Most patients undergoing androgen deprivation therapy relapse eventually and progress to androgen-independent (AI) prostate cancer. Although the mechanisms underlying progression to AI prostate cancer are not well understood, studies suggest that androgen receptor (AR) is still required for AI prostate cancer. Our lab found that Vav3, a Rho GTPase guanine nucleotide exchange factor (GEF), is up-regulated during the progression of androgen-dependent human prostate cancer cells to androgen-independence in vivo and in cell-based experiments. Since Vav3 significantly increases ligand-dependent AR transcriptional activity and this action requires the Vav3 pleckstrin homology (PH) domain but not Vav3 GEF activity, we explored the role of the Vav3 PH domain in ligand-dependent AR coactivation by Vav3. We found that targeting the Vav3 PH mutant into nuclei but not the plasma membrane restored Vav3 PH mutant in AR coactivation. Targeting Vav3 to the plasma membrane eliminated the capacity of Vav3 to coactivate AR. In agreement with nuclear targeting of Vav3 via its PH domain, chromatin immunoprecipitation assays showed that Vav3 enhancement of AR transcriptional activity was accompanied by Vav3 recruitment to AR transcriptional complexes at an AR target gene enhancer. Further, Vav3 increased AR occupancy at the target gene enhancer upon androgen treatment and this may underlie the capacity of Vav3 to enhance AR transcriptional activity. Because Vav3 can also be activated by growth factors (GFs) and GFs activate AR in the absence of androgen (ligand-independent), we investigated the crosstalk between Vav3 and GF activation of AR and found Vav3 strongly enhanced AR transcriptional activity induced by GFs. GEF function and the downstream Rho GTPase, Rac1 were required for constitutively active (Ca) Vav3 activation of AR, which differs from Vav3 activation of AR in the presence of androgen. We also investigated the possible signal pathways contributing to AR activation by Ca Rac1. Ca Rac1 caused ligand-independent activation of AR in part through MAPK/ERK signaling and conferred prostate cancer growth in the absence of androgen in cell culture, soft agar and mouse tumor xenografts. Thus, our findings indicate that Vav3 activates AR in the presence or absence of ligand through two distinct mechanisms, which supports a versatile regulatory effect of Vav3 in AR signaling and prostate cancer progression.

As the most frequently diagnosed cancer in North American men, prostate cancer can progress to the androgen independent stage after initial response to androgen ablation therapy. The molecular mechanisms involved in the hormonal progression of prostate cancer are not completely understood. Here, we analyze changes in the transcriptome of prostate cancer cells at different stages of progression to reveal potential mechanisms. Applying Affymetrix GeneChip technology, we identified the transcriptomes in response to stimulation of androgen and PKA pathways in human prostate cancer cells. In addition to PSA, other common target genes were identified. Genes differentially expressed in response to androgen and stimulation of the PKA pathway in vitro were also differentially expressed during hormonal progression in vivo. Upon androgen stimulation, androgen receptor binds to a functional androgen response element within the promoter region of SESN1, a p53 targeted gene, and represses its expression. The expression of SESN1 was induced by castration in LNCaP xenografts, but the expression was eventually suppressed again in the androgen independent stage of prostate cancer. Knockdown of SESN1 promoted the proliferation of prostate cancer cells. Expression patterns of androgen-regulated genes in androgen independent tumours were revealed to be more similar to that from before castration than to the tumors under androgen ablation. The β-catenin, a potent coactivator of the androgen receptor, and Wnt pathway was deregulated in androgen-independent tumours. There was increased nuclear colocalization and interaction of androgen receptor and β-catenin with hormonal progression of prostate cancer. This study provides insight into hormonal effects on prostate cancer and possible pathways involved in the development of androgen independent disease, as well as potential therapeutic targets.

The mechanism by which prostate cancer progresses from androgen dependence (AD) to androgen independence/castration resistance (AI/CR) is currently a major focus of prostate cancer-related research. Prostate cancers that progress to a state of AI/CR are typically resistant to most standard types of treatments. Due to its primary role in driving normal prostate cell growth and proliferation, the androgen receptor (AR) is believed to play a key role in progression. Coregulators, or any proteins which may either enhance or abrogate AR activity, are considered to be one of the potential mechanisms by which AR function may become impaired. Cyclin G-associated kinase (GAK) was initially identified as a potential coregulator of AR in a Tup 1 repressed transactivation system. A LNCaP cDNA library was screened for proteins which interacted with the NH2-terminus of AR. GAK was isolated from three independent library clones using two different AR baits (AR 1-549 and AR 1-646). This interaction was confirmed via GST pulldown and coimmunoprecipitation experiments, and preliminary luciferase assays suggested that GAK activates AR in a hormone dependent manner. In this study, my objectives were to validate GAK’s role as a coregulator of AR and to determine if overexpressing GAK affects progression to AI. In vitro luciferase assays whereby GAK was either overexpressed or knocked down in both LNCaP and PC3 cells did not significantly affect AR activity. Xenograft experiments utilizing a doxycycline (DOX) inducible lentiviral LNCaP-GAK overexpressing stable cell line demonstrated that while GAK may not play a significant role in modulating AR activity, it may adopt a more subtle role enhancing tumour take and tumour volume growth rate in vivo. While these results could not confirm GAK to be a direct coregulator of AR, it is entirely possible that GAK may influence prostate cancer progression, albeit indirectly. Recent publications report a growing amount of evidence suggesting GAK’s involvement in the critical cellular process of clathrin coated vesicle endocytosis, the dysregulation of which could potentially indirectly affect AR regulated genes.

