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The current standard treatment of prostate cancer by androgen deprivation therapy involves using drugs such as bicalutamide (Casodex) to antagonistically block androgen receptors that are normally present within prostate cells. Usually, the therapy is successful in the short run at limiting the growth of prostate cancer. However, in virtually all cases tumors begin to grow aggressively again after several months of treatment and new therapies must be started. The mechanism by which these prostate cells transform from androgen sensitive to androgen independent and anti-androgen resistant is unclear. In this study, we investigated the role of microRNAs, small 15 to 18 nucleotide regulatory RNAs, in regulating the desensitization of prostate cancer cells to the androgen receptor antagonist drug bicalutamide. In order to identify significant microRNAs, quantitative PCR was used to obtain genome-wide microRNA expression levels of 885 human microRNAs at different timepoints for androgen sensitive LNCaP cancer cells treated with bicalutamide and for untreated control cells in tissue culture. Analysis of microRNA expression by clustering analysis and by statistical comparisons of treatment groups resulted in identification of 28 microRNAs that have altered expression in the progression process. In silico target prediction analysis was performed with the microRNAs shown to have altered expression, and a group of genes predicted to be under microRNA regulatory control during cancer progression to resistance was identified. A microRNA expression profile can be useful in developing more effective prognostic and therapeutic tools for prostate cancer.

The androgen hormone is responsible for the growth of secondary sex characteristics in humans, such as skeletal muscle. Upon an exercise stimulus, the androgen receptor (AR) plays a crucial role in transmitting the androgenic signal to the nucleus which upregulates transcription of target genes related to growth of skeletal muscle. AR content has been implicated in the hypertrophic response between high and low responders following resistance exercise training (RET) in males. Little is known of the impact of AR expression on acute skeletal muscle damage and whether AR may influence the adaptive response to RET in females. This study aimed to investigate acute changes to AR content following a single bout of muscle damage-inducing exercise as well as sex differences during skeletal muscle repair and hypertrophy. A skeletal muscle biopsy from the vastus lateralis was obtained from 26 healthy, young males (n=13) and females (n=13) at baseline and 48 hours following a single bout of 300 eccentric contractions of the quadriceps. Subsequently, participants performed whole-body RET 4 times a week for 10 weeks, followed by a final skeletal muscle biopsy under resting conditions. Females had greater AR mRNA than males at baseline (~53%) and post-damage (~126%; p<0.05) while AR protein content increased in both sexes similarly following a single bout of eccentric exercise (p<0.05). Damage- and RET-induced satellite cell response was associated with percent change in AR protein content in a sex-specific manner (p<0.05). RET-induced percent change in nuclear AR content was ~17% greater in males compared to females (p<0.05). Interestingly, following RET, males experiencing the highest percent change in myofibre cross sectional area (CSA) exhibited greater changes in nuclear-associated AR protein content compared to females with the highest percent change in CSA. Collectively, AR protein content is elevated following acute eccentric exercise and 10 weeks of RET. Findings from this study suggests that males are more reliant on AR-related mechanisms than females to induce skeletal muscle hypertrophy following RET. / Thesis / Master of Science in Kinesiology / Skeletal muscle is crucial for proper function and activities of daily living. Many factors can regulate the amount and quality of skeletal muscle such as the expression of a protein known as the androgen receptor (AR). The AR plays a role in many cellular pathways that can ultimately dictate the growth and size of a particular tissue like skeletal muscle. There is currently minimal research about AR during skeletal muscle damage and in female skeletal muscle. Understanding how exercise increases AR content in males and females could progress our knowledge of how muscle adapts differently to exercise between sexes. Therefore, the purpose of this study was to understand how the AR behaves in the muscle, in males and females, after a single session of exercise that damages the muscle or after long term resistance exercise (RE). We observed that the AR gene is more abundant in females than males at rest and following damaging exercise. Furthermore, we show that AR protein content increases in both sexes following a single session of damaging exercise and after chronic RE. Muscle stem cells are a component of the muscle that helps to heal muscle after exercise has been performed. In the current study, we demonstrate that AR has a closer relationship to muscle stem cells in males relative to females. Further, AR seems to be more closely linked to muscle growth in males than females. Altogether, AR is a component of the muscle that adapts to exercise differently in males and females. This study may, in part, explain how skeletal muscle responds differently between sexes after exercise.

