School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 211198, Nanjing, China.
Department of Pharmacy, the First Affiliated Hospital of Xinjiang Medical University, 830054, Urumqi, China.
Cell Death Dis. 2020 Aug 20;11(8):670. doi: 10.1038/s41419-020-02908-w.
Recent studies have demonstrated that acquisition of cancer stem-like properties plays an essential role in promoting epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) resistance in non-small cell lung cancer (NSCLC); however, how to regulate cancer stem-like properties and EGFR-TKI resistance is largely unclear. In this study, we discovered that increased iroquois-class homeodomain protein 4 (IRX4) was related to gefitinib resistance in NSCLC cells. Knockdown of IRX4 inhibited cell proliferation, sphere formation, and the expression of CD133, ALDH1A1, NANOG, Sox2 and Notch1, and the transcriptional activity of NANOG promoter. IRX4 overexpression increased the protein level of NANOG and CD133 in PC-9 cells. Combination of knocking-down IRX4 with gefitinib increased cell apoptosis and decreased cell viability and the expression of p-EGFR and NANOG in PC-9/GR cells. IRX4 knockdown in a PC-9/GR xenograft tumor model inhibited tumor progression and the expression of NANOG and CD133 more effectively than single treatment alone. Knockdown of NANOG inhibited the expression of CD133 and restored gefitinib cytotoxicity, and NANOG overexpression-induced cancer stem-like properties and gefitinib resistance could be obviously reversed by knocking-down IRX4. Further, we found that 1,25-dihydroxyvitamin D3 (1,25(OH)D) reduced obviously the expression of IRX4 and NANOG by inhibiting the activation of TGF-β1/Smad3 signaling pathway; moreover, combination of 1,25(OH)D and gefitinib decreased cell viability and proliferation or tumor progression and the expression of IRX4 and NANOG compared with single treatment alone both in PC-9/GR cells and in a PC-9/GR xenograft tumor model. These results reveal that inhibition of IRX4-mediated cancer stem-like properties by regulating 1,25(OH)D signaling may increase gefitinib cytotoxicity. Combination therapy of gefitinib and 1,25(OH)D by targeting IRX4 and NANOG, could provide a promising strategy to improve gefitinib cytotoxicity.
最近的研究表明，获得癌症干细胞样特性在促进非小细胞肺癌（NSCLC）中表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKIs）耐药中起着至关重要的作用；然而，如何调节癌症干细胞样特性和 EGFR-TKI 耐药性在很大程度上尚不清楚。在这项研究中，我们发现，iroquois-class homeodomain protein 4（IRX4）的增加与 NSCLC 细胞中的吉非替尼耐药有关。IRX4 的敲低抑制了细胞增殖、球体形成以及 CD133、ALDH1A1、NANOG、Sox2 和 Notch1 的表达，以及 NANOG 启动子的转录活性。IRX4 的过表达增加了 PC-9 细胞中 NANOG 和 CD133 的蛋白水平。IRX4 的敲低与吉非替尼联合使用增加了 PC-9/GR 细胞中的细胞凋亡，降低了细胞活力以及 p-EGFR 和 NANOG 的表达。IRX4 在 PC-9/GR 异种移植肿瘤模型中的敲低比单独治疗更有效地抑制肿瘤进展以及 NANOG 和 CD133 的表达。敲低 NANOG 抑制了 CD133 的表达并恢复了吉非替尼的细胞毒性，并且通过敲低 IRX4 可以明显逆转 NANOG 过表达诱导的癌症干细胞样特性和吉非替尼耐药性。此外，我们发现 1,25-二羟维生素 D3（1,25(OH)D）通过抑制 TGF-β1/Smad3 信号通路的激活明显降低了 IRX4 和 NANOG 的表达；此外，与单独治疗相比，1,25(OH)D 和吉非替尼联合使用降低了 PC-9/GR 细胞中的细胞活力和增殖或肿瘤进展以及 IRX4 和 NANOG 的表达，在 PC-9/GR 异种移植肿瘤模型中也是如此。这些结果表明，通过调节 1,25(OH)D 信号抑制 IRX4 介导的癌症干细胞样特性可能会增加吉非替尼的细胞毒性。通过针对 IRX4 和 NANOG 的吉非替尼和 1,25(OH)D 的联合治疗，为提高吉非替尼的细胞毒性提供了一种有前途的策略。
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