FoxO proteins are transcriptional factors acting downstream of the tumor suppressor PTEN. Their activity is negatively regulated by AKT-mediated phosphorylation. Our previous studies showed a mutual suppression between PTEN and the androgen receptor (AR) in regulating growth and apoptosis in prostate cancer (PCa) cells. We hypothesize that nuclear FoxO proteins are involved in mediating this mutual antagonism. In this dissertation, we report that PTEN inhibits AR activity through FoxO1 and provide evidence for the involvement of FoxO factors in the androgen-mediated suppression of PTEN-induced apoptosis. Our studies identify a novel mechanism for AR inhibition by FoxO1 and demonstrated the participation of FoxO1 in AR inhibition by PTEN. Ectopic expression of active FoxO1 decrease the transcriptional activity of the AR as well as androgen-induced cell proliferation and production of prostate-specific antigen in PCa cells. FoxO1 knock down by RNA interference increased the transcriptional activity of the AR in PTEN intact cells and relieved its inhibition by ectopic PTEN in PTEN null cells. Mutational analysis revealed that FoxO1 region 150-655, which contains the fork head box and C-terminal activation domain, was required for AR inhibition. Mammalian two-hybrid assays demonstrated that the inhibition of AR activity by PTEN through FoxO1 involved the interference of androgen-induce interaction of the N- and C- termini of the AR and the recruitment of the p160 coactivators to the AR N-terminus. In addition to the inhibition of AR by FoxO1, we also demonstrated that PTEN-induced apoptosis is mediated through FoxO factors and that AR inhibited FoxO1 activity by yet-to-be identified downstream target gene. Mutation of AR DNA binding domain partially relieved the inhibition of FoxO1 trasnscriptional activity by androgens. Inhibiton of new protein synthesis abolished the AR-mediated decrease in the mRNA level of FoxO1 target gene. Overall, these studies reveal novel mechanisms for the mutual inhibition of AR and FoxO1 activity and establish FoxO proteins as important nuclear factors that mediate the mutual antagonism between AR and PTEN tumor suppressor in PCa cells.

Androgens are critical for the development and maintenance of adult male characteristics such as muscle mass and sexual function. Consequently, the established decline with age of serum testosterone (T) in males has major health implications. While the androgen receptor (AR) is the major mediator of genomic androgen action and is required for the development of the male phenotype, reproductive organs and the maintenance of male secondary sexual characteristics, it is the entrance of androgens into the cell that mediates the activation of the AR and the subsequent modulation of expression of androgen regulated genes. Testosterone, biologically the most important androgen in male serum, circulates either free, loosely bound to albumin or tightly bound to sex hormone binding globulin (SHBG). Each of these forms of serum T have different abilities to enter cells, and which proportion of serum T is capable of entering cells and initiating the androgen signalling cascade, thereby leading to the activation of the AR has not been precisely defined. The AR amino terminal domain (NTD) is responsible for the majority of the ability of the AR to activate genes but the relative roles of the two activation functions in the AR NTD (activation functions 1 and 5; AF1 and 5) have not been precisely defined while the role of the AF2 surface which forms in the ligand binding domain upon agonist binding is responsible for interactions with key coregulators and also with the NTD in the amino-carboxyl (N/C) interaction. Our laboratory has recently identified a region within AF5 between amino acids 500-535 to which somatic mutations in castrate resistant prostate tumour samples collocate. Due to the lack of functional information on the AF5 region and the NTD in general, the function of this region and the functional consequences of the mutations remain to be defined. The objectives of this thesis were to develop a specific mammalian cell based bioassay capable of reliable measuring T in serum and to determine the ability of this bioassay to measure a physiologically relevant fraction of T in serum. Additionally, this thesis aimed to determine the relative contributions and roles of the activation functions of the AR to overall AR transcriptional activity along with the functional consequences for AR signalling of prostate cancer mutations which have previously been identified in the AF5 region of the AR NTD. The mammalian-cell based bioassay developed in this thesis is capable of sensitively and reliably measuring serum T. However, evaluation of this bioassay utilising approximately 1000 serum samples from the Florey Adelaide Male Aging Study reveals that this bioassay measures a fraction of T in serum that most closely relates to serum T. Furthermore, this measure does not correlate more strongly with grip strength, sexual function or waist circumference than the existing immunoassay-based measures of serum T, highlighting the limitations of utilising a static mammalian cell-based androgen bioassay to measure physiological levels of serum T in males. The investigation of the roles of the activation functions in the AR in this thesis have revealed that while the AF1 domain is responsible for the majority of the transactivation activity of the AR, AF5 and AF2 govern the sensitivity and cellular response of the AR to androgens by providing protein and interdomain interaction interfaces. Furthermore, the evidence in this thesis demonstrates that the AR requires interdomain communication for sensitive AR signalling. Finally, the findings in this thesis demonstrate that the AF5 surface is required for the N/C interaction and coregulator interactions while advanced prostate cancer mutations identified within this region confer increased transactivation activity of the AR in the presence of high cellular levels of coregulators. Collectively, the findings in this thesis provide several novel insights into the mechanism of action of serum androgens and challenges several long held assumptions of androgenic action in males. These findings also delineate a mechanism of treatment failure in advanced prostate cancer, provide a novel model for the events leading to sensitive AR transactivation and contribute to the understanding of physiologically relevant levels of serum T. / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008