外泌体蛋白Annexin A6在三阴性乳腺癌吉西他滨耐药中的作用及其机制研究

Triple-negative breast cancer (TNBC) is a special molecular subtype of breast cancer with the poorest prognosis and lacks therapeutic targets. Despite of the significant clinical efficacy of gemcitabine in the first-line therapy in metastatic TNBC, nearly all patients will eventually develop drug resistance. Therefore, exploring the mechanism of gemcitabine resistance has a very important significance. Our prior study demonstrated that exosomes derived from MDA-MB-231 gemcitabine-resistant cells increased gemcitabine resistance in MDA-MB-231 gemcitabine-sensitive cells. The up-regulation of exosomal Annexin A1 and A6, especially exosomal Annexin A6, derived from MDA-MB-231 gemcitabine-resistant cells was confirmed by iTRAQ mass spectrometry and western blot, which may promote chemoresistance in TNBC. So the study is aiming to explore the correlations between exosomal Annexin A6 and gemcitabine resistance and to elucidate the detailed mechanism of the drug resistance in the TNBC in vitro and in vivo. In addition, the study is aiming to explore the possibilities of exosomal Annexin A6 from peripheral blood serum as noninvasive liquid biopsy to predict gemcitabine resistance in patients with TNBC. Through this study, we are able to analyze the molecular mechanism which could impact the treatment outcomes and provide backgrounds for precision medicine in the future.

三阴性乳腺癌是乳腺癌中预后最差的一种亚型，缺乏治疗靶点，疾病复发转移后基于吉西他滨的化疗方案常作为一线治疗方案，但是大部分患者对吉西他滨原发性耐药和/或继发性耐药，因此探索吉西他滨的耐药机制具有非常重要的意义。我们的前期研究发现三阴性乳腺癌吉西他滨耐药细胞株的外泌体可以增强敏感细胞株对吉西他滨的抵抗，采用iTRAQ质谱筛选和Western Blot验证，发现外泌体蛋白Annexin A1和Annexin A6在耐药细胞株外泌体中的表达明显上调，且外泌体蛋白Annexin A6的表达上调更为明显，提示外泌体蛋白Annexin A6在吉西他滨耐药的过程中可能起着关键作用。本课题拟探索外泌体蛋白Annexin A6在三阴性乳腺癌吉西他滨耐药过程中发挥的分子机制，并利用外泌体的无创液态活检优势，探索外周血中外泌体蛋白Annexin A6预测三阴性乳腺癌吉西他滨耐药的可能性，实现精准治疗。

外泌体是调节肿瘤微环境中细胞间信息的一个载体，它们通过各种机制（如转运RNA分子和蛋白质）在肿瘤耐药中起着至关重要的作用。但是目前外泌体对三阴性乳腺癌（TNBC）吉西他滨耐药的影响尚不清楚。在本课题研究中，我们通过实验检测外泌体对TNBC细胞活力、克隆形成、细胞凋亡和Annxin A6（ANXA6）/EGFR表达的影响,并探索了外泌体在吉西他滨耐药性中的作用及其潜在机制。本课题的研究结果表明，耐药细胞株源性外泌体能够促进TNBC细胞增殖和克隆形成，抑制TNBC细胞凋亡。耐药细胞株源性外泌体诱导TNBC吉西他滨耐药的机制是外泌体作为载体工具将ANXA6蛋白从耐药细胞株运输给敏感细胞株。iTRAQ质谱和Western blotting显示，ANXA6在耐药癌细胞及其外泌体中表达上调。当ANXA6稳定过表达时，敏感细胞株在吉西他滨的作用下，细胞增殖能力上升、克隆形成增多以及细胞凋亡减少。相反，敲低ANXA6则可以恢复TNBC细胞对吉西他滨的敏感性。免疫共沉淀表达证明外体ANXA6和EGFR可以相互作用，泛素化实验表明外泌体蛋白ANXA6可以抑制EGFR泛素化，而拉帕替尼可逆转外泌体蛋白ANXA6诱导的吉西他滨耐药。此外，与相对耐药的TNBC患者相比，相对敏感的TNBC患者血清外泌体蛋白Annexin A6在基线时低表达，两组间存在统计学差异。总之，耐药细胞株源性外泌体通过运输外泌体蛋白ANXA6抑制EGFR泛素化和降解，从而诱导吉西他滨耐药。TNBC患者示基线血清外泌体蛋白Annexin A6的表达水平或可预测一线含有吉西他滨化疗方案的疗效