Fracture healing requires a series of events including inflammatory response and callus formation, callus remodeling and bone healing. Fracture healing is a complex process, there are several overlapping phases , including inflammation , cartilage formation and bone remodeling, there are many internal or external factors could impact on fracture healing, leading to delayed bone healing or non healing. The global androgen receptor knockout (GARKO) mice has been know to reduce bone mass in endochondral bone and osteoblast mineralization, but the impact for callus formation in fracture healing is still unclear. The goals of study is to investigate the role of androgen and androgen receptor in wild-type (WT) mice and GARKO mice after fracture healing during callus formation and bone mineralization and bone remodeling. Therefore, long-term animal experiments observed by micro-computed tomography to study the roles of androgen and androgen receptor on the process and mechanisms of fracture healing is necessary. We applied in vivo micro-computer tomography (Micro-CT) to build up the three-dimensional model images at different time points for wild-type mice and GARKO mice after fracture healing and observe the bone healing process of micro-structure of the development of callus during fracture healing. The callus tissue morphology observed by histological staining to study the proportion and position of collagen, fibrous tissue and bone. The results show that the healing of WT mice is better than GARKO mice. GARKO mice develop smaller callus size and less bone volume and show delayed healing. In general, orchiectomy (ORX) decreases callus size in WT mice but not in GARKO mice. However, the healing rate of elderly GARKO mice is not obvious in comparison with young GARKO mice. Together, our study demonstrated that the androgen and androgen receptor regulate fracture healing and play an important role in bone repair and healing. Our mouse model may be used for the therapeutic drug screening of bone fractures caused by osteoporosis.

No description available.

Steroid receptors are important in the pathogenesis of a variety of disease states and modulate cellular processes through differential gene expression. Therefore, understanding the regulation of steroid receptors is essential. Environmental sodium arsenite, a toxin associated with male infertility, and arsenic trioxide, a possible prostate cancer therapeutic agent, are inorganic trivalent semimetals. The mechanism of arsenic action in male urogenital tract tissues is not clear. Since androgen receptor (AR) plays an important role in spermatogenesis and prostate cancer, we explored the possibility that trivalent arsenic regulates AR function. We found that arsenic inhibited AR transcriptional activity in prostate cancer and Sertoli cells by inhibiting AR recruitment to an AR target gene enhancer in vivo. Consistent with a deficiency in AR chromatin binding, arsenic disrupted AR amino and carboxyl-termini interaction. Furthermore, ATO caused a significant decrease in prostate cancer cell proliferation that was more pronounced in cells expressing AR compared to cells depleted of AR. Thus, arsenic-induced male infertility may be due to inhibition of AR activity and arsenic may serve as an effective therapeutic option in prostate cancer. Rac1, a Rho GTPase, modulates a variety of cellular processes and is hyperactive in cancer. Estrogen receptor (ER) regulates genes associated with cell proliferation, tumor development, and survival in breast cancer. Therefore, we examined the possibility of crosstalk between Rac1 and ER signaling. We found that Rac1 enhanced ER transcriptional activity in breast cancer cell lines. Vav3, a Rho guanine nucleotide exchange factor, was an upstream activator and P21/Cdc42/Rac1 activating kinase-1 (PAK-1) was a downstream effector of Rac1 enhancement of ER activity. These results suggest that Rac1 may be a beneficial therapeutic target. To test this hypothesis, we used EHT 1864, a small molecule Rac1 inhibitor. EHT 1864 inhibited ER transcriptional activity and estrogen-induced breast cancer cell proliferation. Furthermore, EHT 1864 inhibited ER activity by downregulation of ER mRNA and protein levels. Since ER plays a critical role in the pathogenesis of breast cancer and EHT 1864 inhibits ER activity and breast cancer cell proliferation, Rac1 inhibition is a novel and compelling therapeutic target in breast cancer.

