Submitted by: Pia D Bagamasbad 2017-07-18 00:00:00 Last Updated by: Jeverly Ann S. Principe 2017-07-19 09:40:35 Export to PDF

Promoter-Based Drug Screen for Prostate Cancer Therapy

PHRR170719-001629

FP170021

Unspecified

Promoter-Based Drug Screen for Prostate Cancer Therapy

Prostate cancer (PCa) is a major health problem worldwide and is one of the leading causes of cancer-related death in men. Based on studies conducted by the World Health Organization, PCa has the highest 5-year prevalence (12220 cases), third highest incidence rate (4858 per 100,000), and the fourth highest mortality rate (2580 per 100,000) of all cancers in Filipino men. Growth and development of the prostate is highly dependent on androgen and aberrant androgen and androgen receptor (AR) signaling is the key driver in the pathology and progression of prostate cancer [1]. In early stage disease, androgen deprivation therapy (ADT), through depletion of androgens and blocking AR function, has been effective in slowing disease progression[2]. However, despite the use of second generation drugs that more potently block androgen synthesis (e.g abiraterone) or inhibit AR function (e.g enzalutamide), disease ultimately progresses to a therapy-resistant state called Castration Resistant PCa (CRPC) [1-4]. Remarkably, residual androgens that remain after castration and reactivation of AR signaling drives the development of CRPC and its continued growth. Based on AR-driven gene expression profiles at different disease stages, AR function switches from maintaining homeostasis to driving tumor formation [5-8].

 

The AR belongs to the nuclear hormone receptor superfamily that functions as ligand-activated transcription factors. The full-length AR has four functional domains composed of a divergent N-terminal domain (NTD), a central and highly conserved DNA-binding domain (DBD), a short hinge region, and a moderately conserved C-terminal ligand-binding domain (LBD) <!--[if supportFields]>

Start Date Duration in Months Target Completion Date Actual Completion Date
2017-07-15 36 2020-07-15 0000-00-00

Ongoing

Institution Classification Region LTO #
University of the Philippines - Diliman, National Institute of Molecular Biology and Biotechnology Public Higher Education Institution - State Universities and Colleges NCR
Institution Classification Region LTO #
Pascual Pharma Corp. Private Business NCR
Institution Amount Region
Philippine Council for Health Research and Development, Department of Science and Technology N/A NCR
Name Expertise Affiliation
Pia Bagamasbad, PhD Molecular Endocrinology University of the Philippines - Diliman, National Institute of Molecular Biology and Biotechnology
Isagani D. Padolina, PhD Chemistry and Drug Discovery Pascual Pharma Corp.
Chromewell Augustin Mojica Molecular Biology University of the Philippines - Diliman, National Institute of Molecular Biology and Biotechnology
Romie Angelo Azur Molecular Biology University of the Philippines - Diliman, National Institute of Molecular Biology and Biotechnology

Prostate Cancer
Castration Resistant Prostate Cancer

The broader goal of this study is to form an integrated strategy in screening natural products for prostate cancer therapy, recognizing that the therapeutic effect may modulated or potentiated by the different principal components in the extract. While it would be a worthwhile objective to produce a drug product during the course of this study, the practical intention is to establish a lentiviral facility capable of expressing cell lines specific to variants under study, a screening process utilizing a natural product library, and an assessment platform characterizing potency, specificity and mechanism of action, that can be integrated and applied to other screening studies.
 
Prostate cancer development and its progression into a castration resistant state is driven by androgen receptor (AR) overexpression and AR mutant variants. Given that AR functions as a transcription factor that directly binds to DNA to regulate the transcription of its target genes and based on the hypothesis that genes related by function share DNA response element signatures, a primary goal of this proposal is to identify compounds that can selectively modulate full length AR and inhibit mutant AR activity. Prior to drug screening, genetically engineered cell lines overexpressing wild-type and mutant AR variants will be generated to establish a bioassay that can identify selective AR modulators. Candidate compounds from the pilot screen provide proof of concept that drugs can affect AR response element recognition and promoter elements/signatures can distinguish pro-proliferative from pro-differentiative genes. Selective AR modulators (SARMs) can confer selective gene expression with the potential of reducing resistance, and may be useful as an adjuvant therapy against AR reactivation. Compounds that will inhibit mutant AR variants may be developed as therapeutic agents against castration resistant tumors that arise from the use of currently approved PCa therapies.
 
The specific aims of this proposal are as follows:
 
Aim 1: To establish a lentiviral vector facility to facilitate the generation of cell lines that will stably express mutant AR variants found in CRPC. Lentiviral mediated transduction will be used to stably integrate mutant AR variants into hard to transfect prostate cancer (LNCaP, VCaP or LAPC4), normal prostate epithelium and HeLa cells. Transgenic cell lines stably expressing mutant AR variants will be used in a compound screen similar to the drug screen performed using HeLa cells stabling expressing the full length AR.
 
Aim 2: To establish a bioassay that can identify potential selective androgen receptor modulators for use in prostate cancer therapy and perform a compound screen for SARM and AR mutant inhibitors using the compound/extract from Pascual Pharma Corp. The genetically engineered cell lines from Aim1 will be used in a bioassay can be used to screen any compound library to identify potential drugs for prostate cancer therapy. Initially the bioassay will be used to perform a compound screen using the compounds/extracts from the herbal extract library of Pascual Pharma Corp. developed in collaboration with the UPLB Natural Products Development Program. The extracts and its principal components are quantified using validated LC/MS/MS assay methods to ensure data traceability and reproducibility of the extraction profile. The reproducibility of the extraction profile, including the quantitation of the principal components for each plant extract, will enable proper carryover of preclinical and clinical test materials throughout the study. 
 
Aim 3: To validate compounds from pilot screen and characterize the potency, specificity and mechanisms by which they confer promoter selective gene regulation and mutant AR inhibition. Genetic, genomic and biochemical assays will be used to define mechanisms by which primary screen hits confer differential control of full length AR- and mutant AR-driven pathways.

- Establishment of a working BSL2 cell culture facility
- Acquisition of reagents and cell lines for the lentiviral core facility
- Generation of lentiviral constructs of AR mutants and variants for packaging in HEK293T cells
- Preparation of high concentration – high purity plasmids for packaging transfections
- Assembly of compounds/extracts for drug screening
- Generation and propagation of prostate cancer and HeLa cell lines stably expressing AR mutants and variants
- Standardized and optimized protocol for efficient transfection of HeLa and prostate cancer cell lines
- Validation of screening procedure using agonist, antagonist, and prior hit
- Identification of lead compounds for dose response analysis and subsequent validation in secondary biochemical assays
 
- Maintenance and long-term cryogenic storage of cell line stocks
- Identification of effects of compound on AR expression, localization, and ligand binding
- Validation of the ability of compounds to inhibit cell proliferation and/or promote apoptosis
- Establishment of the effect of compounds on expression of genes associated with proliferation and differentiation
- Standardized and optimized protocol for AR chromatin immunoprecipitation (ChIP) in prostate cancer cell lines
- Validation of the effect of compounds on recruitment of AR wild-type and mutant/variant forms to target genes
- Identification of the effects of compounds on recruitment of co-regulators, histone-modifying proteins, and RNA Pol II to genes differentially affected by compounds

Recruiting

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15 Jul 2017

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