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

Abstract

BackgroundAnoikis 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.MethodsThe β1-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 β1-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.ResultsDifferential association among Timp1, CD63 and β1-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.ConclusionsOur results show that Timp1 is assembled in a supramolecular complex containing CD63 and β1-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.

Signaling Pathways Altered During the Metastatic Progression of Melanoma

“…Previous data from our laboratory show a progressive increase in the TIMP1 expression along the melanoma genesis, and this increase is related to resistance to anoikis and to a more aggressive phenotype in vivo [76]. We also observed that soluble TIMP1 in non-tumorigenic lineage melan-a confers anoikis resistance and it is differentially associated with CD63 and β1-integrin along the melanoma genesis [77]. CD63, β1-integrin and TIMP1 are not interacting in the murine melan-a melanocytes.…”

“…Interestingly, the expression of their inhibitors, TIMPs, is also generally increased in several cancers. Among them, TIMP1 has been associated with poor prognosis in metastatic melanoma, suggesting promising value of TIMP1 as a prognostic marker of the tumor [76][77]. In our laboratory, a model that allows us to study different stages of melanoma progression was developed.…”

“…In the premalignant 4C cell line, TIMP1 associates with CD63, but not with β1-integrins, and CD63 interacts with β1-integrins. In 4C11-and 4C11+ melanoma cell lines, the formation of CD63/β1-integrin/TIMP1 complex occurs, which could be related to a more efficient activation of cell survival signals [77].…”

Les tétraspanines dans la physiopathologie de la peau

“…Les cellules qui expriment fortement CD63 présentent des niveaux d'expression de E-cadhérine 5 élevés et une diminution de N-cadhérine et de Snail 6 , suggérant que la tétraspanine CD63 pourrait inhiber la pseudo-transition épithélio-mésenchymateuse des cellules de mélanome [39]. Des expériences de co-immunoprécipitation 7 montrent qu'au niveau de la membrane plasmique, CD63 interagirait avec l'intégrine β1 et la protéine TIMP1 (metalloproteinase inhibitor 1), un membre de la famille des inhibiteurs de MMP, dont l'expression est fortement augmentée en fonction de la progression tumorale, dans un modèle murin de mélanome [41]. Nous avons identifié une nouvelle tétraspanine, la tétraspanine 8 (Tspan8), qui joue un rôle central dans l'acquisition du phénotype invasif des cellules de mélanome.…”

Immunological Processes in Cancer: A Link Between Inflammation and Immunity

“…Moreover, in this model, PI3K/AKT and ERK1/2 signaling pathways seem to be involved in the cell proliferation. Toricelli et al (2013) reported a Tissue Inhibitor of Metalloproteinase-1 (TIMP1) -CD63-β1 integrins containing-supramolecular complex in cell surface of melanoma cells. The over-expression of TIMP1 was shown to be involved in the acquisition of anoikis-resistant phenotype in melanoma cells through PI3K signaling pathway independently of AKT activation.…”