Elevated c-Src and c-Yes expression in malignant skin cancers

A total of 8 normal skin tissues and 24 malignant skin tumor tissues were obtained from patients who underwent surgery between July 2009 and September 2009 in the Departments of Plastic and Reconstructive Surgery at Hanyang University Guri Hospital and Soonchunhyang University Hospital in South Korea. Informed consent was obtained from the patients before surgery. The malignant skin tumor tissues, including 8 MM, 8 SCC, and 8 BCC, were obtained from patients who were treated with excisional surgery. All tumor tissues were examined using both conventional histopathological confirmation and immunohistochemical studies to confirm the diagnosis. Clinical and histopathological data are shown in Table 1. A portion of the specimens were frozen in liquid nitrogen immediately after resection and stored at -80°C degrees for subsequent western blot analysis. The human malignant melanoma cell line G361, obtained from the American Type Culture Collection (CRL 1424; Rockville, MD, USA), served as a positive control for c-Src and c-Yes expression.

Tissue samples were homogenized in WCE buffer [25 mM HEPES (pH 7.7), 0.3 M NaCl, 1.5 mM MgCl₂, 0.2 mM ethylenediamine tetraacetic acid (EDTA), 0.1% Triton X-100, 0.5 mM dithiothreitol (DTT), 20 mM-glycerolphosphate, 0.1 mM Na₃VO₄, 2 g per mL leupeptin, 2 g per mL aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail tablet (Boehringer Mannheim)]. The tissue suspension was rotated at 4°C for 10 minutes. Supernatants were collected and then kept at -70°C and used for western blotting. Proteins from the tissue were separated by SDS-PAGE using NuPAGE 4-12% bis-Tris gels (Invitrogen, NP0335Box) and then transferred to Immobilon-P membranes. The membrane was blocked using 5% BSA in TBS-T (20 mM Tris, pH 7.6, 130 mM NaCl, and 0.1% Tween 20) solution. 6 MM, 6 SCC, 6 BCC and 6 normal skin tissues were then reacted with the primary antibody, Src (36D10) rabbit mAb (Cell Signaling technology^®, #2109) and Yes antibody (Cell Signaling technology^®, #2734) diluted to 1:1,000 concentration, at 4°C for 16 hours. This was then followed by washing buffer and TBST buffer washes (10 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.05% Tween 20), 4 times for 10 minutes, 10 minutes, 15 minutes and 15 minutes and then reacted with anti-rabbit IgG (Cell Signaling technology^®, #7074) -horseradish peroxidase-linked species-specific whole antibody dilutes to 1:2,000 for 1 hour. After the reaction with the secondary antibody, it was washed 4 times for 10 minutes, 10 minutes, 15 minutes and 15 minutes. Proteins on the membrane were detected using an enhanced chemiluminescence solution kit (Amersham, UK). The membranes were stripped and reblotted with anti-actin antibody (catalog number sigma A5441). Western blotting analysis with mouse monoclonal antibody specific for phospho-Src (Calbiochem-Novabiochem, San Diego, CA) and mouse monoclonal antibody specific for phospho-Yes (WAKO, Osaka, Japan), were carried out on the 2 MM, 2 SCC, 2 BCC and 2 normal skin as described.

For immunohistochemical studies, the stored formalin-fixed, paraffin-embedded samples that included, 16 MM, 16 SCC, and 16 BCC were used. Parraffin sections (4 μm) were deparaffinized in xylene, rehydrated in 10 mM citrate buffer (pH 6.0), and then heated in a microwave oven for 15 minutes to restore antigens. To suppress endogenous peroxidase within the tissues, the samples were treated with 3% peroxide for 5 minutes, then with blocking solution for 30 minutes. Slides were incubated with primary Src (36D10) rabbit mAb and Yes antibody in a humid chamber for 60 minutes. Tissue staining was visualized with 3,3'-Diaminobenzidine (DAB) (ScyTek, USA) substrate chromogen solution.

The amount of expression in western blotting was measured with TINA software (Version 2.10e). Measured amount of expression of malignant skin tumors was compared to that of normal skin.

The data from the TINA score were analyzed using the nonparametric Mann-Whitney test. A p < 0.05 was considered statistically significant.

Western blot analysis was performed to determine the expression of c-Src and c-Yes in 18 malignant skin tumors and 6 normal skin tissues. c-Src was expressed in all malignant skin tumors but not expressed in normal skin tissues, while c-Yes was expressed in MM and SCC and not in BCC and normal skin (Fig. 1) (data not shown for M-5, M-6, S-5, S-6, B-5, B-6, N-5, N-6). The expression amount score in western blotting was measured by TINA software (Version 2.10e), and the average TINA score of c-Src was 0.006 in normal skin, 1.143 in MM, 1.027 in SCC, and 0.590 in BCC. The average TINA score of c-Yes was 0.011 in normal skin, 0.374 in MM, 1.054 in SCC, and 0.012 in BCC. There were significant differences in the TINA scores of c-Src between malignant skin tumors and normal skin (p = 0.002). Of the tumor tissues, significant differences in c-Src score among the tumors (p = 0.002) were noted. With regard to c-Yes expression, there were significant differences in the TINA scores between two skin tumors (MM and SCC) and normal skin (p = 0.002), and there was no significant difference between BCC and normal skin (p = 0.818). The expression amount score based on western blotting is graphed in Fig. 2. To confirm the expression in phosphate form, western blot analysis with phospho-Src and phospho-Yes was also performed in 2 MM, 2 SCC, 2 BCC and 2 normal skin tissues. Phospho-Src was expressed in all malignant skin tumors and not expressed in normal skin tissues and phospho-Yes was expressed in MM and SCC but not in BCC and normal skin (Fig. 3).

Immunohistochemical study showed that the staining pattern of c-Src and c-Yes in MM, SCC and BCC correlated with western blot analysis. c-Src protein was expressed in MM and SCC with moderate positivity, and BCC with mild positivity (Fig. 4). c-Yes was expressed in MM with moderate positivity and SCC with strong positivity, but not in BCC (Fig. 5).