Laboratory of Biochemistry, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Polyclinic, Palermo, Italy.
Associazione Siciliana per la Lotta contro i Tumori (ASLOT), Palermo, Italy.
J Cell Physiol. 2019 Aug;234(10):18432-18447. doi: 10.1002/jcp.28479. Epub 2019 Mar 25.
Triple-negative breast cancer (TNBC) is a form of BC characterized by high aggressiveness and therapy resistance probably determined by cancer stem cells. MCL1 is an antiapoptotic Bcl-2 family member that could limit the efficacy of anticancer agents as recombinant human tumor necrosis factor related apoptosis-inducing ligand (rh-TRAIL). Here, we investigated MCL1 expression in TNBC tissues and cells. We found MCL1 differentially expressed (upregulated or downregulated) in TNBC tissues. Furthermore, in comparison to the human mammary epithelial cells, we found that MDA-MB-231 cells show similar messenger RNA levels but higher MCL1 protein levels, whereas it resulted downregulated in MDA-MB-436 and BT-20 cells. We evaluated the effects of rh-TRAIL and A-1210477, a selective MCL1 inhibitor, on cell viability and growth of MDA-MB-231 cells. We demonstrated that the drug combination reduced the cell growth and activated the apoptotic pathway. Similar effects were observed on three-dimensional cultures and tertiary mammospheres of MDA-MB-231 cells. In MDA-MB-231 cells, after MCL1 silencing, rh-TRAIL confined the cell population in the sub-G0/G1 phase and induced a drop in the mitochondrial transmembrane potential. To understand the molecular mechanism by which the loss of MCL1 function sensitizes the MDA-MB-231 cells to rh-TRAIL, we analyzed by real-time reverse transcription polymerase chain reaction, the expression of genes related to apoptosis, stemness, cell cycle, and those involved in epigenetic regulation. Interestingly, among the upregulated genes through MCL1 silencing or inhibition, there was TNFRSF10A (DR4). Moreover, MCL1 inhibition increased DR4 protein levels and its cell surface expression. Finally, we demonstrated MCL1-DR4 interaction and dissociation of this complex after A-1210477 treatment. Overall, our findings highlight the potential MCL1-roles in MDA-MB-231 cells and suggest that MCL1 targeting could be an effective strategy to overcome TNBC's rh-TRAIL resistance.
三阴性乳腺癌（TNBC）是一种侵袭性和治疗抵抗性较高的乳腺癌，可能由癌症干细胞决定。MCL1 是一种抗凋亡的 Bcl-2 家族成员，可能会限制抗癌药物如重组人肿瘤坏死因子相关凋亡诱导配体（rh-TRAIL）的疗效。在这里，我们研究了 TNBC 组织和细胞中的 MCL1 表达。我们发现 MCL1 在 TNBC 组织中差异表达（上调或下调）。此外，与人类乳腺上皮细胞相比，我们发现 MDA-MB-231 细胞显示出相似的信使 RNA 水平，但 MCL1 蛋白水平较高，而 MDA-MB-436 和 BT-20 细胞则下调。我们评估了 rh-TRAIL 和 A-1210477（一种选择性 MCL1 抑制剂）对 MDA-MB-231 细胞活力和生长的影响。我们证明，药物联合降低了细胞生长并激活了凋亡途径。在 MDA-MB-231 细胞的三维培养和三级类器官中也观察到类似的效果。在 MDA-MB-231 细胞中，沉默 MCL1 后，rh-TRAIL 将细胞群局限在 sub-G0/G1 期，并导致线粒体跨膜电位下降。为了了解失去 MCL1 功能使 MDA-MB-231 细胞对 rh-TRAIL 敏感的分子机制，我们通过实时逆转录聚合酶链反应分析了与凋亡、干性、细胞周期和表观遗传调节相关的基因的表达。有趣的是，通过 MCL1 沉默或抑制，上调的基因中有 TNFRSF10A（DR4）。此外，MCL1 抑制增加了 DR4 蛋白水平及其细胞表面表达。最后，我们证明了 MCL1-DR4 相互作用和 A-1210477 处理后复合物的解离。总之，我们的研究结果强调了 MCL1 在 MDA-MB-231 细胞中的潜在作用，并表明针对 MCL1 可能是克服 TNBC 对 rh-TRAIL 耐药性的有效策略。
