BACKGROUND Although EGFR tyrosine kinase inhibitors (EGFR-TKIs) are beneficial to lung adenocarcinoma patients with sensitive EGFR mutations, resistance to these inhibitors induces a cancer stem cell (CSC) phenotype. Here, we clarify the function and molecular mechanism of shisa3 as a suppressor that can reverse EGFR-TKI resistance and inhibit CSC properties. METHODS The suppresser genes involved in EGFR-TKI resistance were identified and validated by transcriptome sequencing, quantitative real-time PCR (qRT-PCR) and immunohistochemistry. Biological function analyses, cell half maximal inhibitory concentration (IC50), self-renewal, and migration and invasion capacities, were detected by CCK8, sphere formation and Transwell assays. Tumorigenesis and therapeutic effects were investigated in nonobese diabetic/severe combined immunodeficiency (nod-scid) mice. The underlying mechanisms were explored by Western blot and immunoprecipitation analyses. RESULTS We found that low expression of shisa3 was related to EGFR-TKI resistance in lung adenocarcinoma patients. Ectopic overexpression of shisa3 inhibited CSC properties and the cell cycle in the lung adenocarcinoma cells resistant to gefitinib/osimertinib. In contrast, suppression of shisa3 promoted CSC phenotypes and the cell cycle in the cells sensitive to EGFR-TKIs. For TKI-resistant PC9/ER tumors in nod-scid mice, overexpressed shisa3 had a significant inhibitory effect. In addition, we verified that shisa3 inhibited EGFR-TKI resistance by interacting with FGFR1/3 to regulate AKT/mTOR signaling. Furthermore, combinational administration of inhibitors of FGFR/AKT/mTOR and cell cycle signaling could overcome EGFR-TKI resistance associated with shisa3-mediated CSC capacities in vivo. CONCLUSION Taken together, shisa3 was identified as a brake to EGFR-TKI resistance and CSC characteristics, probably through the FGFR/AKT/mTOR and cell cycle pathways, indicating that shisa3 and concomitant inhibition of its regulated signaling may be a promising therapeutic strategy for reversing EGFR-TKI resistance.

中文翻译：

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Shisa3通过抑制癌症干细胞特性来制动对肺腺癌中EGFR-TKI的抗性。

背景技术尽管EGFR酪氨酸激酶抑制剂（EGFR-TKI）对于具有敏感EGFR突变的肺腺癌患者是有益的，但是对这些抑制剂的抗性诱导了癌症干细胞（CSC）表型。在这里，我们阐明了shisa3作为可以逆转EGFR-TKI耐药性并抑制CSC特性的抑制剂的功能和分子机理。方法通过转录组测序，定量实时荧光定量PCR（qRT-PCR）和免疫组化方法鉴定并验证了EGFR-TKI耐药相关的抑制基因。通过CCK8，球形成和Transwell分析检测了生物学功能分析，细胞半数最大抑制浓度（IC50），自我更新以及迁移和侵袭能力。在非肥胖/严重合并免疫缺陷（点头）小鼠中研究了肿瘤发生和治疗效果。通过蛋白质印迹和免疫沉淀分析探索了潜在的机制。结果我们发现，在肺腺癌患者中，shisa3的低表达与EGFR-TKI耐药有关。shisa3的异位过表达抑制了吉非替尼/奥西替尼耐药的肺腺癌细胞的CSC特性和细胞周期。相反，抑制shisa3会促进CSC表型和对EGFR-TKIs敏感的细胞的细胞周期。对于点头小鼠中TKI耐药的PC9 / ER肿瘤，过表达的shisa3具有明显的抑制作用。此外，我们证实shisa3通过与FGFR1 / 3相互作用来调节AKT / mTOR信号传导而抑制EGFR-TKI耐药性。此外，FGFR / AKT / mTOR抑制剂与细胞周期信号传导的组合给药可以克服与shisa3介导的CSC体内能力相关的EGFR-TKI抗性。结论总的来说，shisa3可能通过FGFR / AKT / mTOR和细胞周期途径被鉴定为对EGFR-TKI耐药性和CSC特性的制动，这表明shisa3及其伴随的调控信号的抑制可能是一种有希望的逆转治疗策略EGFR-TKI耐药性。