Steroid receptor coactivators modulate the final outcome of hormone induced gene transcription by steroid receptors. E6-associated protein (E6-AP), an E3 ubiquitin ligase, acts as a coactivator of steroid receptors, including estrogen receptor (ER). In this study, we elucidated the contribution of E6-AP to ER-dependent gene transcription in breast cancer cells. siRNA-mediated knockdown of E6-AP abrogates transcription of classic ER target genes, GREB1 and pS2, suggesting that E6-AP is essential for normal transactivation function of ER. In order to understand the global influence of E6-AP in ER-dependent gene transcription, we used gene expression microarrays under E6-AP knockdown conditions to identify ER target genes which are regulated by E6-AP. Our microarray analysis revealed 455 genes which are differentially regulated by E6-AP. Pathway analysis revealed that E6-AP regulated genes were involved in cell cycle. Cell cycle profiling at various time points of estrogen treatment reveals that under E6-AP knockdown conditions, breast cancer cells progress slowly through S phase and eventually fail to proliferate. Knockdown of E6-AP has no effect on ovarian and uterine cells, suggesting that E6-AP has cell specific roles. Our analysis suggests that knockdown of E6-AP reduces the levels of early (C-Myc and Cyclin-D1), mid (E2F1, E2F2 and E2F7) and late (BUB1, BUBR1, MAD2, NDC80, NUF2 and CASC5) estrogen-dependent cell cycle genes. Overall our data indicate that E6-AP is a major regulator of cell cycle in breast cancer cells. E6-AP also acts as a coactivator for androgen receptor (AR) and we studied the role of E6-AP in prostate gland development. We report the generation of transgenic mice which specifically over expresses E6-AP in the prostate gland. Prostate glands in these mice are larger when compared with its wild-type litter mates, corroborating our observations that knockout of E6-AP in mice leads to impaired prostate gland development. E6-AP transgenic mice also develop prostatic intra epithelial neoplasia after 18 months of age. In addition to these observations, we also show that over expression of E6-AP in the prostate gland leads to increased Akt signaling. In order to understand the mechanism by which E6-AP regulates prostate gland growth, we generated LNCaP cells that stably overexpress E6-AP protein. Data from these cell lines show that the levels of phosphatidylinositol 3-kinase, total Akt, phosphorylated Akt (active Akt) and its down-stream target protein, GSKβ are elevated, suggesting that E6-AP regulates the PI3K-Akt signaling pathway. We further show that E6-AP modulates PI3K-Akt signaling by regulating the protein levels of RhoA, a small GTPase, which is a negative regulator of the Akt signaling pathway. In addition, we show that stable overexpression of E6-AP in prostate cancer cells results in increased proliferation. Overall our data suggests that E6-AP regulates the PI3K-Akt pathway in prostate cells which results in increased prostate cell growth, proliferation and tumorigenesis.

Background: Bone metastases occur in most patients with advanced hormone-refractory prostate cancer causing pain, pathologic fractures, and spinal cord compression. Few studies specifically address surgical treatment of metastatic spinal cord compression (MSCC) in prostate cancer. Criteria for identifying patients who may benefit from surgery are poorly defined. Most of the current knowledge regarding tumor biology in prostate cancer is based on studies of primary tumors or soft tissue metastases. The mechanisms regulating growth of bone metastases are not fully established. Aims: a) to evaluate outcome after surgery for MSCC in prostate cancer and to identify prognostic factors for survival and functional recovery; b) to evaluate current practice for referral of prostate cancer patients with MSCC; c) to analyze expression of androgen receptor (AR), cell proliferation, apoptosis, and prostate-specific antigen (PSA) in bone metastases with regard to survival after surgery for complications of bone metastases. Patients and Methods: We retrospectively evaluated the hospital records of 68 consecutive patients operated for metastatic spinal cord compression. Tumor tissue from bone metastases was obtained on spinal surgery (54 patients), fracture surgery (4 patients) and biopsy (2 patients), and analyzed by immunohistochemistry. Results: Study I: Mortality and complication rate after surgery was high. Patients with hormone-naïve disease and those with hormone-refractory disease with good performance status and without visceral metastases had more favorable survival. The ability to walk after surgery was related to better survival. Study II: A new score for prognosis of survival after surgery for spinal cord compression includes: hormone status of prostate cancer, Karnofsky performance status, evidence of visceral metastasis, and preoperative serum PSA. The score is simple, tumor specific, and easy to apply in clinical practice. Study III: Our results suggest that delays in diagnosis and treatment may have negative impact on functional outcome. Pretreatment ability to walk, hormone status of prostate cancer, and time from loss of ambulation influenced neurological recovery after surgery for spinal cord compression. Study IV: High nuclear AR immunostaining in bone metastases and high preoperative serum PSA were associated with a poor outcome after metastasis surgery in patients with hormone-refractory prostate cancer. Short-term effect of castration therapy disclosed that nuclear AR immunostaining was decreased and apoptosis was increased, but cell proliferation remained largely unaffected. Conclusion:  Prostate cancer patients with metastatic spinal cord compression represent a heterogeneous group. We identified prognostic factors for survival and functional outcome, which may help clinicians in making decisions about treatment. Our results also implicate the need for development of local and regional guidelines for treatment of patients with spinal cord compression, as well as the importance of information to patients at risk.

