Abstract：
Triple-negative breast cancer (TNBC) poses significant challenges for treatment given the lack of targeted therapies and increased probability of relapse. It is pertinent to identify vulnerabilities in TNBC and develop newer treatments. Our prior research demonstrated that transcription factor EB (TFEB) is necessary for TNBC survival by regulating DNA repair, apoptosis signaling, and the cell cycle. However, specific mechanisms by which TFEB targets DNA repair and cell cycle pathways are unclear, and whether these effects dictate TNBC survival is yet to be determined. Here, we show that TFEB knockdown decreased the expression of genes and proteins involved in DNA replication and cell cycle progression in MDA-MB-231 TNBC cells. DNA replication was decreased in cells lacking TFEB, as measured by EdU incorporation. TFEB silencing in MDA-MB-231 and noncancerous MCF10A cells impaired progression through the S-phase following G1/S synchronization; however, this proliferation defect could not be rescued by co-knockdown of suppressor RB1. Instead, TFEB knockdown reduced origin licensing in G1 and early S-phase MDA-MB-231 cells. TFEB silencing was associated with replication stress in MCF10A but not in TNBC cells. Lastly, we identified that TFEB knockdown renders TNBC cells more sensitive to inhibitors of Aurora Kinase A, a protein facilitating mitosis. Thus, inhibition of TFEB impairs cell cycle progress by decreasing origin licensing, leading to delayed entry into the S-phase, while rendering TNBC cells sensitive to Aurora kinase A inhibitors and decreasing cell viability. In contrast, TFEB silencing in noncancerous cells is associated with replication stress and leads to G1/S arrest.
摘要：
三阴性乳腺癌(TNBC)由于缺乏靶向治疗和增加的复发概率而对治疗提出了重大挑战。确定TNBC中的漏洞并开发更新的治疗方法是相关的。我们先前的研究表明，转录因子EB（TFEB）通过调节DNA修复是TNBC存活所必需的，凋亡信号，和细胞周期。然而,TFEB靶向DNA修复和细胞周期通路的具体机制尚不清楚，这些影响是否决定了TNBC的生存还有待确定。这里,我们发现在MDA-MB-231TNBC细胞中，TFEB敲除降低了参与DNA复制和细胞周期进程的基因和蛋白的表达。缺乏TFEB的细胞中DNA复制减少，由EdU公司衡量。非癌性MCF10A和MDA-MB-231细胞中的TFEB沉默在G1/S同步后通过S期的进展受损;然而，这种增殖缺陷不能通过抑制因子RB1的共敲减来挽救。相反，TFEB敲低降低了G1和早期S期MDA-MB-231细胞中的起源许可。TFEB沉默与MCF10A的复制应激相关，但与TNBC细胞无关。最后,我们发现TFEB敲低使TNBC细胞对Aurora激酶A的抑制剂更敏感，一种促进有丝分裂的蛋白质。因此，抑制TFEB通过减少来源许可而损害细胞周期进程，导致延迟进入S阶段，同时使细胞对极光激酶A抑制剂敏感并降低TNBC细胞活力。相比之下,非癌细胞中的TFEB沉默与复制应激相关并导致G1/S阻滞。
