Department of Radiation Oncology, Columbia University College of Physicians and Surgeons, New York, NY, USA.
Department of Oncology, Albert Einstein College of Medicine of Yeshiva University/Montefiore Medical Center, Bronx, NY, USA.
Sci Rep. 2022 Jun 16;12(1):10140. doi: 10.1038/s41598-022-14448-8.
Lung cancer has been the most common cancer worldwide for several decades. The outcomes of patients with locally advanced lung cancer remain dismal, and only a minority of patients survive more than 5 years. However, tumor therapeutic resistance mechanisms are poorly studied. Identification of therapeutic resistance pathways in lung cancer in order to increase the sensitivity of lung tumor cells to therapeutic agents is a crucial but challenging need. To identify novel genes that modulate the response to platinum-based therapy, we performed a genome-wide high-throughput ribonucleic acid interference (RNAi) screen via transfection of human lung cancer (PC9) cells with a viral short hairpin RNA (shRNA) library. We further validated a potential target via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic survival assays on PC9 and A549 lung tumor cells transfected with small interfering RNAs (siRNAs) to successfully downregulate protein expression and then treated with increasing doses of cisplatin or X-ray radiation. We determined protein expression by immunohistochemistry (IHC) after chemoradiotherapy and analyzed gene expression-based survival outcomes in two cohorts of human non-small-cell lung cancer (NSCLC) patients. The screen identified several targets involved in epithelial-to-mesenchymal transition (EMT), including Smurf1, Smurf2, YAP1, and CEBPZ, and glycolytic pathway proteins, including PFKFB3. Furthermore, we found that the small molecule proteasome inhibitor bortezomib significantly downregulated Smurf2 in lung cancer cells. The addition of bortezomib in combination with cisplatin and radiation therapy in PC9 and A549 cells led to an increase in deoxyribonucleic acid (DNA) double-strand breaks with increased numbers of γ-H2AX-positive cells and upregulation of apoptosis. Finally, we found that Smurf2 protein expression was upregulated in situ after treatment with cisplatin and radiation therapy in a relevant cohort of patients with stage III NSCLC. Additionally, Smurf2 gene expression was the strongest predictor of survival in patients with squamous NSCLC after chemotherapy or chemoradiotherapy. We successfully identified and validated Smurf2 as both a common modulator of resistance and an actionable target in lung cancer. These results suggest the urgent need to investigate clinical Smurf2 inhibition via bortezomib in combination with cisplatin and radiation for patients with locally advanced NSCLC.
几十年来，肺癌一直是全球最常见的癌症。局部晚期肺癌患者的预后仍然不佳，只有少数患者能存活 5 年以上。然而，肿瘤治疗耐药机制的研究还很不完善。为了提高肺癌肿瘤细胞对治疗药物的敏感性，确定肺癌中的治疗耐药途径是一个至关重要但具有挑战性的需求。为了鉴定调节顺铂治疗反应的新基因，我们通过用病毒短发夹 RNA (shRNA) 文库转染人肺癌 (PC9) 细胞，进行了全基因组高通量核糖核酸干扰 (RNAi) 筛选。我们进一步通过三-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴盐 (MTT) 和克隆存活测定在转染小干扰 RNA (siRNA) 的 PC9 和 A549 肺肿瘤细胞上验证了一个潜在的靶标，成功下调了蛋白表达，然后用递增剂量的顺铂或 X 射线辐射处理。我们通过化学放射治疗后的免疫组织化学 (IHC) 测定蛋白表达，并分析了两个非小细胞肺癌 (NSCLC) 患者队列的基于基因表达的生存结果。该筛选鉴定了几个参与上皮间质转化 (EMT) 的靶标，包括 Smurf1、Smurf2、YAP1 和 CEBPZ，以及糖酵解途径蛋白，包括 PFKFB3。此外，我们发现小分子蛋白酶体抑制剂硼替佐米可显著下调肺癌细胞中的 Smurf2。在 PC9 和 A549 细胞中，硼替佐米与顺铂和放射治疗联合使用，导致脱氧核糖核酸 (DNA) 双链断裂增加，γ-H2AX 阳性细胞增多，细胞凋亡上调。最后，我们发现在 III 期 NSCLC 相关患者队列中，顺铂和放射治疗后 Smurf2 蛋白在原位表达上调。此外，在接受化疗或放化疗的鳞状非小细胞肺癌患者中，Smurf2 基因表达是生存的最强预测因子。我们成功地鉴定并验证了 Smurf2 是肺癌中常见的耐药调节剂和可操作的靶标。这些结果表明，迫切需要通过硼替佐米联合顺铂和放射治疗，对局部晚期 NSCLC 患者进行临床 Smurf2 抑制研究。
