Takegahara Kyoshiro, Usuda Jitsuo, Inoue Tatsuya, Sonokawa Takumi, Matsui Takuma, Matsumoto Mitsuo
Department of Thoracic Surgery, Nippon Medical School, Tokyo 113-8603, Japan.
Oncol Lett. 2021 May;21(5):418. doi: 10.3892/ol.2021.12679. Epub 2021 Mar 28.
Epithelial-mesenchymal transition (EMT) is considered to serve an important role in the metastatic/invasive ability of cancer cells, in the acquisition of drug resistance, and in metabolic reprogramming. In the present study, it was hypothesized that the Klotho gene is involved in the metastatic/invasive ability of lung cancer. We previously reported an association between Klotho expression and overall survival in patients with small cell lung cancer and large cell neuroendocrine cancer. We also found that Klotho expression was associated with EMT-related molecules in lung squamous cell carcinoma. The present study aimed to analyze the function of the Klotho gene and to elucidate its relevance to the regulation of the EMT. For this purpose, GFP-Klotho plasmids were transfected into lung adenocarcinoma cells (A549) and cell lines with stable expression (A549/KL-1 and A549/KL-2) were established. A549/KL-1 cells expressed higher levels of Klotho protein by western blot analysis compared with A549/KL-2 cells. In western blotting of A549 and A549/KL-1 cells, the expression of the mesenchymal marker N-cadherin was found to be completely inhibited in A549/KL-1 cells suggesting that Klotho expression may regulate the EMT in cancer cells via the inhibition of N-cadherin. The results of the sensitivity tests demonstrated that A549/KL-1 cells were significantly more sensitive to pemetrexed compared with A549 cells (IC A549/KL-1 vs. A549 cells, 0.1 µM vs. 0.7 µM). The results of the microarray analysis demonstrated that a very high level of lipocalin-2 (LCN2) expression was induced in the A549/KL-1 cells. Klotho overexpression completely suppressed the expression of mesenchymal markers, such as N-cadherin and Snail1 (Snail). The results of the present study suggested that there may be a new mechanism of action for the antitumor effects of pemetrexed, namely, LCN2-mediated modulation of N-cadherin expression. Klotho expression during cancer treatment has great potential as a predictor for efficacy of pemetrexed and as a factor in the selection of personalized medicine for postoperative adjuvant chemotherapy.
上皮-间质转化（EMT）被认为在癌细胞的转移/侵袭能力、耐药性的获得以及代谢重编程中发挥重要作用。在本研究中，我们假设Klotho基因与肺癌的转移/侵袭能力有关。我们之前报道了Klotho表达与小细胞肺癌和大细胞神经内分泌癌患者的总生存期之间的关联。我们还发现Klotho表达与肺鳞状细胞癌中与EMT相关的分子有关。本研究旨在分析Klotho基因的功能，并阐明其与EMT调节的相关性。为此，将绿色荧光蛋白-Klotho质粒转染到肺腺癌细胞（A549）中，并建立了稳定表达的细胞系（A549/KL-1和A549/KL-2）。通过蛋白质印迹分析，与A549/KL-2细胞相比，A549/KL-1细胞中Klotho蛋白表达水平更高。在A549和A549/KL-1细胞的蛋白质印迹分析中，发现间充质标志物N-钙黏蛋白在A549/KL-1细胞中的表达完全受到抑制，这表明Klotho表达可能通过抑制N-钙黏蛋白来调节癌细胞中的EMT。敏感性测试结果表明，与A549细胞相比，A549/KL-1细胞对培美曲塞明显更敏感（A549/KL-1与A549细胞的半数抑制浓度，分别为0.1 μM和0.7 μM）。微阵列分析结果表明，A549/KL-1细胞中诱导了非常高水平的lipocalin-2（LCN2）表达。Klotho过表达完全抑制了间充质标志物如N-钙黏蛋白和Snail1（Snail）的表达。本研究结果表明，培美曲塞抗肿瘤作用可能存在一种新的作用机制，即LCN2介导的N-钙黏蛋白表达调节。癌症治疗期间的Klotho表达作为培美曲塞疗效的预测指标以及术后辅助化疗个性化药物选择的一个因素具有巨大潜力。
