BIRC6在三阴性乳腺癌发生发展过程中的作用及其靶向策略研究

Triple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer with the characteristics of high grade, metastasis, increased lymphocytic infiltration and poor survival outcome. Due to the absence of well-characterized molecular targets, chemotherapy is still the first line choice for TNBC treatment. In this study, we found that BIRC6 was stabilized at the posttranslational level by EGFR activation in TNBC cell lines. Importantly, BIRC6 was highly expressed in 29/34 EGFR-positive TNBC tumor samples, and the high mRNA expression level of BIRC6 was positively correlated with poor survival outcome of TNBC patients. Using Immunoprecipitation-Mass Spectrometry analysis, we identified that HECTD1, an E3 ligase, mediated the protein degradation of BIRC6 in TNBC cells, which could be antagonized by EGFR activation. Loss of BIRC6 dramatically induced apoptosis and decreased the viability, colongenicity and sphere formation in both MDA-MB-468 and Hs578T cells, which could be fully rescued by SMAC inhibition. Of note, shRNA knockdown of BIRC6 eradicated the tumor growth of MDA-MB-468 xenograft in vivo, which surpassed EGFR inhibition. Thus, we have identified that BIRC6 is a key regulator of the survival of TNBC cells, which could be stabilized by EGFR activation, and inhibition of BIRC6 selectively reduced the tumor growth of TNBC cells in vitro and in vivo, suggesting it might be a potential therapeutic target for treating TNBCs. In this study, we'll further testify the therapeutic efficacies of IAP inhibitors or SMAC mimics for treating BIRC6-postive TNBCs, and provide potential valuable lead compounds for further development.

三阴性乳腺癌（TNBC）的治疗一直是乳腺癌治疗的难点和热点。除了极少部分BRCA1或BRCA2缺陷的TNBC可以通过PARP抑制剂进行治疗以外，临床上极度缺乏有效的靶向治疗药物。已知EGFR在20％的TNBC中高表达，但靶向EGFR的小分子抑制剂或单抗药物对TNBC的临床治疗效果不显著。我们研究发现EGFR可以在翻译后修饰水平增加BIRC6的蛋白稳定性。免疫共沉淀-质谱联用实验表明这一过程是E3泛素酶HECTD1依赖的并可以被EGFR激活所阻断。BIRC6敲低可以有效抑制TNBC细胞的体内外生长、克隆形成、抑制乳腺癌干细胞并完全阻断其体内裸小鼠皮下移植瘤生长，效果明显优于抑制EGFR。机制研究表明BIRC主要通过泛素化降解SMAC阻断TNBC发生细胞凋亡，提示BIRC6是潜在的三阴性乳腺癌治疗新靶点。对IAP拮抗剂或SMAC类似物的药物筛选，将有助于找到靶向BIRC6的TNBC治疗药物。

三阴性乳腺癌的治疗一直是乳腺癌治疗的难点和热点。目前除了极少部分BRCA1或BRCA2纯合缺陷的三阴性乳腺癌可以通过PARP抑制剂进行治疗以外，临床上极度缺乏有效的靶向治疗药物。在本研究中，我们发现了丝裂霉素不仅能有效抑制BRCA2纯合缺失肿瘤，而且对BRCA2杂合缺失肿瘤也有极为显著的抑制效果(Chen X, et al. Cell Death & Disease, 2020)。这一作用对包括三阴性乳腺癌在内的多种BRCA2杂合或纯合缺失肿瘤都有类似的治疗效果。基于这一研究成果，我们正在和杭州市第一人民医院合作开展相关临床实验，目前已提出了患者入组筛选标准并正在开展伦理审批；同时，我们还通过全基因组敲除筛选工作发现了MYC扩增是TYMS抑制剂药物敏感标志物，其分子机制与MYC作为TYMS转录因子有关(Liu T, et al. EBioMedicine, 2019)。这一工作为TYMS抑制剂临床应用提供了新的使用标准，可能使MYC扩增三阴性乳腺癌患者受益。对于EGFR高表达三阴性乳腺癌，我们发现BIRC6敲低可以有效抑制三阴性乳腺癌细胞的体内外生长、克隆形成和乳腺癌干细胞微球形成，效果明显优于EGFR抑制本身， 提示BIRC6作为潜在三阴性乳腺癌治疗新靶点值得进一步研发。最后，我们还发现了pERK2-p53复合物是KRAS突变胃肠道肿瘤核心事件(Wang X, et al. BioRxiv, 2020; PNAS in revision)。鉴于KRAS突变是导致EGFR靶向药物临床治疗失败的重要原因，这一工作为EGFR靶向药物或BIRC6抑制剂与MEK抑制剂联用用药提供了科学依据。综上，我们的研究从多个方面解析了三阴性乳腺癌治疗靶点及药物的精准化应用，为三阴性乳腺癌临床治疗提供了重要参考依据。