The rat androgen receptor gene promoter

The androgen receptor (AR) is activated upon binding of testosterone or dihydrotestosterone and exerts regulatory effects on gene expression in androgen target cells. To study transcriptional regulation of the rat AR gene itself, the 5' genomic region of this gene was cloned from a genomic library and the promoter was identified. S1-nuclease protection analysis showed two major transcription start sites, located between 1010 and 1023 bp upstream from the translation initiation codon. The area surrounding these start sites was cloned in both orientations in a CAT reporter plasmid. Upon transfection of the constructs into COS cells, part of the promoter stimulated transcription in an orientation-independent manner, but the full promoter showed a higher and unidirectional activity. In the promoter/reporter gene constructs, transcription initiated from the same positions as in the native gene. Sequence analysis showed that the promoter of the rat AR gene lacks typical TATA and CCAAT box elements, but one SP1 site is located at about 60 bp upstream from the major start site of transcription. Other possible promoter elements are TGTYCT sequences at positions -174 to -179, -434 to -439., -466 to -471, and -500 to -505, resembling half-sites of the glucocorticoid-responsive element (GRE). Furthermore, a homopurine stretch containing a total of 8 GGGGA elements and similar to sequences that are present in several other GC-rich promoters, is located between -89 and -146 bp upstream from the major start site of transcriptio

Eighty years of targeting androgen receptor activity in prostate cancer: the fight goes on

Prostate cancer (PCa) is the most common cancer in men in the West, other than skin cancer, accounting for over a quarter of cancer diagnoses in US men. In a seminal paper from 1941, Huggins and Hodges demonstrated that prostate tumours and metastatic disease were sensitive to the presence or absence of androgenic hormones. The first hormonal therapy for PCa was thus castration. In the subsequent eighty years, targeting the androgen signalling axis, where possible using drugs rather than surgery, has been a mainstay in the treatment of advanced and metastatic disease. Androgens signal via the androgen receptor, a ligand-activated transcription factor, which is the direct target of many such drugs. In this review we discuss the role of the androgen receptor in PCa and how the combination of structural information and functional screenings is continuing to be used for the discovery of new drug to switch off the receptor or modify its function in cancer cells

Molecular Mechanisms of Androgen Action : Characterization of the Androgen Regulated Gene KLK4 and the Role of Nuclear Dynamics in Androgen Receptor-Mediated Transcription

Androgens are important for the normal development of the male sexual characteristics, but are also involved in pathological conditions such as prostate cancer. The biological effects of androgens are mediated by the androgen receptor (AR), which is a transcription factor that regulates the expression of a wide range of target genes. In this thesis, KLK4 was identified as an androgen regulated gene that is specific to the prostate for expression. Surprisingly, KLK4 has a different gene structure than the other closely related members of the kallikrein family, resulting in an intracellular protein. Analysis of KLK4 expression in prostate tissue specimens showed that KLK4 was overexpressed in malignant prostate as compared to normal prostate glands. Experiments in prostate cancer cells furthermore demonstrated that KLK4 is a proliferative factor, possibly through the alteration of cell cycle regulatory gene expression. These data suggest that KLK4 plays an important role in prostate cancer, thus having potential utility as a diagnostic marker or therapeutic target in prostate cancer therapy. Using advanced fluorescence microcopy techniques, the interaction between AR and its target genomic sites in living cell were also studied. In contrast to the traditional view, the interaction was found to be both transient and highly dynamic, and depended on the nature of ligand. Importantly, there were intramolecular interactions between the N- and C-termini of promoter-bound AR in its active state which were important for transcriptional activity. Finally, increased AR transcriptional activity, induced by histone deacetylase inhibitors, resulted in reduced mobility of AR at its target promoter. These data support the more recent view of transcription factor–chromatin interactions as a highly dynamic system in continuous change, thus providing a kinetic and mechanistic basis for regulation of gene expression by androgens and anti-androgens in living cells

Intratumoral androgen biosynthesis in prostate cancer: Evidence from preclinical models and clinical specimens

