Marianne Sadar
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Graduate Student Supervision
Doctoral Student Supervision
Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
Currently, there is no cure for patients with castration-resistant prostate cancer (CRPC). Most CRPC continues to be driven by androgen receptor (AR) signalling for growth and survival, despite androgen-deprivation therapy maintaining castrate levels of serum androgen. AR transcriptional activity is mediated entirely by its intrinsically disordered N-terminal domain (NTD) and believed to involve transiently induced structures within Tau-5 of the NTD to promote interaction with basal transcriptional machinery. The AR NTD harbours six putative binding sites for Pin1, a proline isomerase that regulates protein conformation at specific phosphorylated-Ser/Thr-Pro motifs. Since induced folding within the AR NTD is supposed to be required for transactivation, perturbation of its structure may be a promising approach to block its activity. The purpose of this study was to elucidate the role of Pin1 in regulating the transcriptional activity of AR and to test Pin1 inhibitors for CRPC. We hypothesized that Pin1 interacts with the AR NTD to regulate transactivation and that targeting Pin1 would enhance the inhibitory response of EPI compounds which specifically bind to Tau-5 in the AR NTD. We assessed Pin1 inhibitors, juglone and ATRA, for their effects on AR function using various cell-based assays. We evaluated the efficacy of ATRA in vivo, as monotherapy and combination therapy with EPI-7170, using human CRPC xenograft models. We found that targeting the isomerase activity of Pin1 reduced the transcriptional activity of AR. Pin1 interacted with the AR NTD and regulated ligand-independent transactivation by interleukin-6. The Pin1 inhibitor ATRA prevented cell proliferation driven by the constitutively active AR splice variant, AR-V7, and was synergistic with EPI compounds for inhibiting AR transcriptional activity. Furthermore, combination therapy of ATRA and EPI-7170 was effective in reducing the in vivo growth of CRPC xenografts. In summary, the isomerase function of Pin1 was essential for transactivation of the AR NTD and targeting Pin1 enhanced the potency of AR NTD inhibitors. Since there are currently no effective treatments for lethal CRPC, elucidating the molecular mechanisms which regulate the transcriptional activity of AR could aid in the development of more effective therapies for prostate cancer.
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Background: Inhibition of the androgen receptor (AR) is the mainstay treatment for advanced prostate cancer. While initially effective, the disease ultimately progresses to metastatic castration-resistant prostate cancer, which is lethal. Ralaniten specifically targets the AR N-terminal domain (NTD). This allows for sustained inhibition in the context of constitutively active AR-splice variants and mutations which often drive resistance to current therapies. Ralaniten acetate is the pro-drug of ralaniten, and the first AR-NTD inhibitor to enter clinical trials (NCT02606123, ESSA Pharma, Inc.). Here we generated and characterized a model of acquired ralaniten resistance to aid in the development of next generation AR-NTD inhibitors.Methods: A resistant cell line (LNCaP-RALR) was created by serially passaging parental LNCaP cells in the presence of ralaniten. Resistant cells were challenged with ralaniten and existing AR antagonists to assess growth and sensitivity of the resistant line in vitro and in vivo. LNCaP-RALR cells were subject to global microarray profiling, and findings were validated using qRT-PCR, western blot and functional assays.Results: Ralaniten displayed antitumour activity in LNCaP but not LNCaP-RALR xenografts due to its reduced ability to block AR mediated gene transcription. Interestingly, LNCaP-RALR cells retained sensitivity to antiandrogens and AR knockdown by targeted siRNA, implying growth remains driven by AR signalling. Interrogation of microarray data revealed candidate genes (UGT2B family) associated with ralaniten resistance. These genes are involved in drug metabolism (glucuronidation), and knockdown of UGT2B isoforms was sufficient to restore sensitivity to ralaniten in resistant cells. LC/MS and ion spectra data from serum samples collected from patients, revealed that ralaniten is glucuronidated in humans. EPI-045 (which is resistant to glucuronidation) significantly inhibited AR mediated transcription and proliferation in LNCaP-RALR cells - both in vitro and in vivo.Conclusions: We have generated a model of acquired ralaniten resistance, and demonstrated that selective modification of ralaniten can reduce its glucuronidation. LNCaP-RALR cells remain dependent upon AR signalling, and are sensitive to both EPI-045 and antiandrogens used clinically. This work highlights the potential for combination or sequential therapy following ralaniten resistance, and provides a model with which to test next generation AR-NTD inhibitors.
