MUSASHI-2 RNA binding protein confers resistance to an epidermal growth factor receptor-tyrosine kinase inhibitor Osimertinib in lung adenocarcinoma

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective in non-small cell lung cancer (NSCLC) patients harboring EGFR mutations. However, due to the acquired resistance to EGFR-TKIs, even third generation Osimertinib, the patients suffer poor prognosis. The resistance mechanisms are still not fully understood. Here, we demonstrate that increased expression of MUSASHI-2 (MSI2), an RNA binding protein, is novel mechanisms for resistance to EGFR-TKIs. We found that after long-exposure of gefitinib, the first generation EGFR-TKI, lung cancer cells harboring the EGFR-TKI-sensitive mutations became resistant to not only gefitinib but also Osimertinib. Although other mutations in EGFR were not found, expression levels of Nanog, a stemness core protein, and activities of aldehyde dehydrogenase (ALDH) were increased, suggesting that cancer stem-like properties were increased. Transcriptome analysis revealed that MSI2 was among the top list of the stemness-related genes upregulated in the EGFR-TKI-resistant cells. Knockdown of MSI2 reduced cancer stem-like properties, including expression levels of Nanog a core stemness factor. We demonstrate that knockdown of MSI2 restored sensitivity to Osimertinib or gefitinib in the EGFR-TKI-resistant cells to the similar levels of the parental cells in vitro. RNA immunoprecipitation (RIP) assay revealed that antibodies against MSI2 bound to Nanog mRNA, suggesting that MSI2 increases Nanog expression by binding to Nanog mRNA. Moreover, overexpression of MSI2 or Nanog conferred resistance to Osimertinib or gefitinib in parental cells. Finally, knockdown of MSI2 greatly increased sensitivity to Osimertinib in vivo. Collectively, our findings provide proof-of-principle that targeting MSI2-Nanog axis in combination with EGFR-TKIs would effectively prevent emergence of acquired resistance. Comaprison in ALDH-high cell populations between PC9 cells and PC9M2 cells.There are four biological replicates for each conditions.

表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKIs）对携带EGFR突变的非小细胞肺癌（NSCLC）患者具有显著临床疗效。然而，受EGFR-TKIs获得性耐药困扰，即便第三代奥希替尼也无法改善此类患者的不良预后，且目前该耐药机制尚未完全阐明。本研究证实，RNA结合蛋白Musashi-2（MSI2）的表达上调是EGFR-TKIs获得性耐药的全新机制。研究发现，在长期暴露于第一代EGFR-TKI吉非替尼后，携带EGFR-TKI敏感突变的肺癌细胞不仅对吉非替尼产生耐药，同时也对奥希替尼产生交叉耐药。尽管未检测到EGFR其他继发突变，但干性核心蛋白Nanog的表达水平以及乙醛脱氢酶（ALDH）的活性均显著上调，提示细胞的癌症干细胞样特性增强。转录组分析显示，在EGFR-TKI耐药细胞中，MSI2是上调最为显著的干性相关基因之一。敲低MSI2可抑制癌症干细胞样特性，包括干性核心因子Nanog的表达水平。体外实验证实，敲低MSI2可使EGFR-TKI耐药细胞对奥希替尼或吉非替尼的敏感性恢复至亲本细胞的相似水平。RNA免疫沉淀（RIP）实验显示，靶向MSI2的抗体可结合Nanog mRNA，提示MSI2通过结合Nanog mRNA上调其表达。此外，在亲本细胞中过表达MSI2或Nanog可使其对奥希替尼或吉非替尼产生耐药性。最后，体内实验证实，敲低MSI2可显著增强细胞对奥希替尼的敏感性。综上，本研究结果证实，联合靶向MSI2-Nanog轴与EGFR-TKIs可有效预防获得性耐药的发生，为相关治疗策略提供了原理验证依据。PC9细胞与PC9M2细胞的ALDH高表达细胞群比较实验中，每组实验条件均设置4次生物学重复。