Amina Zoubeidi

The treatment landscape of advanced prostate cancer continues to evolve, and the implementation of highly potent next-generation androgen receptor pathway inhibitors (ARPIs), such as Abiraterone and Enzalutamide, has prolonged the survival of men with advanced disease. However, prolonged treatment can alter the archetypal course of the disease, as highlighted by an increased incidence of aggressive disease variants that have lost their luminal identity. These variants often display histological dedifferentiation and alterations of the lineage. Clinically, these patients develop progression in the setting of low/non-rising serum prostate-specific antigen levels, elevated serum neuroendocrine markers, carcinoembryonic antigen levels, and/or metastases. Despite similar genetic profiles, only a few genetic variations, such as loss of RB1 and TP53, reliably distinguishes adenocarcinoma from neuroendocrine prostate cancer (NEPC), suggesting that epigenomic events are key contributing factors to cell plasticity and the acquired treatment resistance. While studies presented in this thesis and work published by others point to an epigenetic basis for emergent NEPC, there is still much unknown about the fundamental cellular and molecular interactions that bias lineage specification following ARPI therapies. We mapped genome-wide chromatin accessibility using an assay for transposase-accessible chromatin using sequencing across naturally occurring castration-resistance prostate cancer and NEPC cell lines. We found that ARPIs induce a rapid epigenetic alteration mediated by large-scale chromatin remodelling to support the activation of stem cell-like/neuronal transcriptional programs. We discovered the DNA binding motif for the neural lineage transcription factor ASCL1 to be disproportionally enriched in accessible chromatin regions in t-NEPC compared to adenocarcinoma. Matched RNAseq data confirmed ASCL1 expression and transcriptional activity were upregulated in t-NEPC cell lines and patient tumours. Silencing ASCL1 prevented the emergence of NEPC in vitro and in vivo. Targeting ASCL1 switches the neuroendocrine lineage back to the luminal epithelial state. This effect is modulated by the disruption of the Polycomb Repressive Complex-2 (PRC2) through the UHRF1/AMPK axis and changes the chromatin architecture in favour of the luminal phenotype. We showed that these epigenetic alterations are governed by ASCL1. Furthermore, this work provides proof of the principle of targeting ASCL1 to reverse neuroendocrine phenotype, support luminal conversion and re-addict tumours to ARPIs.

View record

While effective, resistance to 1st generation and 2nd generation androgen receptor (AR) pathway inhibitors is inevitable, creating a need to study the mechanisms by which prostate cancer (PCa) cells become resistant to these treatments. At its core, resistance can be categorized into AR driven and non-AR driven. The research presented in this thesis explores the hypothesis that non-AR driven resistance mechanisms exploit cellular plasticity to gain a survival advantage. Previous research has demonstrated that in response to 1st generation anti-androgens, epithelial PCa cells can undergo an epithelial-mesenchymal transition (EMT); a process implicated in metastatic dissemination of cancer cells throughout the body. Augmenting prior research demonstrating LYN tyrosine kinase’s role in resistance to 1st generation anti-androgens, our research demonstrates LYN promoting both EMT and metastasis. Specifically, we discovered that signaling cascade downstream of LYN alters the sub-cellular localization and therefore stability of key EMT promoting transcription factors Slug and Snail. While LYN does contribute to both treatment resistance and metastasis in PCa, its role in EMT was a pan-cancer mechanism observed in both breast and bladder cancer. Our research also explored the increased incidence of neuroendocrine PCa (NEPC) as a resistance mechanism to more potent 2nd generation anti-androgens (enzalutamide and abiraterone). Interrogating a model of in vivo derived enzalutamide resistance, our research uncovered the crucial role of neuronal transcription factor brain derived 2 (BRN2) in the transformation of epithelial prostate cells to neuroendocrine (NE) phenotype. Importantly, we discovered that AR inhibition relives its’ suppression of BRN2; thereby increasing BRN2 expression and initiating NE differentiation of epithelial PCa cells. Moreover, targeting BRN2 not only reduced the enzalutamide-induced NE differentiation but also reduced proliferation of enzalutamide resistant PCa cells and terminal NEPC cells. Building on this data, our research outlines the discovery of small molecule BRN2 inhibitors (BRN2i) that recapitulate the functional results demonstrated by conventional knockdown techniques. These BRN2i display specificity to BRN2 and selectivity in compromising growth of NEPC cells. Altogether, this thesis demonstrates two possible mechanisms how PCa cells alter their identity as epithelial cells to more mesenchymal (LYN) and/or neuroendocrine (BRN2) and gain resistance to AR pathway inhibitors.

