need for the development of novel therapeutics. Altered metabolism in the form of elevated aerobic glycolysis is a common cancer characteristic. Here we propose a novel conceptual understanding for the central, functional role of excessive cancer-generated lactic acid. In particular, the acidification of the tumor microenvironment via increased MCT4-mediated lactic acid secretion can facilitate multiple crucial cancer-promoting processes, including proliferation, tissue invasion/metastasis, angiogenesis, and suppression of local anticancer immunity. As such, the inhibition of MCT4 could be an effective therapeutic strategy broadly impacting multiple downstream lactate-associated tumour-promoting processes. Experimentally, we were able to confirm the clinical relevance of elevated glycolysis and increased lactic acid production in various advanced PCa patient-derived xenograft (PDX) models and patient tumours using a novel metabolic pathway score. In particular, NEPC tumours appear to rely much more heavily on elevated aerobic glycolysis and MCT4-mediated lactic acid secretion. In a proof-of-concept study using MCT4-specific antisense oligonucleotides (ASOs), reduced MCT4 expression is able to reduce proliferation, invasion/migration, and glucose metabolism of advanced PCa cells in vitro. More importantly, we demonstrated in two distinct in vivo models containing residual functional immune cells that MCT4 inhibition enhanced anticancer immunity. Finally, a state-of-the-art in silico drug discovery pipeline was employed in the first steps towards developing a potent and specific MCT4 small molecule inhibitor. Computer modeling of MCT4 structure, virtual molecular docking, and downstream experimental validation identified a promising hit series based on the chemical scaffold of VPC-25009 as a potential second therapeutic modality for MCT4 inhibition. Taken together, we were able to provide experimental support for our novel hypothesis regarding the central tumour-promoting and immunosuppressive role of cancer-generated lactic acid. A therapeutic approach blocking lactic acid secretion by targeting MCT4 function could thus inhibit multiple downstream lactate-associated processes for effective treatment of advanced PCa and other highly glycolytic cancers.

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The lack of effective therapy for advanced prostate cancer (PCa) remains a major unmet clinical need. Recently approved therapeutics, such as enzalutamide (ENZ), have only delayed the inevitable progression of castration-resistant PCa (CRPC), as resistance will typically emerge following treatment. Although increased apoptosis-resisting ability of cancer cells represents a fundamental mechanism for the onset of treatment resistance, no relevant agents have yet been developed. Preliminary work in our laboratory has revealed an association between elevated expression of BIRC6, an Inhibitor of Apoptosis (IAP) protein, and advanced PCa. The overall objective of this doctoral study is to investigate the roles of BIRC6 in advanced PCa, and to assess the therapeutic efficacy of a novel anti-BIRC6 agent. Firstly, I evaluated the clinical relevance of BIRC6 using patients’ PCa specimens, and the functional importance of BIRC6 using cell line-based PCa models. A significant correlation was found between elevated BIRC6 protein expression in clinical PCa and poor patient prognostic factors. Functional assays validated the importance of BIRC6 in PCa cell proliferation and apoptosis suppression. Next, I designed BIRC6-based, dual IAP-targeting antisense oligonucleotides (dASOs) to inhibit BIRC6 and an additional IAP. Two dASOs, 6w2 and 6w5 targeting BIRC6+cIAP1 and BIRC6+survivin, showed substantial inhibition of CRPC cell proliferation in vitro and in vivo. Functional studies showed that both dASOs significantly induced apoptosis, cell cycle arrest and suppression of NFκB activation in CRPC cells. Finally, I assessed the growth-inhibitory efficacy of dASO-6w2 in ENZ-resistant CRPC, which has become an increasingly prominent problem in the clinic. The efficacy of dASO-6w2 was studied using both ENZ-resistant PCa cell lines and a clinically relevant, transplantable patient-derived xenograft PCa tissue model, designated LTL-313BR, which exhibits primary ENZ resistance. Importantly, I showed that treatment with dASO-6w2 markedly suppressed the growth of LTL-313BR xenografts. The dASO-6w2 was also found to increase tumour apoptosis and inhibit the expression of several pro-survival genes that were up-regulated in the LTL-313BR line. In conclusion, this doctoral study has established the clinical relevance and functional importance of BIRC6 in advanced PCa, and has also presented new BIRC6-targeting agents that markedly suppress the growth of advanced PCa.

