Marianne Sadar


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Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - Nov 2019)
A model of acquired resistance to the novel AR-NTD inhibitor ralaniten reveals resistance occurs via a selective metabolism pathway (2019)

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Development of novel small molecule inhibitor of androgen receptor to treat castration-resistant prostate cancer (2015)

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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Crosstalk between signaling pathways in hormonal progression of prostate cancer (2009)

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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Gene expression in prostate cancer (2008)

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 (2010 - 2018)
EPI-002 accelerates ligand dissociation from androgen receptor by disrupting N-terminus to C-terminus interaction (2013)

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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