View record

Prostate cancer causes morbidity and mortality for thousands of Canadian men each year. Castration remains the primary treatment for recurrent or metastatic prostate cancer. However, castration is never curative, and the cancer inevitably recurs as castration resistant prostate cancer (CRPC). Newer androgen receptor (AR) antagonists such as enzalutamide(ENZ) demonstrate significant benefit in CRPC patients, but these agents remain non-curative. Therefore, the goal of this thesis was to explore novel strategies to target ENZ-resistant prostate cancer using previously developed models of resistance. Our models suggest that similar resistance patterns to CRPC may be found in ENZ-resistance, including the upregulation of steroidogenesis and activation of survival pathways such as the PI3K/Akt pathway. Unlike inhibition of the AR pathway, we found that inhibition of different nodes of the PI3K/Akt pathway had limited efficacy as monotherapy and we therefore focused on combination strategies to target this prominent survival pathway. We found that combined Akt and MEK pathway inhibition demonstrates only moderate synergy in AR-positive models of prostate cancer, and this did not appear significantly greater in ENZ-resistant than ENZ-sensitive prostate cancer. However, we did find that blockade of Akt signaling in combination with ENZ significantly delays the development of resistance to ENZ through a very significant induction of apoptosis and cell cycle arrest. Further, co-targeting of the Akt and AR pathways appears more effective at earlier stages of prostate cancer progression, when the tumours are still castrate-sensitive. Finally, to further evaluate the combination of PI3K/Akt pathway and AR blockade, we investigated the pre-clinical rationale for the use of bromodomain inhibitors, which indirectly targets both the AR and myc transcription factors. Upregulation of myc was observed following PI3K/Akt inhibition, but overall our studies did not support the combination of bromodomain inhibitors in combination with PI3K/Akt inhibition in prostate cancer models. Taken together, our pre-clinical results highlight several treatment strategies and pitfalls in targeting ENZ-resistant prostate cancer. These studies enhance our understanding of therapeutic approaches to target resistant prostate cancer with several novel agents and combination strategies. Further evaluation in clinical trials is warranted and ongoing.

View record

Prostate Cancer (PCa) is the most common male cancer and the second leading cause ofcancer‐related deaths in North America. While many gains have been made in early detectionand treatment of localized PCa, many men still die of the metastatic disease. Androgen ablationtherapy remains the most effective therapy for patients with advanced disease. While ~80% ofpatients respond initially to this treatment, most patients progress to Castrate ResistantProstate Cancer (CRPC) stage. Current literature indicates that CRPC tumours are not uniformlyhormone refractory and may remain sensitive to therapies directed against the AR axis.Therefore, several new classes of AR‐targeting agents are now in clinical development,including more potent AR antagonists (MDV3100) and inhibitors of steroidogenesis(abiraterone). Although enthusiasm for this approach remains high, prostate tumourheterogeneity and the inevitable development of resistance dictates a critical need to betterunderstand the mechanisms of resistance in which AR remain active.In the current doctoral thesis role of heat shock protein (Hsp) 27 and Lyn tyrosine kinasein regulation of AR protein expression was investigated. The central hypothesis is that increasedexpression and activity of Hsp27 and Lyn kinase stabilizes and activates AR protein and leads toprostate cancer progression through promoting prostate cancer cell survival.Three specific objectives were accomplished in this thesis. First, the relationshipbetween Hsp27 and AR was studied. To this end, it was demonstrated that expression and activity of Hsp27 via a nongenomic mechanism regulates AR protein stability, shuttling andtranscriptional activity. In the next step, the underlying molecular mechanisms through whichLyn tyrosine kinase regulates AR activity in the castrated environment was investigated. Theexperiments demonstrated that Lyn kinase regulates AR protein expression and transcriptionalactivity and that it plays a key role in PCa progression to the castrated resistant stage. Finally, anovel role for Lyn kinase in Epidermal Growth Factor‐mediated (EGF‐mediated) AR activity wasdefined.The results obtained in this thesis defined new pathways involved in regulation of ARprotein stability and justified further investigation of Hsp27 and Lyn kinase as therapeutictargets for CRPC.

View record