Integrated analysis of ALK higher expression in human cancer and downregulation in LUAD using RNA molecular scissors
Integrated analysis of ALK higher expression in human cancer and downregulation in LUAD using RNA molecular scissors
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DOI：
10.1007/s12094-022-02835-6
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发表时间：
2022-04-29
影响因子：
3.4
通讯作者：
Tsukahara, Toshifumi
中科院分区：
文献类型：
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作者：
Saifullah;Tsukahara, Toshifumi
间变性淋巴瘤激酶（ALK）是ALK重排癌（包括肺腺癌）的一个被认可的分子靶点。然而，使用可药物抑制剂靶向ALK的临床优势几乎普遍受到耐药性发展的限制。因此，对抗ALK过表达的策略对于ALK驱动的癌症仍然是至关重要的。方法采用综合多维方法系统分析ALK的过表达模式及其临床后果、基因改变及其在肿瘤标志基因中的意义和相关性。利用LwCas13a RNA分子剪刀，利用肺腺癌（LUAD）细胞中的两个靶标引导RNA下调alk重排。免疫细胞化学、免疫印迹和MTT检测证实下调。结果我们发现，在包括LUAD在内的几种恶性肿瘤中，ALK水平高于正常组织。与低ALK表达患者相比，高ALK表达患者的生存期较差或较短。我们在LUAD患者的ALK中发现了许多遗传改变，这些改变可能会改变癌症标志基因，包括STAT1和CTSL。接下来，我们观察到与对照组相比，LwCas13a分子剪刀在LUAD细胞中显著下调磷酸化和总ALK嵌合体蛋白的表达。此外，我们发现下调ALK嵌合体蛋白可显著抑制细胞活力并诱导细胞死亡，包括凋亡。结论我们的研究结果为ALK作为预后生物标志物提供了基础，LwCas13a分子剪刀成功下调了肿瘤共同驱动的ALK重排蛋白，这将为开发新的治疗ALK驱动癌症的策略铺平道路。
Purpose Anaplastic lymphoma kinase (ALK) is an endorsed molecular target in ALK-rearranged carcinomas, including lung adenocarcinoma. However, the clinical advantage of targeting ALK using druggable inhibitors is almost universally restricted by the development of drug resistance. Therefore, a strategy for combating ALK overexpression remains paramount for ALK-driven cancer. Methods We systemically analyzed the overexpression pattern of ALK and its clinical consequences, genetic alterations, and their significance in cancer hallmark genes, and correlation using integrated multidimensional approaches. The LwCas13a RNA molecular scissors was used to downregulate ALK-rearrangement by leveraging two target guide RNAs in lung adenocarcinoma (LUAD) cells. Immunocytochemistry, immunoblotting, and MTT assays were conducted to validate the downregulation. Results We found elevated levels of ALK in several malignancies, including LUAD, than in normal tissues. Higher expression of ALK was significantly associated with worse or shorter survival than patients with lower expression. We identified numerous genetic alterations in ALK, which potentially alter the cancer hallmark genes, including STAT1 and CTSL, in patients with LUAD. Next, we observed that the LwCas13a molecular scissors robustly downregulated both phosphorylated and total ALK chimera protein expression in LUAD cells compared to the control. Furthermore, we found that downregulation of ALK chimera protein substantially inhibited cell viability and induced cell death, including apoptosis. Conclusion Our findings suggest a basis for ALK as a prognostic biomarker and the LwCas13a molecular scissors successfully downregulated the onco-driver ALK-rearrangement protein, which will potentially pave the way toward the development of novel therapeutic strategies for ALK-driven cancer.