Understanding and studying the normal bone growth and development is causal. Bone and cartilage tissue provide in addition to their mechanical support, they provide a protection for vital organs such as heart, lung and brain. Longitudinal growth is regulated by the activity of chondrocytes in the epiphyseal growth plates of long bones. Many hormones and growth factors are involved in the regulation of this process. Among these, sex steroids are of crucial importance, especially during puberty. In long bones, endochondral bone formation occurs at the growth plate, a region of developing cartilage located between the epiphysis and the metaphysic. The process of endochondral ossification is regulated in part by sex steroid hormones. Androgens stimulate endochondral bone growth and elongation, while estrogen is known to suppress longitudinal bone growth and accelerate growth plate closure. Studies using rat costochondral growth plate chondrocytes as a model show that the effects of 17β-estradiol (E₂) on apoptosis are found in both male and female cells and the same mechanism is involved. In contrast, E₂ causes rapid activation of PKC in female cells but not in male cells. Dihydroxytestosterone (DHT) also has direct effects on growth plate chondrocytes, increasing matrix synthesis including sulfated glycosaminoglycan production, and enhancing cell maturation by increasing alkaline phosphatase enzymatic activity. Short stature and abnormally slow increase in height is one of the main reasons for referral to endocrinologist. Excessive growth and abnormally tall is also a problem, especially because it increase risk for the trunk abnormalities. Furthermore until now a few growth-promoting therapies are available for clinical use. Therefore future therapies for treating the growth disorders are essential. The overall goal of this project is to investigate the sexual-dimorphic effect of the sex steroid hormone in rat growth plate chondrocytes, the cellular signaling pathways mediating these actions, and their physiological role. The information gleaned from this study will provide new information about the role of sex steroid hormones in chondrogenesis and has implications in the development of new therapies for the treatment of bone fracture healing, and growth plate disorders. The central hypothesis was that sex steroid would play an important and sex-specific role in regulating chondrocytes as a main regulator of longitudinal bone growth.

H2A.Z is an essential histone variant that has multiple chromosomal functions. One such role is transcriptional regulation. However, its role in this process is complex since it has been reported to function both as a repressor and activator. Earlier work in our lab showed that H2A.Z can be post-translationally modified with monoubiquitin (H2A.Zub1) and this form of H2A.Z is linked to transcriptional silencing. We further predicted that changes in the H2A.Z ubiquitylation status directly modulated its function in transcription. Furthermore, H2A.Z-containing nucleosomes possess a unique set of post-translational modifications (PTMs), compared to H2A nucleosomes, many of which are linked to transcriptional activation. The central aim of this thesis was to characterize the role of PTMs on H2A.Z nucleosomes in transcriptional regulation. To this end, I have provided the first evidence linking H2A.Z deubiquitylation to transcriptional activation. I demonstrated that ubiquitin specific protease 10 (USP10) is a deubiquitylase that targets H2A.Z in vitro and in vivo. Moreover, I found that both H2A.Z and USP10 are required for activation of androgen-receptor (AR)-regulated genes, and that USP10 regulates the levels of H2A.Zub1 at these genes. To understand how H2A.Z engages downstream effector proteins, in the nucleosome context, we used a mass spectrometry approach to identify H2A.Z-nucleosome-interacting proteins. Many of the identified proteins contained conserved structural motifs that bind post-translationally modified histones. For example, we found that Brd2 contains tandem bromodomains that engage H2A.Z nucleosomes through acetylated H4 residues. To investigate the biological relevance of this interaction, I present evidence that Brd2 is recruited to AR-regulated genes in a manner dependent on H2A.Z and the bromodomains of Brd2. Consistent with this observation, chemical inhibition of Brd2 recruitment greatly inhibited AR-regulated gene expression. Collectively, these studies have defined how H2A.Z mediates transcriptional regulation through multiple mechanisms and pathways.