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Androgen receptor (AR), a transcription factor, is a validated therapeutic target for prostate cancer. All current AR-targeting therapies inhibit the growth of prostate cancer cells by blocking the ligand-binding domain (LBD), where androgen binds to activate the receptor. Unfortunately, these therapies fail to maintain a durable clinical effectiveness, as patients eventually succumb to castration-resistant prostate cancer (CRPC). The clinical onset and progression of most CRPC is accompanied by rising levels of serum prostate specific antigen (PSA), a gene transcriptionally regulated by AR. This indicates aberrant AR transcriptional activity is involved in driving CRPC and conferring therapy-resistance. Therefore, it is imperative to continue the research and development of novel AR inhibitors that can overcome molecular mechanisms underlying aberrant AR transcriptional activity. This dissertation presents three research projects: 1) Discovery of novel AR inhibitors; 2) Evaluation of EPI-002, an AR N-terminal domain (NTD) antagonist; and 3) Generation of a prostate cancer cell line model with resistance to EPI-002. To discover novel AR inhibitors, candidate compounds identified from high throughput screening were characterized by fluorescent ligand binding assays, AR-driven reporter assays, qPCR gene expression analyses, and proliferation assays. AR NTD inhibitor EPI-002 was evaluated against several mechanisms believed to cause aberrant AR transcriptional activity, including coactivator overexpression, AR gain-of-function mutations, and constitutively active AR splice variants with truncated LBD. To generate a prostate cancer cell line model that developed resistance to EPI-002, LNCaP human prostate cancer cells were cultured under chronic EPI-002 exposure. First, spongian diterpenoids were discovered as novel antiandrogens that bind to the AR LBD. The diterpenoids blocked androgen-dependent AR transcriptional activity with a structure-activity relationship, reduced androgen-regulated gene expression, and inhibited the proliferation of androgen-sensitive prostate cancer cells. Secondly, EPI-002 was effective against aberrant AR transcriptional activity caused by overexpressed coactivators; AR gain-of-function mutations; and constitutively active AR splice variants such as AR-V7. Importantly, EPI-002 inhibited the growth of CRPC cells driven by AR-V7, whereas antiandrogens had no effect. Finally, a human prostate cancer cell line model resistant to growth inhibition by EPI-002 was generated, allowing future studies to investigate mechanisms of resistance against AR inhibition through the NTD.
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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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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.
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Development and maintenance of the prostate is dependent on androgens and the androgenreceptor. The androgen pathway continues to be important in prostate cancer. Here, we evaluatedthe transcriptome of prostate cancer cells in response to androgen using Long Serial Analysis ofGene Expression (L0ngSAGE) libraries. We identified 35 genes with novel associations toandrogen signalling and validated 24 of these genes using quantitative real time-polymerasechain reaction (qRT-PCR). These genes were: ARL6IF5, BL VRB, C]9orf48, C]orfJ22,C6orf66, CAMK2NJ, CCNI, DERA, ERRFI], GLUL, GOLFH3, HMJ3, HSP9OB], MANEA,NANS, NIPSNAP3A, SLC4JA], SOD], SVIF, TAOK3, TCP], TMEM66, USP33, andVTAJ. The physiological relevance of these expression trends was evaluated in vivo using theLNCaP Hollow Fibre model.There is no cure for castration-recurrent prostate cancer (CRPC), and the mechanisms underlyingthe disease are not known. To address this problem, we assayed the transcriptome of LNCaPhuman prostate cancer