Proliferating cell nuclear antigen in regulation of androgen receptor signalings in castration-resistant prostate cancer cells

全长雄激素受体(AR-FL)的过度表达和/或结构性活性AR剪接 变异体(AR-V)，如AR-V7和ARv567缺乏全部或部分配体结合结构域，有助于 耐去势前列腺癌(CRPC)的发生发展。雄激素剥夺疗法 (ADT)对过度表达AR-V的CRPC无效，甚至可诱导AR-V过度表达。 增殖细胞核抗原是一种非致癌性抗原，在所有肿瘤细胞中优先过表达。 DNA复制和修复以及细胞生长和存活所必需的蛋白质。天然的增殖细胞核抗原是一个环形的 同源三聚体主要分布在核质中。为了发挥作用，增殖细胞核抗原必须线性化或单体，以 重新定位到染色质、细胞质或细胞膜，并作为其功能的平台和执行 通过与含有增殖细胞核抗原相互作用蛋白盒(PIP-box)和其他基序的伙伴蛋白相互作用。 Pi‘s小组鉴定了AR N-末端的PIP-box的一致序列。增殖细胞核抗原复合体 AR-FL和AR-V7，可被PIP盒特异性抑制剂T2AA减弱。增殖细胞核抗原也与ARv567结合 并直接转化为重组AR蛋白。增殖细胞核抗原-I1S是由Pi‘s小组开发的一种小分子增殖细胞核抗原抑制物， 结合在两个单体的界面上，稳定三聚体结构，并减弱与 染色质。增殖细胞核抗原-I1S和T2AA抑制CRPC中AR转录活性和AR靶基因表达 LNCaP-AI和22Rv1细胞。抑制增殖细胞核抗原的表达可减少双氢睾酮刺激的AR 并取消增殖细胞核抗原-I1S对AR活性的抑制作用。更重要的是，AR- PI实验室新近开发的特异性PIP-box多肽抑制剂R9-AR-PIP可与增殖细胞核抗原结合并抑制AR 转录活性、AR靶基因的表达和AR阳性细胞的生长。基于这些 观察到，PI假设增殖细胞核抗原通过AR PIP盒与AR-FL和AR-V相互作用 增强AR转录活性，靶向PCNA-AR相互作用将减弱AR-FL- AR-V介导的信号转导和抑制过表达AR-FL和/或AR-VS的CRPC的生长。二 提出了具体的目标。目标1的研究将进一步阐明AR PIP-box在相互作用中的作用 AR-FL和AR-VS与增殖细胞核抗原的结合，研究增殖细胞核抗原-I1S，T2AA， 和R9-AR-PIP对增殖细胞核抗原与AR-FL和AR-VS共定位及染色质结合的作用 增殖细胞核抗原-AR相互作用在AR-FL和AR-VS向染色质募集中的作用及R-受体选择性抑制作用 9-AR-PIP对AR-增殖细胞核抗原相互作用的影响AIM 2的研究将确定增殖细胞核抗原-I1S、T2AA、 和R9-AR-PIP对AR-FL和AR-VS雄激素反应元件占有率的影响 AR-FL、AR-V7活性及AR-V特异性基因表达。此外，R9-AR-R的细胞毒作用也明显增强。 PIP对培养的CRPC细胞的影响，R9-AR-PIP对小鼠CRPC移植瘤的治疗作用 我们将研究R9-AR-PIP对CRPC肿瘤中AR信号的选择性抑制作用。

The overexpression of the full-length androgen receptor (AR-FL) and/or the constitutively active AR splicing variants (AR-Vs), such as AR-V7 and ARv567es lacking all or part of the ligand binding domain, contributes to the development and progression of castration resistant prostate cancers (CRPC). Androgen deprivation therapy (ADT) is not effective in CRPC overexpressing the AR-Vs and can even induce AR-V overexpression. Proliferating cell nuclear antigen (PCNA), preferentially overexpressed in all tumor cells, is a non-oncogenic protein essential for DNA replication and repair as well as cell growth and survival. Native PCNA is a ring-shaped homotrimer located mainly in the nucleoplasm. To be functional, PCNA must be linearized or monomerized for relocalization to chromatin, cytoplasm, or cell membrane, and serves as a platform for and executes its function through interaction with partner proteins containing the PCNA-interacting protein box (PIP-box) and other motifs. The PI’s group identified a consensus sequence of the PIP-box at the N-terminus of AR. PCNA complexes with AR-FL and AR-V7, which can be attenuated by a PIP-box-specific inhibitor T2AA. PCNA also binds to ARv567es and directly to recombinant AR protein. PCNA-I1S, a small molecule PCNA inhibitor developed by PI’s group, binds at the interface of two monomers, stabilizes trimer structure, and attenuates PCNA association to chromatin. PCNA-I1S and T2AA inhibit AR transcriptional activity and expression of AR target genes in CRPC LNCaP-AI and 22Rv1 cells. The knockdown expression of PCNA reduces dihydrotestosterone-stimulated AR transcriptional activity and abolishes the inhibitory effects of PCNA-I1S on AR activity. More importantly, an AR- specific PIP-box peptide inhibitor R9-AR-PIP, developed recently in the PI’s lab, binds to PCNA and inhibits AR transcriptional activity, expression of AR target genes, and growth of AR-positive cells. Based on these observations, the PI hypothesizes that PCNA interacts with AR-FL and AR-Vs through the AR PIP-box and enhances AR transcriptional activities and that targeting the PCNA-AR interaction will attenuate AR-FL- and AR-V-mediated signaling and inhibit growth of CRPC overexpressing AR-FL and/or AR-Vs. Two specific aims are proposed. Studies in aim 1 will further elucidate the role of the AR PIP-box in the interaction of AR-FL and AR-Vs with PCNA by protein mutagenesis and investigate the inhibitory effects of PCNA-I1S, T2AA, and R9-AR-PIP on the colocalization and chromatin association of PCNA with AR-FL and AR-Vs, the role of PCNA-AR interaction in recruitment of AR-FL and AR-Vs to chromatin, and the selective inhibitory effects of R- 9-AR-PIP on AR-PCNA interaction. Studies in aim 2 will determine the inhibitory effects of PCNA-I1S, T2AA, and R9-AR-PIP on the occupancy of the androgen response elements by AR-FL and AR-Vs, the transcriptional activity of AR-FL and AR-V7, and expression of AR-V-specific genes. Moreover, the cytotoxic effects of R9-AR- PIP on CRPC cells in culture, the therapeutic effects of R9-AR-PIP against CRPC xenograft tumors in mice, and the selective inhibitory effects of R9-AR-PIP on AR signaling in the CRPC tumors will be investigated.