Author: Yabin Zhou

Zhou, Yabin, 2011 ANDROGEN RESPONSIVE ELEMENTS IN TRPM8 GENE IN PROSTATE CANCER CELLS, Flinders University, School of Medicine

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On the basis of previous knowledge of the androgen receptor regulation of other genes in prostate epithelial cells and of prostate specific antigen gene expression, it was hypothesized that regulation of TRPM8 gene expression by the androgen receptor is through a genomic pathway by binding at least two AREs at the TRPM8 promoter: one in the proximal promoter initiates TRPM8 transcription; the other one at a distal promoter site enhances the TRPM8 transcription activity. The aim of this thesis was to test this hypothesis. Transient Receptor Potential Maelastatin 8 (TRPM8) non-selective cation channel is a member of the TRP family of cation channels. The expression of TRPM8 is regulated by the androgen receptor. It has been proposed that the functions of TRPM8 may be to act as a cold sensor and/ or to regulate regulation of ion and protein secretion in the prostate gland. TRPM8 may also be involved in the regulation of prostate epithelial cell proliferation or apoptosis. In the benign prostate and in the early stages of prostate cancer, TRPM8 expression increases whereas its expression in advanced stages of prostate cancer decreases. In hormone refractory prostate cancer TRPM8 expression is lost. These observations indicate a potential role of TRPM8 in the progression of prostate cancer, especially the early stages of the cancer. The distinct expression patterns of TRPM8 in different stages of prostate cancer also make it a potential marker for diagnosis and for prediction programs for prostate cancer. The bioinformatic search for ARE in the TRPM8 promoter was performed in human, chimpanzee and baboon. In the 6 kb TRPM8 promoter, four putative AREs have been identified and are conserved across the three species. The 6 kb TRPM8 promoter was subsequently cloned into a luciferase reporter vector (pGL3 Basic) to investigate its androgen responsiveness. However, insignificant androgen response was found. The endogenous TRPM8 mRNA expression pattern during androgen depletion and re-addition was also investigated by RT-qPCR. This was to predict the potential response pattern of the 6 kb TRPM8 promoter during androgen treatment in the luciferase experiments. The endogenous TRPM8 mRNA expression study also provided information to optimize the conditions for the luciferase experiments. The search for AREs was then expanded to the region from +1 kb to -30 kb (relative to the transcription start site) of the 5' regulatory region and the 5' untranslated region of TRPM8 using ChIP -qPCR analysis to directly detect the in vivo androgen receptor binding sites. 32 pairs of qPCR primers were designed and synthesized. They were validated by PCR-gel electrophoresis followed by sequencing confirmation. The ChIP-qPCR has identified an in vivo AR binding site, designated as Androgen Receptor Binding Site 1 which contains a putative ARE, located at around -28 kb of the 5' regulatory region of the TRPM8 gene. The following functional test of Androgen Receptor Binding Site 1 by luciferase reporter gene assay showed that with androgen stimulation, it specifically inhibited luciferase expression in a luciferase reporter construct driven by the 6 kb TRPM8 promoter. In conclusion, we have detected a potential ARE that serves as a silencer in the 30 kb 5' regulatory region of the TRPM8 gene. This indicated a more complicated mechanism of androgen receptor regulation of TRPM8 gene transcription: the androgen receptor may bind to some other ARE(s) to initiate and /or enhance TRPM8 gene transcription; under certain condition, AR may repress the TRPM8 gene transcription through the binding to the silencer within Androgen Receptor Binding Site 1. However, further experiments are needed to test this hypothesis. In future experiments, it would be helpful for our understanding of AR regulation of TRPM8 gene expression to 1) identify the ARE sequence within the Androgen Receptor Binding Site 1; 2) further test the four putative AREs identified by cross species analysis; 3) search for AREs in the 5' untranslated region, introns of the TRPM8 gene; 4) identify the region in the TRPM8 promoter that the Androgen Receptor Binding Site 1 interacts with to exert its regulatory function.

Keywords: Androgen responsive element (ARE),AR,ChIP assay,DHT,LNCaP,pGL3,Promoter,Prostate cancer (PCa),TRPM8

Subject: Medical Biochemistry thesis

Thesis type: Doctor of Philosophy
Completed: 2011
School: School of Medicine
Supervisor: Professor Greg Barritt