Antiaging Gene Klotho Regulates Epithelial-Mesenchymal Transition and Increases Sensitivity to Pemetrexed by Inducing Lipocalin-2 Expression

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 (IC50 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.