Timp1 interacts with beta-1 integrin and CD63 along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway independently of Akt phosphorylation.

BACKGROUND: Anoikis resistance is one of the abilities acquired along tumor progression. This characteristic is associated with metastasis development, since tumorigenic cells must survive independently of cell-matrix interactions in this process. In our laboratory, it was developed a murine melanocyte malignant transformation model associated with a sustained stressful condition. After subjecting melan-a melanocytes to 1, 2, 3 and 4 cycles of anchorage impediment, anoikis resistant cells were established and named 1C, 2C, 3C and 4C, respectively. These cells showed altered morphology and PMA independent cell growth, but were not tumorigenic, corresponding to pre-malignant cells. After limiting dilution of 4C pre-malignant cells, melanoma cell lines with different characteristics were obtained. Previous data from our group showed that increased Timp1 expression correlated with anoikis-resistant phenotype. Timp1 was shown to confer anchorage-independent growth capability to melan-a melanocytes and render melanoma cells more aggressive when injected into mice. However, the mechanisms involved in anoikis regulation by Timp1 in tumorigenic cells are not clear yet. METHODS: The ß1-integrin and Timp1 expression were evaluated by Western blotting and CD63 protein expression by flow cytometry using specific antibodies. To analyze the interaction among Timp1, CD63 and ß1-integrin, immunoprecipitation assays were performed, anoikis resistance capability was evaluated in the presence or not of the PI3-K inhibitors, Wortmannin and LY294002. Relative expression of TIMP1 and CD63 in human metastatic melanoma cells was analyzed by real time PCR. RESULTS: Differential association among Timp1, CD63 and ß1-integrins was observed in melan-a melanocytes, 4C pre-malignant melanocytes and 4C11- and 4C11+ melanoma cells. Timp1 present in conditioned medium of melanoma cells rendered melan-a melanocytes anoikis-resistant through PI3-K signaling pathway independently of Akt activation. In human melanoma cell lines, in which TIMP1 and beta-1 integrin were also found to be interacting, TIMP1 and CD63 levels together was shown to correlate significantly with colony formation capacity. CONCLUSIONS: Our results show that Timp1 is assembled in a supramolecular complex containing CD63 and ß1-integrins along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway, independently of Akt phosphorylation. In addition, our data point TIMP1, mainly together with CD63, as a potential biomarker of melanoma.

Sentinel lymph node biopsy in cutaneous melanoma: analysis of 240 consecutive cases.

BACKGROUND: The objective of this study was to evaluate practical rules for sentinel lymph node biopsy for melanoma and discuss the indications and outcomes of 240 patients. METHODS: A prospective, nonrandomized analysis was performed on 240 patients in a referral cancer center. The median patient age was 51 years, and the median Breslow thickness was 1.60 mm. Ulceration was found in 30.4 percent of the cases. The median follow-up was 27.81 months. The sentinel lymph node biopsy was performed in 240 patients with cutaneous melanoma thicker or equal to 1 mm. The operation was performed with preoperative lymphoscintigraphy and postoperative immunohistochemistry. A statistical analysis was performed comparing the need for a gamma probe in each location, the value of the experience, the need for immunohistochemistry, positivity compared with Breslow thickness, reasons for the success of the lymph node localization, and evolution. RESULTS: A total of 263 lymph node basins were identified (160 in the axilla, 86 in the inguinal region, and 17 in less common locations, including the popliteal, epitrochlear, and cervical regions). In every lymph node basin, the success of localization was directly related to use of the probe. The success rate for finding the sentinel lymph node increased year by year. Lymph node analysis disclosed positivity of 12.5 percent with hematoxylin and eosin staining and 17.5 percent with immunohistochemistry (excluding the sentinel lymph node not found disclosed 13.2 percent with hematoxylin and eosin and 18.5 percent with HMB45). Immunohistochemistry increased positivity by 40 percent. Positivity was directly related to Breslow thickness (p < 0.001). CONCLUSIONS: This study shows the importance of the gamma probe in all lymph node basins but mainly in the axilla and unusual basins, as well as the importance of experience and immunohistochemistry. As a new procedure, it was possible to recognize the pattern of recurrence in the follow-up.