Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University, 06800, Ankara, Turkey.
Department of Drug Discovery and Biomedical Sciences, University of South Carolina, Columbia, SC, 29208, USA.
Breast Cancer Res Treat. 2022 Jun;193(2):331-348. doi: 10.1007/s10549-022-06569-5. Epub 2022 Mar 25.
Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer that is frequently treated with chemotherapy. However, many patients exhibit either de novo chemoresistance or ultimately develop resistance to chemotherapy, leading to significantly high mortality rates. Therefore, increasing the efficacy of chemotherapy has potential to improve patient outcomes.
Here, we performed whole transcriptome sequencing (both RNA and small RNA-sequencing), coupled with network simulations and patient survival data analyses to build a novel miRNA-mRNA interaction network governing chemoresistance in TNBC. We performed cell proliferation assay, Western blotting, RNAi/miRNA mimic experiments, FN coating, 3D cultures, and ChIP assays to validate the interactions in the network, and their functional roles in chemoresistance. We developed xenograft models to test the therapeutic potential of the identified key miRNA/proteins in potentiating chemoresponse in vivo. We also analyzed several patient datasets to evaluate the clinical relevance of our findings.
We identified fibronectin (FN1) as a central chemoresistance driver gene. Overexpressing miR-326 reversed FN1-driven chemoresistance by targeting FN1 receptor, ITGA5. miR-326 was downregulated by increased hypoxia/HIF1A and ECM stiffness in chemoresistant tumors, leading to upregulation of ITGA5 and activation of the downstream FAK/Src signaling pathways. Overexpression of miR-326 or inhibition of ITGA5 overcame FN1-driven chemotherapy resistance in vitro by inhibiting FAK/Src pathway and potentiated the efficacy of chemotherapy in vivo. Importantly, lower expression of miR-326 or higher levels of predicted miR-326 target genes was significantly associated with worse overall survival in chemotherapy-treated TNBC patients.
FN1 is central in chemoresistance. In chemoresistant tumors, hypoxia and resulting ECM stiffness repress the expression of the tumor suppressor miRNA, miR-326. Hence, re-expression of miR-326 or inhibition of its target ITGA5 reverses FN1-driven chemoresistance making them attractive therapeutic approaches to enhance chemotherapy response in TNBCs.
三阴性乳腺癌（TNBC）是乳腺癌中最具侵袭性的亚型，常采用化疗进行治疗。然而，许多患者表现出原发性化疗耐药，或最终对化疗产生耐药，导致死亡率显著升高。因此，提高化疗疗效有可能改善患者预后。
本研究通过全转录组测序（包括 RNA 和小 RNA 测序），结合网络模拟和患者生存数据分析，构建了一个新的 miRNA-mRNA 相互作用网络，以调控 TNBC 的化疗耐药性。我们进行了细胞增殖实验、Western blot 实验、RNAi/miRNA 模拟实验、FN 包被、3D 培养和 ChIP 实验，以验证网络中的相互作用及其在化疗耐药性中的功能作用。我们构建了异种移植模型，以测试所鉴定的关键 miRNA/蛋白在体内增强化疗反应的治疗潜力。我们还分析了几个患者数据集，以评估我们研究结果的临床相关性。
我们鉴定出纤维连接蛋白（FN1）是一个重要的化疗耐药驱动基因。过表达 miR-326 通过靶向 FN1 受体 ITGA5 逆转了 FN1 驱动的化疗耐药性。miR-326 的表达下调是由耐药肿瘤中缺氧/HIF1A 和细胞外基质硬度增加引起的，导致 ITGA5 上调和下游 FAK/Src 信号通路激活。miR-326 的过表达或 ITGA5 的抑制通过抑制 FAK/Src 通路在体外克服了 FN1 驱动的化疗耐药性，并增强了体内化疗的疗效。重要的是，化疗治疗的 TNBC 患者中 miR-326 表达较低或预测 miR-326 靶基因水平较高与总生存期较差显著相关。
FN1 是化疗耐药的核心。在化疗耐药肿瘤中，缺氧和由此产生的细胞外基质硬度抑制肿瘤抑制 miRNA miR-326 的表达。因此，miR-326 的重新表达或其靶基因 ITGA5 的抑制逆转了 FN1 驱动的化疗耐药性，使其成为增强 TNBC 化疗反应的有吸引力的治疗方法。
