Klotho expression is correlated to molecules associated with epithelial‑mesenchymal transition in lung squamous cell carcinoma

Abstract

Abstract. Klotho is known as an anti-aging gene. We previously reported that the expression of Klotho is a postoperative prognostic factor for patients with lung large cell neuroendocrine carcinoma and lung small cell carcinoma. Recently, Klotho was shown to suppress the epithelial-mesenchymal transition (EMT). In the present study, we examined the association between the expression of Klotho and the regulation of EMT in lung squamous cell carcinoma. We immunohistochemically examined the expression of Klotho in patients with lung squamous cell carcinoma who had undergone surgical resection or photodynamic therapy. The immunohistochemical analysis showed that Klotho expression was observed not only in normal bronchial epithelial cells, but also in centrally located early lung cancers, which were all carcinomas in situ and were treated using PDT. However, in lung cancer patients with invasive and or advanced squamous cell carcinoma who had undergone a complete surgical resection, Klotho expression was observed in only 4 patients (13%). To elucidate the associations between the expression of Klotho and the expressions of EMT-related proteins, such as E-cadherin, N-cadherin, vimentin, and Snail, we transiently transfected GFP-Klotho plasmid DNA into the human squamous lung cancer cell line SQ5 and examined the expressions of these proteins of GFP-positive cells after sorting using flow cytometry. In SQ5 cells overexpressing GFP-Klotho, the expression of N-cadherin, which is a mesenchymal marker, was completely inhibited, compared with that in SQ5 cells transfected with the GFP vector. The overexpression of Klotho did not affect the regulation of either other mesenchymal markers (such as vimentin and Snail) or the regulation of an epithelial marker (E-cadherin). We concluded that the expression of Klotho was related to the degree of cancer invasiveness and that Klotho inhibits the expression of N-cadherin and regulates the EMT in lung cancer.

Alpha‐Klotho, a critical protein for lung health, is not expressed in normal lung

“…There is controversy concerning whether αKlotho present in the lung is produced by resident lung cells or derived from the circulation. Several lung cell lines show αKlotho mRNA expression by RT‐PCR but none express αKlotho protein . On the other hand, Kuro‐o and colleagues discovered that αKlotho could not detect αKlotho transcript in the intact lung, a finding independently reproduced by our group .…”

“…On the other hand, Kuro‐o and colleagues discovered that αKlotho could not detect αKlotho transcript in the intact lung, a finding independently reproduced by our group . Despite the absence of mRNA, αKlotho protein expression was reported in lungs and large airways by several groups using commercial antibodies . The discrepant in vitro and in vivo results, complicated by uncertain sensitivity and specificity of the various anti‐αKlotho antibodies used in different studies, significantly impede progress in the understanding of αKlotho biology.…”

“…Several published reports described αKlotho expression in lung parenchyma by IHC and very intense labeling particularly in the airways using various commercial antibodies . As in the case of IB and IP (Figure ), no αKlotho labeling was detected in wild‐type mouse lung by IHC using Antibody 1 or 2, contradicting the widespread labeling observed with Antibody 3, 4, or, 5 (Figure A, upper row).…”

“…Endogenous αKlotho transcripts have been detected in cultured lung cells transfected with αKlotho cDNA; yet none of the lung cell lines express αKlotho at baseline . Li et al performed IHC using an unspecified polyclonal anti‐αKlotho antibody, and reported co‐localization with alveolar macrophages and decreased staining in lungs of smokers and patients with chronic obstructive pulmonary disease (COPD); however, control specimens showed high background intensity, and specificity of staining was not established.…”

“…The highly enriched αKlotho content in human induced pluripotent stem cell secretome significantly contributes to protection of lung cells and lungs from hyperoxic injury . Multiple laboratories have shown direct or indirect αKlotho actions in the lung using in vitro systems . Cumulative literature unequivocally supports a pivotal cytoprotective role of αKlotho in the lung.…”