Cancer is a leading cause of human mortality worldwide, and is expected to soon become the overall leading cause of death in the United States. Some cancers are hormone-related, including the sex-specific cancers of the breast (predominantly in women) and prostate (in men). In both cases, early stage tumors are responsive to inhibitory endocrine-based therapies. However, both cancers progress to hormone-nonresponsive states and this is in part due to altered properties of the primary nuclear hormone receptor signaling pathway (estrogen receptor [ER] in breast; androgen receptor [AR] in prostate). Other nuclear receptors are thus being investigated as therapeutic targets due to their crosstalk with hormone receptor pathways and these include the aryl hydrocarbon receptor (AhR), peroxisome proliferator activated receptor γ(PPARγ, retinoic acid receptor and retinoid X receptor (RAR/RXR), and vitamin D receptor (VDR). Previous studies have demonstrated that the AhR mediates chemoprotective, antiestrogenic, and tumoristatic effects in experimental models, and relatively non-toxic selective aryl hydrocarbon receptor modulators (SAhRMs) have been developed. Studies in this dissertation have investigated the therapeutic properties of a new class of compounds related to the SAhRM 3,3‘-diindolylmethane (DIM) in models of breast cancer. Additionally, the potential therapeutic role of the AhR in human prostate cancer cells has been investigated. Several ring- and methylene-substituted DIMs exhibited antiestrogenic and tumoristatic activities in breast cancer cells and in carcinogen-induced rat mammary tumors. At least some of the methylene-substituted DIMs act through PPARγ. The AhR is expressed in LNCaP and iv 22Rv1 prostate cancer cells and AhR agonists inhibit cell growth and AR-induced transactivation through pathways independent of androgen receptor downregulation.

The present study evaluated the prevalence, severity, and psychosocial correlates of hot flashes in men receiving androgen deprivation therapy for prostate cancer. Seventy-two men completed a detailed packet of questionnaires prior to the initiation of treatment and again three-months later. Results indicated that the extent to which hot flashes interfered with patients' daily functioning significantly contributed to changes in depressive symptoms. Changes in fatigue were found to mediate the relationship between hot-flash related interference and depressive symptomatology, suggesting that increases in fatigue were responsible for the concurrent increases in symptoms of depression. The coping strategy of catastrophizing moderated the relationship between hot flash-related interference and cancer-related distress, such that levels of cancer-related distress in men reporting greater use of catastrophizing were dependent upon levels of hot flash-related interference. Men who did not engage in catastrophizing reported uniformly low levels of cancer-related distress regardless of the extent to which hot flashes interfered with daily functioning. Expected relationships between hot flashes and sexual functioning or masculine self-image were not confirmed. These findings provide valuable information regarding the experience of hot flashes in this population. Results indicate that reduction of fatigue may lead to reductions in depressive symptoms, while reducing patients' use of catastrophizing may alleviate cancer-related distress.