Androgens regulate the growth and development of normal prostate and prostate cancer. Blocking the production and effects of androgens, either by castration or medication, has been the most efficient strategy for treating metastatic prostate cancer for decades. Although most patients respond to the therapy, in many of them the disease progresses to castration-resistant prostate cancer (CRPC) that cannot be cured with current therapies. Intratumoral androgen biosynthesis has been identified as one of the mechanisms leading to castration resistance. Recent studies have confirmed that prostate tumors can synthesize androgens by themselves to maintain tumor growth in androgen-deprived conditions. Thus, suppressing intratumoral androgen biosynthesis has become an attractive option for drug development. To understand the mechanisms of intratumoral androgen biosynthesis and develop new CRPC therapies, better preclinical models for CRPC are needed. In this study, we developed an orthotopic VCaP xenograft model suitable for studying the progression of CRPC and the mechanisms of intratumoral androgen biosynthesis in vivo. The VCaP model exhibited the clinical features of CRPC, including the activation of intratumoral androgen biosynthesis and the overexpression of androgen receptor (AR) and its splice variants. Furthermore, novel antiandrogens enzalutamide and ARN-509 reduced intratumoral androgen levels and altered steroidogenic enzyme expression in the VCaP model. In addition to preclinical studies, androgen levels were analyzed in prostate and serum specimens obtained from prostate cancer patients. Intraprostatic androgen levels in cancerous and benign samples were highly variable between the patients. Distinct intratumoral androgen levels and altered AR target gene expression were associated with TMPRSS2-ERG fusion gene expression

Hormonal control of spermatogenesis: expression of FSJH receptor and androgen receptor genes

FSH and testosterone are the main hormonal regulators of spermatogenesis. The actions of androgens and FSH are mediated by their respective receptors. Receptor gene expression (mRNA and protein). is an important determinant of hormone action. Biochemical aspects of the regulation of androgen and FSH receptor gene expression in the testis were chosen as the subject of the studies described in this thesis. Regulation of the expression of the receptor genes was studied at the level of gene transcription, and at the level of mRNA and protein expression. In Chapters 2-4, a detailed characterization is given of the effects of FSH on androgen and FSH receptor mRNA and protein expression in cultured immature Sertoli cells. For the androgen receptor, these findings were extended by measurements of androgen receptor gene transcription initiation rate in cultured immature Sertoli cells and LNCaP Oymph node carcinoma of the prostate) cells (Chapter 5). Preliminary results concerning a putative paracrine factor, produced by Sertoli cells and affecting androgen receptor mRNA expression in peritubular myoid cells, are presented in Chapter 6. The effects of testosterone deprivation iD. vivo on androgen receptor mRNA and protein expression in the adult rat testis were examined as described in Chapter 7.ffi vitro effects of testosterone on androgen receptor gene expression in cultured testicular cells and LNCaP cells are described in the Chapters 2, 3 and 5. In the General Discussion (Chapter 8) we have considered some aspects of regulation of spermatogenesis by FSH and testosterone and have discussed them in relation to our experimental data as well as in a broader perspective. This way, we hope that the results which we have presented, and discussions which we have tried to initiate, may contribute to research concerning hormonal control of spermatogenesis, now and in the futur

Hormonal control of spermatogenesis: expression of FSJH receptor and androgen receptor genes

FSH and testosterone are the main hormonal regulators of spermatogenesis. The actions of androgens and FSH are mediated by their respective receptors. Receptor gene expression (mRNA and protein). is an important determinant of hormone action. Biochemical aspects of the regulation of androgen and FSH receptor gene expression in the testis were chosen as the subject of the studies described in this thesis. Regulation of the expression of the receptor genes was studied at the level of gene transcription, and at the level of mRNA and protein expression. In Chapters 2-4, a detailed characterization is given of the effects of FSH on androgen and FSH receptor mRNA and protein expression in cultured immature Sertoli cells. For the androgen receptor, these findings were extended by measurements of androgen receptor gene transcription initiation rate in cultured immature Sertoli cells and LNCaP Oymph node carcinoma of the prostate) cells (Chapter 5). Preliminary results concerning a putative paracrine factor, produced by Sertoli cells and affecting androgen receptor mRNA expression in peritubular myoid cells, are presented in Chapter 6. The effects of testosterone deprivation iD. vivo on androgen receptor mRNA and protein expression in the adult rat testis were examined as described in Chapter 7.ffi vitro effects of testosterone on androgen receptor gene expression in cultured testicular cells and LNCaP cells are described in the Chapters 2, 3 and 5. In the General Discussion (Chapter 8) we have considered some aspects of regulation of spermatogenesis by FSH and testosterone and have discussed them in relation to our experimental data as well as in a broader perspective. This way, we hope that the results which we have presented, and discussions which we have tried to initiate, may contribute to research concerning hormonal control of spermatogenesis, now and in the futur