PO.MCB07.01 · 分子与细胞生物学
探究PBX1扰动对恩杂鲁胺耐药CRPC抗增殖作用的影响
Exploring the anti-proliferative impact of PBX1 perturbation in enzalutamide-resistant CRPC
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
晚期前列腺癌（PCa）常由雄激素受体（AR）活性上调所驱动，临床上采用恩杂鲁胺（Enzalutamide）等第二代AR拮抗剂进行靶向治疗。尽管患者初期对治疗有响应，但许多患者会产生耐药，导致疾病进展和预后不良。我们聚焦于一种机制，即随着PCa产生恩杂鲁胺耐药，AR顺式作用元件组（cistrome）从经典型向非经典型发生转换。由于协同蛋白伙伴驱动转录活性，我们在恩杂鲁胺耐药的PCa细胞系模型中运用核心调控回路分析，以识别可能介导非经典AR顺式作用元件组重编程的新型转录因子（TF）候选者，其中包括前B细胞白血病同源框1（PBX1），一种TALE同源结构域转录因子。我们此前已证明，与敏感型模型相比，PBX1在去势抵抗型和恩杂鲁胺耐药型（EnzaR-CRPC）细胞系模型中高表达。我们还证明，在高表达PBX1的EnzaR-CRPC细胞系中抑制PBX1具有抗增殖效应，并在持续恩杂鲁胺治疗下观察到协同作用。在本研究中，我们通过shRNA（PBX1 KD）和CRISPR-Cas9（PBX1 KO）在一个EnzaR-CRPC细胞系中扰动PBX1表达。Western印迹、RT-qPCR和基因组DNA分析验证了PBX1蛋白和基因水平的显著降低。与此前PBX1抑制剂的观察结果类似，PBX1 KD和PBX1 KO细胞相比各自对照组表现出初期显著的增殖下降。有趣的是，PBX1 KO克隆经过数次传代后恢复了增殖增强。相反，PBX1 KD细胞持续表现出抗增殖。正在进行的转录组学和全基因组DNA分析将清晰阐明受PBX1扰动影响的生物学通路，最重要的是揭示在PBX1低水平（PBX1 KD）与PBX1完全消除（PBX1 KO）状态下AR和PBX1顺式作用元件组、启动子及增强子格局的变化。这将有助于更好地洞察：（i）AR-PBX1转录轴以及PBX1在促进治疗耐药型晚期PCa中的作用，（ii）PBX1抑制的长期影响，以及（iii）同时靶向PBX1和AR以在晚期治疗耐药型CRPC中获得持久积极疗效的最佳治疗策略。
查看英文原文 English abstract
Advanced prostate cancer (PCa) is often driven by upregulated activity of the androgen receptor (AR) and is clinically targeted by second-generation AR-antagonists such as Enzalutamide. Though patients initially respond to treatment, many develop resistance, leading to disease advancement and poor prognosis. We have focused on a mechanism involving an AR cistrome switch from a canonical to a noncanonical cistrome as PCa becomes Enzalutamide-resistant. As collaborating protein partners drive transcriptional activity, we employed core regulatory circuitry analysis in Enzalutamide-resistant PCa cell line models to identify novel TF candidates that may mediate non-canonical AR cistrome reprogramming, including Pre-B-cell leukemia homeobox 1 (PBX1), a TALE homeodomain TF. We previously showed that PBX1 is highly expressed in castrate-resistant and Enzalutamide-resistant (EnzaR-CRPC) cell line models compared with sensitive models. We have also demonstrated that PBX1 inhibition has an anti-proliferative effect in a high-PBX1-expressing EnzaR-CRPC cell line, with synergism observed with continuous Enzalutamide treatment. In the current study, we perturbed PBX1 expression in an EnzaR-CRPC cell line by shRNA (PBX1 KD) and CRISPR-Cas9 (PBX1 KO). Western blotting, RT-qPCR, and genomic DNA analysis validated a significant reduction in PBX1 protein and gene levels. Similar to previous observations with PBX1 inhibitors, PBX1 KD and PBX1 KO cells showed an initial, prominent decrease in proliferation compared to their respective controls. Interestingly, PBX1 KO clones after several passages regained increased proliferation. On the contrary, the PBX1 KD cells continue to demonstrate anti-proliferation. Ongoing transcriptomic and genome-wide DNA analysis will provide a clear understanding of the biological pathways affected by PBX1 perturbation and, most importantly, of changes in the AR and PBX1 cistrome, promoter, and enhancer landscape at low levels of PBX1 (PBX1 KD) vs. complete elimination of PBX1 (PBX1 KO). This would provide a better insight into (i) AR-PBX1 transcriptional axis and PBX1's role in promoting treatment-resistant, advanced PCa, (ii) the long-term implications for PBX1 suppression, and (iii) the best treatment strategy for targeting both PBX1 and AR for a durable positive outcome in advanced treatment-resistant CRPC.
利益披露 Disclosure
E. J. Gardner, None..
M. Aust, None.