<p>Understanding the mechanisms whereby the sequence of the human genome is interpreted into diverse cellular phenotypes is a critical endeavor in modern biology. A major determinant of cellular phenotype is the spatial and temporal pattern gene expression, which is regulated in part by epigenomic properties such as histone post-translational modifications, DNA methylation, chromatin accessibility and the 3-dimensional architecture of the genome within the nucleus. These properties regulate the dynamic assembly of transcription factors and their co-regulatory proteins upon chromatin. To properly understand the interplay between the epigenomic framework of a cell and transcription factors, integrated analysis of transcription factor-DNA binding, chromatin status, and transcription is required. This work integrates information about chromatin accessibility, as measured by DNaseI hypersensitivity, transcription factor binding, as measured by chromatin immunoprecipitation, and transcription, as measured by microarray or transcriptome sequencing, to further understand the functional role of two important transcription factors, the androgen receptor (AR) and CTCF, in cancer cell line models. Data gathered from a prostate cancer cell line model demonstrate that the AR does not exclusively bind accessible chromatin upon ligand-activation, and induces significant changes in chromatin accessibility upon binding. Regions of quantitative change in chromatin accessibility contain motifs corresponding to potential collaborators for AR function, and are also significantly associated with AR-regulated transcriptional changes. Furthermore, base pair resolution of the DNaseI cleavage profile revealed three distinct patterns of AR-DNA interaction, suggesting multiple modes of AR interacting with the genome. A novel role for the nuclear receptor REV-ERB&#945; in AR-mediated transcription was explored within the same model system. Though preliminary, results thus far indicate that REV-ERB&#945; is required for AR-induced increases in target gene transcription in a manner that is likely dependent on HDAC3. Genetic knockdown of REV-ERB&#945; resulted in notable changes in chromatin accessibility around AR-target genes both before and after AR activation. The function of CTCF was interrogated using stable knockdown in a breast cancer cell line model. CTCF knockdown led to widespread changes in chromatin accessibility that were dependent on DNA sequence. Further analysis suggested that AP-1 and FOXA1 are involved in CTCF function. Together, the work presented in this dissertation offers novel insight into the behavior of two critical transcription factors in cancer cell lines, and describe a framework of analysis that can be extended and applied to any transcription factor within any desired cellular context.</p> / Dissertation

As the most frequently diagnosed cancer in North American men, prostate cancer can progress to the androgen independent stage after initial response to androgen ablation therapy. The molecular mechanisms involved in the hormonal progression of prostate cancer are not completely understood. Here, we analyze changes in the transcriptome of prostate cancer cells at different stages of progression to reveal potential mechanisms. Applying Affymetrix GeneChip technology, we identified the transcriptomes in response to stimulation of androgen and PKA pathways in human prostate cancer cells. In addition to PSA, other common target genes were identified. Genes differentially expressed in response to androgen and stimulation of the PKA pathway in vitro were also differentially expressed during hormonal progression in vivo. Upon androgen stimulation, androgen receptor binds to a functional androgen response element within the promoter region of SESN1, a p53 targeted gene, and represses its expression. The expression of SESN1 was induced by castration in LNCaP xenografts, but the expression was eventually suppressed again in the androgen independent stage of prostate cancer. Knockdown of SESN1 promoted the proliferation of prostate cancer cells. Expression patterns of androgen-regulated genes in androgen independent tumours were revealed to be more similar to that from before castration than to the tumors under androgen ablation. The β-catenin, a potent coactivator of the androgen receptor, and Wnt pathway was deregulated in androgen-independent tumours. There was increased nuclear colocalization and interaction of androgen receptor and β-catenin with hormonal progression of prostate cancer. This study provides insight into hormonal effects on prostate cancer and possible pathways involved in the development of androgen independent disease, as well as potential therapeutic targets.

The mechanism by which prostate cancer progresses from androgen dependence (AD) to androgen independence/castration resistance (AI/CR) is currently a major focus of prostate cancer-related research. Prostate cancers that progress to a state of AI/CR are typically resistant to most standard types of treatments. Due to its primary role in driving normal prostate cell growth and proliferation, the androgen receptor (AR) is believed to play a key role in progression. Coregulators, or any proteins which may either enhance or abrogate AR activity, are considered to be one of the potential mechanisms by which AR function may become impaired. Cyclin G-associated kinase (GAK) was initially identified as a potential coregulator of AR in a Tup 1 repressed transactivation system. A LNCaP cDNA library was screened for proteins which interacted with the NH2-terminus of AR. GAK was isolated from three independent library clones using two different AR baits (AR 1-549 and AR 1-646). This interaction was confirmed via GST pulldown and coimmunoprecipitation experiments, and preliminary luciferase assays suggested that GAK activates AR in a hormone dependent manner. In this study, my objectives were to validate GAK’s role as a coregulator of AR and to determine if overexpressing GAK affects progression to AI. In vitro luciferase assays whereby GAK was either overexpressed or knocked down in both LNCaP and PC3 cells did not significantly affect AR activity. Xenograft experiments utilizing a doxycycline (DOX) inducible lentiviral LNCaP-GAK overexpressing stable cell line demonstrated that while GAK may not play a significant role in modulating AR activity, it may adopt a more subtle role enhancing tumour take and tumour volume growth rate in vivo. While these results could not confirm GAK to be a direct coregulator of AR, it is entirely possible that GAK may influence prostate cancer progression, albeit indirectly. Recent publications report a growing amount of evidence suggesting GAK’s involvement in the critical cellular process of clathrin coated vesicle endocytosis, the dysregulation of which could potentially indirectly affect AR regulated genes.

