Lirim Shemshedini - US grants

[unreadable] DESCRIPTION (provided by applicant): The androgen receptor (AR) mediates the biological effects of androgens by regulating gene expression. This AR transcriptional activity is regulated by coactivators, among which c-Jun is important for its widespread role in AR-regulated gene expression and its potential role in prostate cancer progression. Cellular proliferation and invasiveness are two important AR biological effects involved in prostate cancer progression, and we have evidence suggesting that c-Jun coactivation of AR mediates both of these cellular processes. On the proliferation side, we have generated a series of prostate cancer cell lines that differ in c-Jun expression and, consequently, cellular proliferation. Interestingly, our data suggest that c-Jun coactivation supports prostate cancer cellular proliferation, but c-Jun transactivation is anti-proliferative. With respect to invasiveness, we have identified Ets Variant Gene 1 (ETV1) as an AR-regulated gene that mediates prostate cancer invasiveness. Importantly, AR induction of ETV1 expression is under c-Jun coactivation, representing the first demonstration of an important role for c-Jun coactivation in prostate cancer invasiveness. Hence, our major hypothesis is that the c-Jun coactivation function on AR mediates prostate cancer cell proliferation, while its transactivation function antagonizes proliferation. Additionally, we hypothesize that c-Jun supports prostate cancer cell invasiveness by enhancing both the expression and activity of ETV1. These hypotheses will be tested in three specific aims. In aim 1, we will analyze the coactivation function of c-Jun on AR by studying phosphorylated and non-phosphorylated c-Jun. The effect of phosphorylation will be mimicked by mutating both Ser63 and Ser73 to the negatively charged glutamic acid (Glu). We will also study the possibility of c-Jun in either phosphorylation form to be recruited with AR to androgen-regulated promoters by chromatin immunoprecipitation (ChIP) assays and to associate with AR in LNCaP cells. In aim 2, we will study the role of c-Jun in regulation of AR induction of ETV1 and of downstream ETV1 target genes in prostate cancer cells. c- Jun and AR will be studied for the ability to regulate ETV1 expression and ETV1 activity in prostate cancer cell invasiveness. In aim 3, we will characterize the mechanism of anti-proliferation by c-Jun transactivation in prostate cancer cells. We have previously provided data suggesting that transactivation function is anti-growth and coactivation is pro-growth. We will utilize a phosphorylation mimic of c-Jun to study its effect on LNCaP cellular proliferation and to determine downstream target genes that are involved in repression of proliferation. This proposed work will allow us to study the anti-proliferative activity of c-Jun in prostate cancer cells, an activity contrary to c-Jun's oncogenic action. In addition, our studies with ETV1 will provide an opportunity to examine the interaction between AR and c-Jun in regulating gene-specific expression. Prostate cancer is the second leading cause of death among men, undergoing a transition from the treatable hormone-dependent to the usually lethal hormone-refractory. The androgen receptor and regulatory factors are key factors in both stages of prostate cancer. We have identified the proto-oncoprotein c-Jun as an important regulatory protein for androgen receptor activity, and we propose here study the role of c-Jun in androgen receptor-regulated gene expression that controls the proliferation and metastasis of prostate cancer cells. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The growth and progression of prostate cancer are dependent on androgens and AR (androgen receptor). The major function of AR in the prostate is to regulate the proliferation and survival of prostate cells and this function is mediated by the ability of AR to modulate gene expression. While many androgen-regulated genes have been identified in the prostate, few have been shown to play a pivotal role in prostate cancer cell growth and progression. This led us to postulate that gene products not yet identified may be important in mediating the AR effects on cellular proliferation. Through gene array studies in LNCaP cells, we identified sGC11 as a novel androgen-regulated gene, whose expression level is directly related to prostate cancer cell proliferation. We base this on several key observations from our laboratory. First, siRNA knock-down of sGC11 severely compromised the proliferation of both androgen-dependent and -independent LNCaP cells. Secondly, sGC11 expression in androgen-independent cells is high and androgen unresponsive. Finally, sGC11 over- expression in androgen-dependent cells is sufficient for reproducing their androgen-induced growth. In addition, preliminary expression data using prostate tissues show that sGC11 is highly expressed in malignant androgen-dependent prostate cancer, and this is significantly elevated beyond this in advanced androgen- independent prostate cancer. Our overall hypothesis is that sGC11 is an important component of AR action in prostate carcinogenesis. Accordingly, the specific aims of this proposal are to (1) study the role of sGC11 in the proliferation, apoptosis, and tumorigenesis of prostate cancer cells, (2) elucidate the signaling pathway that is responsible for pro-proliferative actions of sGC11 in prostate cancer cells, (3) study the mechanism of androgen regulation of sGC11 expression in prostate cancer cells, and (4) measure the expression of sGC11 during the progression of prostate cancer. In view of the importance of AR in the development and progression of prostate cancer, the identification of sGC11 as a novel androgen-regulated gene may provide an important tool for studying the role of androgens and AR in prostate carcinogenesis. Because of its implicated role in the proliferation of prostate cancer cells and recently identified anti-p53 activity, sGC11 may be very important in the initiation and progression of prostate cancer and thus be a potential target of future cancer therapies. PUBLIC HEALTH RELEVANCE: Prostate cancer is the second leading cause of death among men, undergoing a transition from the treatable hormone-dependent to the usually lethal hormone-refractory. The androgen receptor and regulatory factors are key factors in both stages of prostate cancer. We have identified sGC11 as a novel androgen-regulated gene important for the proliferation of both androgen-dependent and, more importantly, androgen-independent prostate cancer cells, and we propose here study the role of sGC11 in the growth and tumorigenesis of prostate cancer cells.

A grant has been awarded to the Department of Biological Sciences at The University of Toledo to support the acquisition of: 1) the Storm 860 Gel and Blot Imaging system; 2) the ImageQuant RT ECL Imaging system; and 3) a SpectraMax M5 Multi-detection plate reader. This instrumentation will support the research programs of the PI and Co-PIs and other members of the Department of Biological Sciences focused on cell signaling and regulation of gene expression in human cells and in model systems (nematodes and plants). In addition, these instruments will also be available to the broader research community in other departments at The University of Toledo. These instruments will provide investigators with more advanced and sensitive tools for conducting assays that they currently do not have the capabilities of performing.

The acquisition of these instruments will have a significant broader impact on the educational mission of the Department of Biological Sciences and The University of Toledo. The PI/Co-PIs as well as the department as a whole are strongly committed to the integration of research and education. The use of these instruments will be available to undergraduate students working on research projects under the direction of a faculty mentor. In addition, the use of these instruments will be incorporated into several upper division undergraduate laboratory courses, in particular the core Molecular Genetics and Cell Biology (taught by the PI) labs. Underrepresented groups, through the department?s commitment to the STARS and Glenn-Stokes programs will also benefit from the acquisition of these instruments.