c-Jun and Androgen Signaling in Prostate Cancer Cells

描述（申请人提供）：雄激素受体（AR）通过调节基因表达介导雄激素的生物学效应。这种AR转录活性受辅激活因子的调节，其中c-Jun因其在AR调节的基因表达中的广泛作用及其在前列腺癌进展中的潜在作用而重要。细胞增殖和侵袭性是前列腺癌进展中涉及的两个重要AR生物学效应，我们有证据表明AR的c-Jun共激活介导了这两个细胞过程。在增殖方面，我们已经产生了一系列在c-Jun表达和因此细胞增殖方面不同的前列腺癌细胞系。有趣的是，我们的数据表明，c-Jun共激活支持前列腺癌细胞增殖，但c-Jun反式激活是抗增殖的。关于侵袭性，我们已经确定Ets变体基因1（ETV 1）作为AR调节的基因，介导前列腺癌的侵袭性。重要的是，ETV 1表达的AR诱导是在c-Jun共激活下进行的，这首次证明了c-Jun共激活在前列腺癌侵袭性中的重要作用。因此，我们的主要假设是c-Jun对AR的共激活功能介导前列腺癌细胞增殖，而其反式激活功能拮抗增殖。此外，我们假设c-Jun通过增强ETV 1的表达和活性来支持前列腺癌细胞的侵袭性。这些假设将在三个具体目标中得到检验。目的1：通过研究c-Jun的磷酸化和非磷酸化，分析c-Jun对AR的共激活作用，并通过将Ser 63和Ser 73突变为带负电荷的谷氨酸（Glu）来模拟磷酸化的作用。我们还将研究c-Jun以任一磷酸化形式通过染色质免疫沉淀（ChIP）测定与AR一起招募到雄激素调节的启动子并与LNCaP细胞中的AR相关联的可能性。在目标2中，我们将研究c-Jun在前列腺癌细胞中调节ETV 1和下游ETV 1靶基因的AR诱导中的作用。将研究c-Jun和AR在前列腺癌细胞侵袭中调节ETV 1表达和ETV 1活性的能力。在目标3中，我们将通过c-Jun反式激活来表征前列腺癌细胞中的抗增殖机制。我们以前提供的数据表明，反式激活功能是抗生长和共激活是促生长。我们将利用c-Jun的磷酸化模拟物来研究其对LNCaP细胞增殖的影响，并确定参与增殖抑制的下游靶基因。这项拟议的工作将使我们能够研究c-Jun在前列腺癌细胞中的抗增殖活性，这与c-Jun的致癌作用相反。此外，我们对ETV 1的研究将提供一个机会来研究AR和c-Jun在调节基因特异性表达方面的相互作用。前列腺癌是男性死亡的第二大原因，正在经历从可治疗的依赖性前列腺癌到通常致命的难治性前列腺癌的转变。雄激素受体和调节因子是前列腺癌两个阶段的关键因素。我们已经确定了原癌蛋白c-Jun作为雄激素受体活性的重要调节蛋白，我们建议在这里研究c-Jun在控制前列腺癌细胞增殖和转移的雄激素受体调节基因表达中的作用。

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.