Biomedical Research Institute, INBIOMED (UBA, CONICET), Department of Biochemistry, School of Medicine, University of Buenos Aires, CABA C1121ABG, Argentina.
Biomedical Research Institute, INBIOMED (UBA, CONICET), Department of Biochemistry, School of Medicine, University of Buenos Aires, CABA C1121ABG, Argentina.
Biochem Pharmacol. 2019 Jan;159:52-63. doi: 10.1016/j.bcp.2018.11.005. Epub 2018 Nov 9.
Acyl-CoA synthetase-4 (ACSL4) is an enzyme implicated in estrogen receptor α (ERα) negative regulation and hormone therapy resistance in breast cancer. In addition, ACSL4 has been associated to certain types of hormone resistance in prostate cancer. Chemotherapeutic treatment of disseminated breast cancer is usually faced with therapy resistance associated to ATP-binding cassette (ABC) transporter expression, which detect and eject anti-cancer drugs from cells. In this context, the aim of the present work was to study the role of ACSL4 in anti-cancer drug resistance and the involvement of ABC transporters in the underlying mechanisms. To this end, we used MCF-7 Tet-Off/ACSL4 and MDA-MB-231 mock cells, which overexpress ACSL4, and control line MCF-7 Tet-Off empty vector, MDA-MB-231 shRNA ACSL4 and MDA-MB-231 wild type cells. Assays were conducted on cell viability (MTT), cell proliferation (BrdU), drug efflux (flow cytometry), ACSL4-responsive drug resistance ABC transporter genes (RNA-Seq), transporter mRNA expression, protein levels and signaling pathway participation (real-time PCR and Western blot). Higher survival rates upon chemotherapeutic treatment were obtained in MCF-7 Tet-Off/ACSL4 and MDA-MB-231 mock cells, an effect counteracted by doxycycline- or shRNA-induced ACSL4 inhibition, respectively. A synergic effect of ACSL4 inhibitor triacsin C and chemotherapeutic drugs was observed on the inhibition of MDA-MB-231 wild type cell proliferation. MCF-7 Tet-Off/ACSL4 cells showed greater doxorubicin, Hoechst 33342 and calcein AM efflux. In contrast, MDA-MB-231 shRNA ACSL4 cells evidenced inhibition of chemotherapeutic drug efflux. ABCG2, ABCC4, and ABCC8 were identified as ACSL4-responsive drug resistance genes whose expression was increased in MCF-7 Tet-Off/ACSL4 cells but inhibited in MDA-MB-231 shRNA ACSL4 cells. Further cell survival assays in the presence of Ko 143 and Ceefourin 1, inhibitors of ABCG2 and ABCC4, respectively, upon chemotherapeutic treatment showed greater participation of ABCG2 in anti-cancer drug resistance in cells overexpressing ACSL4. In addition, ACSL4 inhibition and chemotherapeutic treatment combined with rapamycin-induced mTOR inhibition synergically inhibited proliferation and reduced ABCG2 expression in cells overexpressing ACSL4. In sum, ACSL4 may be regarded as a novel therapeutic target regulating the expression of transporters involved in anticancer drug resistance through the mTOR pathway to restore drug sensitivity in tumors with poor prognosis for disease-free and overall survival.
酰基辅酶 A 合成酶 4（ACSL4）是一种与雌激素受体 α（ERα）负调控和乳腺癌激素治疗耐药性相关的酶。此外，ACSL4 与前列腺癌的某些类型的激素耐药性有关。播散性乳腺癌的化学治疗通常面临与 ATP 结合盒（ABC）转运蛋白表达相关的治疗耐药性，这些转运蛋白可从细胞中检测和排出抗癌药物。在这种情况下，本研究的目的是研究 ACSL4 在抗癌药物耐药性中的作用以及 ABC 转运蛋白在潜在机制中的参与。为此，我们使用 MCF-7 Tet-Off/ACSL4 和 MDA-MB-231 mock 细胞，这些细胞过表达 ACSL4，以及对照 MCF-7 Tet-Off 空载体、MDA-MB-231 shRNA ACSL4 和 MDA-MB-231 野生型细胞。通过 MTT（细胞活力测定）、BrdU（细胞增殖测定）、药物外排（流式细胞术）、ACSL4 反应性耐药性 ABC 转运体基因（RNA-Seq）、转运体 mRNA 表达、蛋白水平和信号通路参与（实时 PCR 和 Western blot）进行检测。在 MCF-7 Tet-Off/ACSL4 和 MDA-MB-231 mock 细胞中，化学治疗后获得更高的存活率，而多西环素或 shRNA 诱导的 ACSL4 抑制分别拮抗了这种作用。在 MDA-MB-231 野生型细胞增殖的抑制中观察到 ACSL4 抑制剂三乙酰精氨酸 C 和化疗药物的协同作用。MCF-7 Tet-Off/ACSL4 细胞表现出更高的阿霉素、Hoechst 33342 和 calcein AM 外排。相比之下，MDA-MB-231 shRNA ACSL4 细胞显示出对化疗药物外排的抑制。鉴定出 ABCG2、ABCC4 和 ABCC8 作为 ACSL4 反应性耐药基因，其在 MCF-7 Tet-Off/ACSL4 细胞中的表达增加，但在 MDA-MB-231 shRNA ACSL4 细胞中受到抑制。在存在 Ko143 和 Ceefourin1（ABCG2 和 ABCC4 的抑制剂）的情况下进行进一步的细胞存活测定，在化疗治疗后，发现 ABCG2 在过表达 ACSL4 的细胞中对抗癌药物耐药性的参与更大。此外，ACSL4 抑制和化疗治疗与雷帕霉素诱导的 mTOR 抑制联合作用，可协同抑制细胞增殖并降低过表达 ACSL4 的细胞中 ABCG2 的表达。总之，ACSL4 可被视为一种新型治疗靶点，通过 mTOR 途径调节参与抗癌药物耐药性的转运体的表达，从而恢复预后不良的肿瘤的药物敏感性，提高无病和总生存的几率。
