title: 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: 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 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.
  
  
  RESULTS
  Differential 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.
  
  
  CONCLUSIONS
  Our 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.
authors.affiliation: Pharmacology Department, Universidade Federal de São Paulo, São Paulo, Brazil.
authors.forename: Mariana
authors.initials: M
authors.lastname: Toricelli
authors.forename: Fabiana H M
authors.initials: FH
authors.lastname: Melo
authors.forename: Giovani B
authors.initials: GB
authors.lastname: Peres
authors.forename: Débora C P
authors.initials: DC
authors.lastname: Silva
authors.forename: Miriam G
authors.initials: MG
authors.lastname: Jasiulionis
mesh_terms: D023102:Anoikis / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000494:pharmacology; D018450:Disease Progression; D015870:Gene Expression; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism*; D058965:MART-1 Antigen / Q000378:metabolism; D008544:Melanocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008545:Melanoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000378:metabolism; D009362:Neoplasm Metastasis; D010641:Phenotype; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D011485:Protein Binding; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015398:Signal Transduction; D060149:Tetraspanin 30 / Q000378:metabolism*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D017077:Culture Media, Conditioned; D019012:Integrin beta1; D058965:MART-1 Antigen; D046912:Multiprotein Complexes; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D060149:Tetraspanin 30; D019715:Tissue Inhibitor of Metalloproteinase-1