For the analysis of non-invasive squamous cell carcinoma, such as carcinoma in situ (cis), at Tokyo Medical University Hospital (Usuda J moved to Nippon Medical School on December 2012) between June 2009 and December 2011, we identified 10 patients with centrally located early lung cancers (CLELC) that had been diagnosed during a bronchoscopy performed because of abnormal sputum production and/or sputum cytological abnormalities in a mass screening. These 10 patients, who received photodynamic therapy using NPe6 (talaporfin sodium), were enrolled in the present study. This retrospective study was conducted with the approval of the Ethics Committee of Tokyo Medical University.

For the analysis of invasive squamous cell carcinoma, among 161 patients who underwent surgical resection for primary lung cancer between April 2007 and December 2008 at Nippon Medical School Hospital, 30 patients with squamous cell carcinoma were enrolled in the present study. Clinical information was extracted from the medical records. The disease stage was based on the TNM classification, 7th edition, using the International Union Against Cancer (UICC) staging system. The all-histological types were diagnosed by experienced pathologists at the Department of Pathology, Nippon Medical School Hospital, according to the histological typing of lung and pleural tumors in the WHO International Histological Classification of Tumors, 4th edition. This retrospective study was conducted with the approval of the Ethics Committee of Nippon Medical School Hospital.

We immunohistochemically examined the expression of Klotho in patients with lung squamous cell carcinoma who had undergone surgical resection or photodynamic therapy. Immunohistochemical staining for Klotho was performed on 4-µM formalin-fixed, paraffin-embedded tissue sections (15–17). The slides were deparaffinized in xylene and dehydrated in a graded ethanol series. Endogenous peroxidase was blocked with 0.3% H₂O₂ in methanol for 30 min. All the slides were heated to 95ºC by exposure to microwave irradiation for 20 min. The slides were then cooled for 1 h at room temperature and washed in phosphate-buffered saline (PBS). Non-specific binding was blocked by pre-incubation with 1% BSA for 30 min. After washing with PBS, the slides were incubated with an anti-Klotho antibody (KM2076; Kyowa Hakko Kirin, Tokyo, Japan) at 4ºC overnight. The slides were then incubated with anti-Klotho antibody for 30 min at room temperature. After washing in PBS, the slides were then incubated with a peroxidase-conjugated secondary antibody. Negative controls were prepared by omitting the primary antibody under the same experimental conditions. Antibody staining was considered positive when at least 10% of the tumor cells were stained, based on the use of a 10% cutoff level in several previous studies (15,16). All the slides were examined by two observers who had no knowledge of the patients' clinical data.

The human squamous lung cancer cell line SQ5 and the human adenocarcinoma cell line A549 were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). We transfected a GFP-Klotho plasmid, which was kindly provided by Dr Nabeshima (Foundation for Biomedical Research and Innovation, Kobe, Japan), into SQ5 cells using Lipofectamin 3000 transfection reagent, according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA).

We transfected the GFP-Klotho plasmid or a GFP vector into SQ5 cells; 24 h later, the cells were pelleted by centrifugation, resuspended in PBS to a final density of ~2.9×10⁶ cells/ml, and then filtered through a nylon membrane to remove cell aggregates. We sorted the GFP-positive cells using flow cytometry and a FACSCanto II (BD Biosciences, CA, USA) with activation at 488 nm and fluorescence emission monitoring at 508 nm (GFP). The data acquisition and analysis were performed using FlowJo software (TreeStar, Ashland, OR, USA). A minimum of 10,000 events were collected for each analysis. We eliminated dead cells and debris from the analysis using forward-scatter and side-scatter parameters, and the remaining cells were then sorted into GFP-positive and GFP-negative populations. The resulting cells were resuspended in PBS. The protein expression levels were then analyzed using an immunoblot analysis.

Cells were harvested by centrifugation and washed twice with ice-cold PBS. The cell pellets were incubated in a lysis buffer (50 mM Tris-HCl, pH 7.6, 120 mM NaCl, 1% Triton X-100, 0.2% sodium-deoxycholate and protease inhibitor). The lysates were cooled on ice for 10 min and then centrifuged at 14,000 × g for 30 min. Each sample of 6.56 µg of total protein was separated using SDS-PAGE on 7.5% gels and transferred to polyvinylidene difluoride membranes.

