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

基本信息

批准号：
10544062
负责人：
Zhongyun Dong
金额：
$ 18.56万
依托单位：
UNIVERSITY OF CINCINNATI
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-01-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10544062
关键词：
Androgen Receptor Androgen Response Element Apoptosis Attenuated Automobile Driving Binding Binding Proteins Biological Assay C-terminal Cell Cycle Progression Cell Survival Cell membrane Cells Chromatin Complex Consensus Sequence DNA Damage DNA Repair DNA biosynthesis Data Development Disease-Free Survival FDA approved Genes Genetic Transcription Gleason Grade for Prostate Cancer Growth 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 Shapes Signal Transduction Stanolone Structure Testing Therapeutic Therapeutic Effect Variant advanced prostate cancer androgen deprivation therapy 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

项目摘要

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）和/或组成性AR剪接的过表达缺少所有或部分配体结合域的变体（AR-V），例如AR-V7和ARV567ES，有助于抑制前列腺癌（CRPC）的发展和进展。雄激素剥夺疗法（ADT）在过表达AR-VS的CRPC中无效，甚至可以诱导AR-V过表达。在所有肿瘤细胞中优选过表达的增殖细胞核抗原（PCNA）是一种非康复性 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是环形的同型聚合物主要位于核局部。为了实用，必须将PCNA线性化或单体化。重新定位到染色质，细胞质或细胞膜，并用作其功能的平台并执行其功能通过与含有PCNA相互作用蛋白盒（PIP-box）和其他基序的伴侣蛋白相互作用。 PI的组确定了在AR的N末端的PIP框共有序列。 PCNA复合物与 AR-FL和AR-V7，可以通过PIP盒特异性抑制剂T2AA减弱。 PCNA还与ARV567ES结合直接以重组AR蛋白。 PCNA-i1s，一种由PI组开发的小分子PCNA抑制剂，在两个单体的界面上结合，稳定触发结构，并使PCNA关联减弱染色质。 PCNA-I1和T2AA抑制AR靶基因在CRPC中的转录活性和表达 LNCAP-AI和22RV1细胞。 PCNA的敲低表达降低了二氢睾丸激素刺激的AR 转录活性并废除PCNA-I1对AR活性的抑制作用。更重要的是，最近在PI实验室中开发的特定Pip-Box抑制剂R9-AR PIP与PCNA结合并抑制AR 转录活性，AR靶基因的表达以及AR阳性细胞的生长。基于这些观察结果，PI假设PCNA通过AR PIP-box与AR-FL和AR-VS相互作用，增强AR的转录活性，靶向PCNA-AR相互作用将减弱AR-FL- AR-V介导的信号传导并抑制过表达AR-FL和/或AR-VS的生长。二提出了具体目标。 AIM 1中的研究将进一步阐明AR PIP盒在相互作用中的作用通过蛋白质诱变与PCNA的AR-FL和AR-VS研究PCNA-I1S T2AA的抑制作用，T2AA， PCNA与AR-FL和AR-VS的共定位和染色质关联的R9-AR-PIP，其作用 PCNA-AR相互作用在AR-FL和AR-VS募集到染色质中的相互作用，以及R-的选择性抑制作用 AR-PCNA相互作用的9-AR-PIP。 AIM 2中的研究将确定PCNA-I1的抑制作用T2AA，T2AA， R9-ar-PIP关于AR-FL和AR-VS的雄激素响应元件的占用，转录 AR-FL和AR-V7的活性，以及AR-V特异性基因的表达。此外，R9-ar-的细胞毒性作用对CRPC细胞在培养中的PIP，R9-AR-PIP对小鼠CRPC Xenographic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。