Antibody information for western blots.

Adenomatous Polyposis Coli (APC) is lost in approximately 70% of sporadic breast cancers, with an inclination towards triple negative breast cancer (TNBC). TNBC is treated with traditional chemotherapy, such as paclitaxel (PTX); however, tumors often develop drug resistance. We previously created APC knockdown cells (APC shRNA1) using the human TNBC cells, MDA-MB-157, and showed that APC loss induces PTX resistance. To understand the mechanisms behind APC-mediated PTX response, we performed cell cycle analysis and analyzed cell cycle related proteins. Cell cycle analysis indicated increased G2/M population in both PTX-treated APC shRNA1 and parental cells, suggesting that APC expression does not alter PTX-induced G2/M arrest. We further studied the subcellular localization of the G2/M transition proteins, cyclin B1 and CDK1. The APC shRNA1 cells had increased CDK1, which was preferentially localized to the cytoplasm, and increased baseline CDK6. RNA-sequencing was performed to gain a global understanding of changes downstream of APC loss and identified a broad mis-regulation of cell cycle-related genes in APC shRNA1 cells. Our studies are the first to show an interaction between APC and taxane response in breast cancer. The implications include designing combination therapy to re-sensitize APC-mutant breast cancers to taxanes using the specific cell cycle alterations.腺瘤性结肠息肉病蛋白（Adenomatous Polyposis Coli，APC）在约70%的散发性乳腺癌中发生缺失，且更偏好累及三阴性乳腺癌（triple negative breast cancer，TNBC）。三阴性乳腺癌目前采用紫杉醇（paclitaxel，PTX）等传统化疗方案进行治疗，但肿瘤常继发化疗耐药。我们此前以人三阴性乳腺癌细胞系MDA-MB-157为模型，构建了APC敲低细胞株（APC shRNA1），并证实APC缺失可诱导紫杉醇耐药。为阐明APC介导的紫杉醇应答背后的分子机制，我们开展了细胞周期分析，并检测了细胞周期相关蛋白的表达水平。细胞周期分析结果显示，经紫杉醇处理的APC shRNA1细胞与亲本细胞均出现G2/M期细胞比例升高的现象，表明APC的表达并未改变紫杉醇诱导的G2/M期阻滞。我们进一步研究了G2/M期转换蛋白细胞周期蛋白B1（cyclin B1）与细胞周期蛋白依赖性激酶1（CDK1）的亚细胞定位情况。结果发现，APC shRNA1细胞中CDK1的表达水平升高，且该蛋白主要定位于细胞质；同时该细胞株的基础CDK6（细胞周期蛋白依赖性激酶6）表达水平也有所上升。为全面解析APC缺失下游的基因表达变化谱，我们开展了RNA测序（RNA-sequencing），并发现APC shRNA1细胞中存在广泛的细胞周期相关基因表达失调。本研究首次揭示了乳腺癌中APC与紫杉烷类药物应答之间的关联，其研究意义在于可针对细胞周期的特异性改变设计联合治疗方案，使携带APC突变的乳腺癌细胞重新对紫杉烷类药物恢复敏感性。