Objectives: Men who receive androgen deprivation therapy (ADT) for prostate cancer (PCa) are at risk of several adverse effects that can be detrimental to both their physical and mental health. Common adverse effects include weight gain, muscle wasting, cardiovascular morbidity, fatigue and impaired quality of life (QOL). This study tested whether a combined aerobic and resistance exercise program can alleviate some of these symptoms in men receiving ADT. Design: Men with PCa, aged 50-80 years, receiving ADT were recruited to participate in this prospective randomized controlled trial. Subjects were assigned to a usual care group (UCG) or an exercise intervention group (EIG). The EIG completed a 16 week combined aerobic and resistance exercise program. Outcomes measures were assessed at baseline, 16 weeks, and 24 weeks and included: cardio-respiratory fitness; muscle strength and endurance; body composition; and reports of QOL, fatigue, mood, partner relations, and exercise behaviour. Results: Fifteen men were recruited to this study, but two participants in the EIG did not finish the study leaving the EIG with an n = 6 and the UCG with an n = 7. The exercise program did not lead to changes in weight, BMI or body fat. There was a small, close to significant, increase in muscle mass in the EIG over the intervention period (p = 0.052). This is encouraging as it demonstrates that exercise can counteract the catabolic effects of ADT. Interestingly, cardio-respiratory fitness improved over the course of the study for both groups. Muscular fitness, however, improved only for the EIG. There was a significant difference in chest press strength (p = 0.041) and leg press strength was bordering significance (p = 0.058). Unexpectedly, QOL declined for both groups during the intervention (p = 0.029). Participants in both groups also reported increased levels of fatigue from baseline to 24 weeks, although these changes were not significant (p = 0.586). Mood worsened over the study period for both groups from baseline to 16 weeks, but this increase in anxiety and depression was reduced at the follow-up period. These changes, too, were not significant (p = 0.364). Reports of partner relationships trended towards lower scores from baseline to 16 weeks. The men’s report in both groups and the women’s report in the EIG improved at the 24 week mark, but women in the UCG experienced further decline. Surprisingly, participants in both groups reported increases in exercise behaviour from baseline to 24 weeks. This could account for the lack of difference found in many of the measures. The power of this study was 0.22. Conclusion: Although this was a small study, it showed that a combined aerobic and resistance exercise program can have some positive benefits for men with PCa who are receiving ADT. Larger trials are needed to further examine the role of exercise in ameliorating the side effects of ADT, particularly in the areas of mood and partner relationships.

Currently, there are no preclinical immunocompetent mouse models that adequately represent all stages of prostate cancer (PC) especially, androgen depletion independent (ADI) and bone metastatic PC. The best characterized, Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model is logistically a difficult model for in vivo assessments and it does not adequately represent all stages of PC. Therefore, the aim of this study was to broaden the TRAMP model to include ADI and bone metastatic PC. Three ADI sublines were derived from androgen-sensitive (AS) TRAMP C1 (TC1) and TRAMP C2 (TC2) parental lines in vitro by dihydrotestosterone (TC1-T5 and TC2-T5) deprivation and in vivo by growing in TRAMP female mice (TC1-F1). The new sublines showed several characteristic features of ADI-PC 1.) faster growth rates in vitro and in vivo 2.) increased invasiveness 3.) androgen depletion independence in vitro and in vivo 4.) variable expression of androgen receptor 5.) downregulation of metastasis suppressor genes, E-cadherin and KAI-1. Genetic and molecular studies of ADI sublines showed alteration of genes representing major cancer related pathways. ADI TC1-T5, that displayed the most aggressive phenotype/genotype was selected to expand the TRAMP model to represent PC metastases Metastatic ability of TC1-T5 to migrate to bone and other soft tissues after intracardiac injections was shown in contrast to AS TC1 cells. Bone metastatic lesions displayed both osteoblastic and osteolytic features in multiple locations. Additionally, unlike AS TC1, the TC1-T5 tumours were able to grow with 100% incidence in the prostate and as lungs pseudometastases. The ADI PC lines were used to explore Aurora Kinases (AKs) as therapeutic targets for ADI PC. Compared to TC1, ADI-TC1-T5 cells showed a significant upregulation of AK-A and AK-B and their downstream regulators, survivin and phosphorylated-histone H3. Enhanced sensitivity of TC1-T5 to AK inhibitor VX680 functionally validated this and together with docetaxel led to enhanced efficacy which correlated with implication of AK-A/B in development of docetaxel-resistance. Thus, TRAMP model now represents ADI-PC that can grow in the bone, lungs and in the prostate; a significant step towards a well rounded preclinical model with greater clinical relevance.