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

增殖细胞核抗原对去势抵抗性前列腺癌细胞雄激素受体信号传导的调节

• 批准号: 10350984
• 负责人: Zhongyun Dong
• 金额: $ 22.72万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10350984
• 关键词: Androgen Receptor; Androgen Response Element; Attenuated; Automobile Driving; Binding; Binding Proteins; Biological Assay; C-terminal; Cell Cycle Progression; Cell Survival; Cell membrane; Cells; Chromatin; Complex; Consensus Sequence; Cytoplasm; DNA Damage; DNA Repair; DNA biosynthesis; Data; Development; Disease-Free Survival; FDA approved; Genes; Genetic Transcription; Gleason Grade for Prostate Cancer; Growth; Inhibition of Apoptosis; Innovative Therapy; Interruption; LNCaP; Legal patent; Length; Ligand Binding Domain; Malignant neoplasm of prostate; Mediating; Metabolism; Mus; Mutagenesis; Nucleoplasm; PSA level; Pathologic; Peptides; Proliferating Cell Nuclear Antigen; Proteins; RNA Splicing; Receptor Signaling; Recombinants; Regulation; Reporter; Research; Research Design; Role; Signal Transduction; Stanolone; Structure; Testing; Therapeutic; Therapeutic Effect; Variant; advanced prostate cancer; androgen deprivation therapy; base; castration resistant prostate cancer; cell growth; cytotoxic; cytotoxicity; efficacious treatment; inhibitor; innovation; knock-down; monomer; neoplastic cell; non-oncogenic; novel; overexpression; prognostic value; prostate cancer cell; prostate cancer model; receptor; receptor-mediated signaling; recruit; small molecule; targeted treatment; tumor; tumor growth; tumor xenograft

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.

全长雄激素受体（AR-FL）的过表达和/或组成性活性AR剪接 缺乏全部或部分配体结合结构域的变体（AR-V），如AR-V7和ARv 567， 去势抵抗性前列腺癌（CRPC）的发展和进展。雄激素剥夺治疗 (ADT)在过表达AR-V的CRPC中无效，甚至可以诱导AR-V过表达。 增殖细胞核抗原（PCNA），优先在所有肿瘤细胞中过表达，是一种非致癌的肿瘤细胞因子。 DNA复制和修复以及细胞生长和存活所必需的蛋白质。天然PCNA是一种环状的 同源三聚体主要位于核质中。为了发挥功能，PCNA必须线性化或单体化， 重新定位于染色质、细胞质或细胞膜，并作为平台并执行其功能 通过与含有PCNA-相互作用蛋白盒（PIP-盒）和其它基序的伴侣蛋白相互作用。 PI的小组在AR的N末端鉴定了PIP盒的共有序列。PCNA复合物， AR-FL和AR-V7，其可以通过PIP盒特异性抑制剂T2 AA减弱。PCNA也与ARv 567 es结合 并直接转化为重组AR蛋白。PCNA-I1 S是PI小组开发的一种小分子PCNA抑制剂， 结合在两个单体的界面，稳定三聚体结构，并减弱PCNA的关联， 染色质PCNA-I1 S和T2 AA抑制CRPC中AR转录活性和AR靶基因表达 LNCaP-AI和22 Rv 1细胞。敲低PCNA表达减少二氢睾酮刺激的AR 转录活性，并消除PCNA-I1 S对AR活性的抑制作用。更重要的是，AR- PI实验室最近开发了一种特异性PIP盒肽抑制剂R9-AR-PIP，它与PCNA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 PI假设PCNA通过AR PIP盒与AR-FL和AR-Vs相互作用， 增强AR转录活性，靶向PCNA-AR相互作用将减弱AR-FL- 和AR-V介导的信号传导并抑制过表达AR-FL和/或AR-V的CRPC的生长。 提出了具体目标。目的1中的研究将进一步阐明AR PIP盒在以下相互作用中的作用： 通过蛋白质突变法将AR-FL和AR-Vs与PCNA共转染，并观察PCNA-I1 S，T2 AA， 和R9-AR-PIP对PCNA与AR-FL和AR-Vs的共定位和染色质缔合的影响， 在AR-FL和AR-Vs募集到染色质中的PCNA-AR相互作用，以及R- 9-AR-PIP对AR-PCNA相互作用的影响。目的2中的研究将确定PCNA-I1 S，T2 AA， 和R9-AR-PIP对AR-FL和AR-Vs占据雄激素反应元件的影响， AR-FL和AR-V7的活性，以及AR-V特异性基因的表达。此外，R9-AR- PIP对培养的CRPC细胞的治疗作用，R9-AR-PIP对小鼠CRPC异种移植肿瘤的治疗作用，以及 将研究R9-AR-PIP对CRPC肿瘤中AR信号传导的选择性抑制作用。