cells as they progress to castration-recurrence in vivo using replicateL0ngSAGE libraries. We identified 96 novel genes consistently differentially expressed inCRPC. The expression profiles support a role for the transcriptional activity of the androgenreceptor genes (CCNH, CUEDC2, FLNA, and FSMA 7), steroid synthesis and metabolism genes(DHCR24, DHRS7, ELOVL5, HSDJ 7B4, and OPRKJ), neuroendocrine cell genes (ENO2,MAOA, OPRK], SJOOA]O, and TRPM8), and proliferation genes (GAS5, GNB2L], MT-ND3,NKX3-], PCGEM], PTGFR, STEAFJ, and TMEM3OA) in castration-recurrence.Screening for prostate cancer using serum levels of prostate-specific antigen has resulted in theover-treatment of indolent disease. Novel diagnostic and prognostic markers for prostate cancerare required. To address this need, the levels of 27 transcripts were investigated with qRT-PCR.Expression of POP3 (100 kb from EST CF140309) was prostate-specific, with restrictedexpression ofADAM2, POP1 (50 kb from AK000023), POP4 (truncated TMEFF2), POP 10(intron ofADAM2), ELOVL5, RAMP], and SPON2. ELO VL5, NGFRAP1, POP5 (intron ofNCAM2), POP8 (intron of EFNA5), RAMP], SPON2, and TMEM66 were differentiallyexpressed between laser microdissected tumour and normal clinical samples of prostatic tissue.These studies suggest that ADAM2, ELOVL5, POP 1, POP3, POP4, POP 10, RAMP], and SPON2may be good candidates for biomarkers of prostate cancer.
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Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Background: Advanced prostate tumors that develop resistance to androgen deprivation therapy are incurable and uniformly fatal. Nonetheless, the manifestation of this lethal form, known as castration-resistant prostate cancer (CRPC), does not preclude most of these tumors from sustained dependence on the androgen receptor (AR) for growth and survival. To inhibit AR pathway signaling, the strategy of all currently approved drugs ultimately converge upon the C-terminus ligand-binding domain (AR-LBD) to target and disrupt AR activation. Ralaniten is a novel, first-in-class drug which binds the AR within its N-terminal domain (AR-NTD). Due to this unique mechanism of action, we predicted that Ralaniten would induce a distinct global response compared to alternative AR-inhibitors. This study initiates the characterization of Ralaniten specific gene expression profiles and unravels the mechanism of induction of an unexpected group of genes from the metallothionein (MT) gene family.Methods: In vitro experiments were performed in 4 human prostate cancer cell lines with experimentally useful genomic and phenotypic features. Preliminary gene expression data were generated by microarray. Pathway and statistical analyses revealed candidate genes for subsequent investigation. Transcriptional data were validated by qPCR and at the protein level by western blot. Reporter assays for gene activity were conducted after transient transfection of plasmids. Transient siRNA- mediated knockdown experiments assessed involvement of potentially relevant transcription factors. AR NTD inhibitors included Ralaniten, EPI-7170, SINT-1, and LPY26, whereas AR LBD inhibitors included the antiandrogens bicalutamide and enzalutamide. AR transactivation was mediated using the synthetic androgen R1881.Results: Microarray analyses revealed the MT family to be the most abundantly induced by Ralaniten in the absence of androgen. Induction was experimentally confirmed to be Ralaniten specific. Knockdown experiments implicate a central role for the transcription factor, MTF-1, in the induction of MT genes by Ralaniten, and have ruled out the requirement for the AR and the redox activated transcription factor, Nrf2, in this mechanism.Conclusions: Ralaniten induced the expression of MT genes by a mechanism independent of expression of AR and Nrf2. MT induction by Ralaniten is exquisitely dependent on the expression of the transcription factor, MTF-1.