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Metastatic prostate cancer is currently incurable. Metastasis is thought to result from changes in the expression of specific metastasis-driving genes, leading to a cascade of activated downstream genes setting the metastatic process in motion. As such, metastasis-driving genes could provide effective therapeutic targets and prognostic biomarkers for improved disease management. In search of potential metastasis-driving genes, genes with elevated expression in patient-derived metastatic LTL-313H prostate cancer tissues, as distinct from non-metastatic LTL-313B tissues, were identified. Among these genes, TIMELESS and DLX1 were promising. Unfortunately, their silencing and overexpression in prostate cancer cells did not lead to inhibition of metastatic properties, indicating that they were not metastasis-driving genes. A different, novel approach was used based on the notion that metastasis-driving genes can activate genes in an amplification cascade fashion. Accordingly, I used the IPA’s Upstream Regulator Analysis tool to analyze the differential gene expression profile of the metastatic and non-metastatic tissues to predict the upstream master regulatory (metastasis-driving) genes accountable for the differential expression. Six candidate genes were identified, including GATA2, a pioneer factor-encoding gene. Elevated GATA2 expression in clinical metastatic prostate cancer specimens correlated with poor patient prognosis. Furthermore, GATA2 gene silencing in human prostate cancer LNCaP cells led to marked reduction in cell proliferation, cell migration, tissue invasion, focal adhesion disassembly and a dramatic change in transcriptional activity, indicating that GATA2 plays a critical role in prostate cancer metastasis. As such, GATA2 could represent a metastasis-driving gene and a potential therapeutic target for inhibiting the growth and metastasis development in prostate cancer. Further analysis of GATA2-regulated genes led to the development of a GATA2-based metastatic gene signature. Its prognostic value was confirmed using two prostate cancer patient cohorts. In addition, it was shown to be a prognostic factor for risk assessment of metastasis development, independent of the widely used D’Amico prognostic classification system. However, a thorough validation is critical and, if successful, the GATA2-based gene signature could lead to a paradigm shift in the management of early prostate cancer. In conclusion, the findings of this study appear to be potentially useful for improved management of metastatic prostate cancer.

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Genistein is an isoflavone found in soy, and its chemotherapeutic effects have been well established from in vitro studies. Recently, however, its therapeutic actions in vivo have been questioned due to contradictory reports from animal studies, which rely on rodent models or implantation of cell lines into animals. Using patient-derived prostate cancer xenograft models, in which clinical prostatectomy samples were grafted into immune deficient mice, this study showed that genistein promoted metastatic progression in vivo. To test if the metatstasis-promoting effects of genistein may be mediated via ERβ activation, we treated the xenografted mice with genistein, an anti-estrogen compound (i.e. ICI 182 780) or a combination of both. The results showed that anti-estrogen treatment significantly decreased metastatic spread compared to genistein, which promoted lung metastasis in a dose-dependent manner. Gene expression analyses showed that genistein and anti-estrogen treatments targeted the same signaling pathway but different molecules, producing opposite effects on tumour biology. Genistein stimulated expression of upstream molecules that reside in the Focal Adhesion Kinase (FAK) pathway, while anti-estrogen down-regulated downstream molecules within the same pathway. Further analysis of the microarray data revealed a unique set of genes that were up-regulated by genistein and also were down-regulated by ICI 182,780. Five out of the six genes identified from this comparison belonged to the metallothionein (MT) gene family. Using qRT-PCR, the changes in expression levels were validated in metastatic and non-metastatic tumour lines of LTL313b, both of which had been derived from the same PCa patient, indicating a strong association between MT gene expression and prostate cancer metastasis.In summary, genistein-activated-ERβ promotes metastasis in two ways; genomic and non-genomic pathways. In the non-genomic pathway, ERβ stimulates kinase signaling pathways, leading to cell survival and increased motility. In the genomic pathway, ERβ increases MT and/or other metastasis-associated gene expression, which can be inhibited by anti-estrogen treatment. This study has demonstrated that genistein elicits cancer promoting effects in vivo and that ERβ is important in metastatic progression of human PCa. The significant inhibition of metastasis by anti-estrogen treatment shown here potentiates a promising new selective estrogen receptor modulator treatment for metastatic patients.

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Metastasis is thought to be based on genetic and epigenetic alterations. The mechanisms underlying prostate cancer metastasis are not clear. Studies aimed at identifying genes with key roles in this process have been impeded by lack of clinically relevant models. The heterogeneity of primary prostate cancer specimens from patients, consisting of non-metastatic and metastatic subpopulations, hampers identification of metastasis-associated genes by direct comparison of primary and secondary cancers. To overcome such hurdles, metastatic and non-metastatic tumor sublines have been developed from one patient’s primary prostate cancer specimen using subrenal capsule grafting into NOD-SCID mice. Chromosomal alterations present in the metastatic subline, but not in non-metastatic counterparts, were identified in a small percentage of cells in the parental tissue, suggesting that metastatic potential of primary cancers can be associated with a small cancer cell subpopulation. Sublines with different metastatic potential derived from same patient’s multifocal primary cancer provide valuable materials for identifying metastasis-associated genes and predictive markers. To identify metastasis-associated genes, differential gene expression analysis of metastatic PCa1-met and non-metastatic PCa2 prostate cancer sublines was carried out. Among various differentially expressed genes identified, ASAP1, a gene not previously associated with prostate cancer, was upregulated in the metastatic subline as confirmed by qRT-PCR and immunohistochemical staining. In clinical specimens, ASAP1 protein staining was elevated in 80% of primary prostate cancers and substantially higher in metastatic lesions compared to benign prostate tissue. Extra ASAP1 gene copies were detected in 58% of primary prostate cancer specimens. Increased ASAP1 protein expression was correlated with prostate cancer metastasis and PSA recurrence. siRNA- and shRNA-induced reduction of levels of ASAP1 protein markedly suppressed in vitro PC-3 cell migration, matrigel invasion and metastasis in vivo. These results indicate that ASAP1 plays an important role in prostate cancer invasion and metastasis and suggest that it provides a potential predictive marker and therapeutic target for the disease. Furthermore, the approach used to identify metastasis-associated genes by comparison of gene profiles of paired metastatic and non-metastatic sublines was validated. The subrenal capsule xenograft system provides a valuable platform for studying various aspects of prostate cancer metastasis.

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