The membranes, after blocking with 5% skimmed milk, were incubated with the following antibodies: rat anti-human Klotho monoclonal antibody (1:500 dilution; Kyowa Hakko Kirin, Tokyo, Japan), rabbit anti-human E-cadherin monoclonal antibody (1:1,000 dilution), rabbit anti-human N-cadherin monoclonal antibody (1:1,000 dilution), rabbit anti-human vimentin monoclonal antibody (1:1000 dilution), or rabbit anti-human Snail monoclonal antibody (1:500 dilution) (all from Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h. After rinsing with PBS containing 0.1% (v/v) Triton X-100, the membranes were incubated with goat anti-rat immunoglobulin-G (IgG)-conjugated horseradish peroxidase (HRP) (1:10,000 dilution; Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) for the anti-Klotho antibody or goat anti-rabbit IgG conjugated HRP (1:2,000 dilution; Cell Signaling Technology, Inc.) for the other antibodies. As a loading control, blots were probed with β-actin (1:1,000 dilution; Cell Signaling Technology, Inc.). The membranes were washed and developed using western blotting enhanced chemiluminescence detection reagents (Bio-Rad Laboratories, Richmond, CA, USA).

The statistical analyses were performed using EZR (Saitama Medical Center, Jichii Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria) (18). The study variables were compared between the study groups using the Fisher's exact test for categorical variables and the Student's t-test for continuous variables. P-values of <0.05 were considered significant.

To elucidate the association between cancer-invasiveness and the expression of Klotho, we first examined the expression of Klotho in lung cancer patients with squamous cell carcinoma in situ (cis) using an immunohistochemical analysis. The clinicopathological characteristics of the patients are listed in Table I. The median patient age at the time of diagnosis was 68 years (range, 57–81 years). All the patients were men and were heavy smokers with a smoking history of >30 pack-years. In all 10 patients with carcinoma in situ, we observed the expression of Klotho as shown in Fig. 1C and D. The expression of Klotho was observed in not only normal bronchial epithelial cells, but also centrally located early lung cancers, which were all carcinoma in situ and had been treated using PDT. The criteria for a centrally located early lung cancer (CLELC) were strictly defined in 1975. The tumor must be located only as far as the segmental bronchi and must be a carcinoma in situ or with only limited invasion into the bronchial wall. Fig. 1C and D shows the typical features of CLELC. In these centrally located early lung cancers, cells that were located closer to the basal membrane epithelial cells had lower expression levels of Klotho. These results suggest that the expression of Klotho may be associated with cancer invasiveness.

Next, to analyze invasive squamous cell carcinoma, we performed immunohistochemical staining for specimens from patients who had undergone surgical resection. The clinicopathological characteristics of the patients with invasive squamous cell carcinoma are listed in Table II. Though the sample number in Klotho positive group is small, no significant differences in the clinicopathological characteristics were observed between the Klotho-positive group and the Klotho-negative group (Table III). However, in lung cancer patients with invasive or advanced squamous cell carcinoma, who had undergone a complete surgical resection, Klotho expression was observed in only 4 patients (13%) (Fig. 2). Therefore, these results suggested that the expression of Klotho may be associated with tumor development in squamous cell lung cancer.

We hypothesized that Klotho expression regulates EMT-related proteins, which are associated with invasion and metastasis. We next studied EMT markers such as E-cadherin, N-cadherin, vimentin and Snail in lung squamous cancer cells. To improve the efficiency of transfection, the SQ5 cells were transfected with a GFP-Klotho expression vector and then sorted into GFP-positive or GFP-negative populations using flow cytometry. Overall, 14.8% of the cells were sorted as GFP-positive cells (Fig. 3A, B).

To determine whether Klotho expression affects the protein expressions of EMT markers, an immunoblot assay was performed (Fig. 4). The GFP-positive sorted cells showed an overexpression of Klotho. Moreover, in SQ5 cells transiently overexpressing GFP-Klotho, the expression of N-cadherin, which is a mesenchymal marker, was completely inhibited, compared with wild-type SQ5 cells or SQ5 cells transfected with only the GFP vector. Namely, the overexpression of Klotho resulted in a loss of N-cadherin expression in the SQ5 cells. The overexpression of Klotho did not affect the regulations of other mesenchymal markers, such as vimentin or Snail, or the epithelial marker E-cadherin. Thus, the present in vitro study confirmed that Klotho expression inhibits N-cadherin expression, which is an EMT marker, in lung squamous cancer cells.