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Development of castration-resistant prostate cancer (CRPC) is thought to be dependent on androgen receptor (AR) transcriptional activity. Resistance to current therapies is linked to constitutively active AR splice variants that lack the ligand-binding domain (LBD). Metastatic tumours heterogeneously express varying levels of AR splice variants to full-length AR (fl-AR). AR splice variant V7 is frequently expressed and correlated with poor prognosis in patients. Since current therapies such as enzalutamide target the fl-AR LBD, which is absent in AR variants, an amino-terminal domain (NTD) inhibitor that blocks transcriptional activities of all AR species may significantly improve survival of CRPC patients. EPI-002 binds to the AR NTD and inhibits transcriptional activity of AR. Hypotheses tested in this thesis include: (1) EPI-002 inhibits transcriptional activity of endogenous and exogenous V7 in human prostate cancer cell lines, and (2) combination therapy of EPI-002 and antiandrogen enzalutamide demonstrates greater inhibition of transcriptional activities of mixed AR populations than each treatment alone. V7 transactivation was measured with EPI-002 treatment in AR-negative PC3 cells. Combined transcriptional activities with probasin-, PSA- and ARR3-luciferase AR-driven reporters were measured in androgen-sensitive LNCaP cells ectopically expressing V7, and androgen-independent LNCaP95 cells endogenously expressing V7. EPI-002 and enzalutamide monotherapies and in combination were evaluated with mixed AR populations in LNCaP and LNCaP95. Cell growth of LNCaP95 was measured with combination treatment. EPI-002 inhibited constitutive transcriptional activity of V7. EPI-002 monotherapy and in combination with enzalutamide demonstrated greater inhibition of transcriptional activities of V7 expressed approximately 1:1 with fl-AR in LNCaP than enzalutamide monotherapy. With low V7 levels compared to fl-AR, enzalutamide monotherapy and in combination with EPI-002 generally showed greater inhibition of AR transcriptional activities in LNCaP95 than EPI-002 monotherapy. It was observed that V7 activity demonstrated differential gene regulation of AR-driven reporters employed. Proliferation of LNCaP95 cells was inhibited by EPI-002 monotherapy and in combination with enzalutamide, with enzalutamide monotherapy having no effect. Data shown here begins to reveal: 1) proof-of-principle that AR NTD inhibitor EPI-002 blocks all AR species in a heterogeneous tumour population; and 2) a potential therapeutic benefit of CRPC patients treated with EPI-002 in combination with enzalutamide.
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Constitutively active splice variants of androgen receptor (AR) lacking the ligand-bindingdomain (LBD) are linked to the development and progression of castration-resistant prostatecancer (CRPC). Recent studies suggest a constitutively active splice variant, ARv⁵⁶⁷es, is capableof interacting with full-length AR, stabilizing and enhancing its ligand-dependent activitiesdespite castrate levels of circulating androgen. EPI-001, an AR antagonist targeting the Nterminusdomain (NTD) prevents N-terminus to C-terminus (N/C) interaction of AR, which isessential for AR antiparallel dimer formation. The ligand-dependent N/C interactions slow thedissociation of ligand from the LBD. Here we examine the effect of EPI-002, the most potentstereoisomer of EPI-001, on ARv⁵⁶⁷es complexed with full-length AR and test the hypothesis thatEPI-002 will cause ligand to dissociate more quickly because it blocks N/C interaction. The aimof this study is two-fold as we first examined the effects of ARv⁵⁶⁷es on the dissociation rate ofthe full-length receptor. Then, we examined the effect of EPI-002 on the ligand dissociation rateof full-length AR with and without the presence of ARv⁵⁶⁷es.We have demonstrated that EPI-002 did not affect binding affinity of wild-type full-length ARnor the time for it to reach binding equilibrium. EPI-002 accelerated the ligand dissociation rateof wild-type full-length AR possibly by disrupting N/C interaction. Co-expression of ectopicARv567es and wild-type full-length AR at 50:50 ratios did not alter the ligand dissociation rate ofwild-type full-length AR but attenuated the effect of EPI-002. However, EPI-002 had minimaleffect on the ligand dissociation rate of endogenous AR in LNCaP prostate cancer cells,consistent with the lack of effect when AR has a mutation in the LBD (T877A) that enhances the N/C interaction and slows the ligand dissociation rate compared to the wild-type AR. Togetherthese data begin to reveal 1) the unique mechanisms of splice variant ARv⁵⁶⁷es on thedissociation rate of full-length AR; and 2) the effect of an AR NTD inhibitor on the dissociationrate of full-length AR with and without the presence of splice variants.
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