LINC00624结合SFPQ复合物促进HER2阳性乳腺癌耐药的临床和机制研究

Although anti-HER2/neu antibody therapy combined with chemotherapy has achieved an outstanding effect, many patients with HER2 positive breast cancer still succumb to cancer due to therapeutic resistance. Therefore, understanding the mechanisms by which HER2-positive breast cancer develop therapeutic resistance is of vital importance. We have identified differentially expressed lncRNAs by RNA-seq between the pCR and the non-pCR group from HER2 positive patients' core needle biopsy tissue. Survival information was extracted from public database，compared with which LINC00624，a lncRNA has not been reported previously, was selected. We confirmed that high LINC00624 expression is associated with poor overall survival in breast cancer patients. The half maximal inhibitory concentration (IC50) of the chemotherapy drug and anti-HER2 antibody in HER2 positive breast cancer cells was increased by ectopic overexpression of LINC00624. Overexpression of LINC00624 reveals significant inhibition of inflammatory and antigen presentation related genes. LINC00624 probably promotes therapy resistance through anti-tumor immunity inhibition in the tumor microenvironment. However, the detailed mechanism remains unknown. Our further research is to investigate the effect of LINC00624 that exerted on HER2 positive breast cancer therapy, and the precise mechanism LINC00624 and its complex intrigue therapy resistance signaling through cell lines, animal xenografts, and patient-derived xenografts models. Taken together, these studies will provide new insights into the prediction value of LINC00624 which could be a novel therapeutic target for personalized treatment in HER2 positive breast cancer.

HER2阳性乳腺癌靶向治疗联合化疗疗效显著。然而，耐药可导致治疗失败和疾病进展。长链非编码RNA在影响肿瘤疗效方面作用显著。课题组前期对11对新辅助治疗不同疗效的HER2阳性乳腺癌患者治疗前空心针标本进行转录组测序，筛选差异表达的长链非编码RNA, 通过数据库初筛选定LINC00624作为耐药潜在靶点，并发现LINC00624可通过抑制凋亡增强HER2阳性乳腺癌细胞对化疗及抗HER2治疗药物的耐受。稳转株转录组测序结合差异基因富集分析及细胞实验发现LINC00624可以显著抑制炎症相关基因表达, 提示LINC00624可能通过肿瘤炎症因子途径引发治疗耐药。本课题拟进一步利用临床独立样本验证其疗效预测价值，细胞实验、动物及PDX模型阐明LINC00624及其复合物调控下游信号通路，影响HER2阳性乳腺癌治疗疗效的分子机制，为HER2阳性乳腺癌治疗疗效预测提供新的思路及干预靶点。

HER2阳性乳腺癌的治疗反应随着靶向药物的应用显著改善，然而仍有近一半的患者无法单纯通过化疗联合靶向药物达到临床完全缓解，标志物和新的靶点仍有待探索。近年来长链非编码RNA（lncRNA）因被发现参与细胞内许多重要的分子机制调控，越来越受到重视。本研究旨在通过对比不同治疗结局的HER2阳性乳腺癌患者的差异表达lncRNA，筛选可能调控耐药相关机制的分子标志物，为未来提供潜在的治疗靶点。在本研究中，我们利用HER2阳性乳腺癌患者新辅助化疗前空芯针穿刺标本，根据术后病理结果分为病理完全缓解（pCR）和非病理完全缓解组（non-pCR），筛选差异表达lncRNA。结果发现LINC00624在non-pCR组差异表达最显著（P<0.001）。LINC00624高表达可缩短患者无复发生存时间。对LINC00624功能进行探索发现其可促进HER2阳性乳腺癌细胞增殖，抵抗细胞对拉帕替尼和紫杉醇的治疗作用，提高药物IC50值。利用RNA-seq对LINC00624影响的下游通路进行分析发现LINC00624可抑制IFN通路和细胞凋亡。利用RNAfold web server发现LINC00624的S3段具有dsRNA结构，且该结构在A3转录本中缺失。RT-PCR及RNA-seq发现LINC00624 dsRNA结构具有A-to-I RNA编辑。过表达LINC00624可抑制细胞内固有免疫受体对dsRNA的感知及干扰素通路的激活。进一步，利用RNA pull down结合银染、质谱发现LINC00624可结合ADAR1。ADAR1可对LINC00624 dsRNA区进行A-to-I RNA 编辑，而LINC00624则稳定ADAR1的表达，通过抑制β-TrCP引发的ADAR1泛素化降解，提高ADAR1蛋白稳定性。 LINC00624可以通过抑制MHCI类抗原呈递，降低CD8+T细胞肿瘤微环境中的浸润，导致免疫检查点阻断抑制和抗HER2治疗耐药，并且这一功能是ADAR1依赖的。这些结果表明LINC00624是一种免疫抑制型lncRNA，通过ASO靶向LINC00624高表达的肿瘤具有巨大的治疗潜力和临床应用前景。