Abstract
VILIP-1, a member of the neuronal Ca++ sensor protein family, acts as a tumor suppressor gene in an experimental animal model by inhibiting cell proliferation, adhesion and invasiveness of squamous cell carcinoma cells. Western Blot analysis of human tumor cells showed that VILIP-1 expression was undetectable in several types of human tumor cells, including 11 out of 12 non-small cell lung carcinoma (NSCLC) cell lines. The down-regulation of VILIP-1 was due to loss of VILIP-1 mRNA transcripts. Rearrangements, large gene deletions or mutations were not found. Hypermethylation of the VILIP-1 promoter played an important role in gene silencing. In most VILIP-1-silent cells the VILIP-1 promoter was methylated. In vitro methylation of the VILIP-1 promoter reduced its activity in a promoter-reporter assay. Transcriptional activity of endogenous VILIP-1 promoter was recovered by treatment with 5′-aza-2′-deoxycytidine (5′-Aza-dC). Trichostatin A (TSA), a histone deacetylase inhibitor, potently induced VILIP-1 expression, indicating that histone deacetylation is an additional mechanism of VILIP-1 silencing. TSA increased histone H3 and H4 acetylation in the region of the VILIP-1 promoter. Furthermore, statistical analysis of expression and promoter methylation (n = 150 primary NSCLC samples) showed a significant relationship between promoter methylation and protein expression downregulation as well as between survival and decreased or absent VILIP-1 expression in lung cancer tissues (p<0.0001). VILIP-1 expression is silenced by promoter hypermethylation and histone deacetylation in aggressive NSCLC cell lines and primary tumors and its clinical evaluation could have a role as a predictor of short-term survival in lung cancer patients.
Highlights
Visinin-like protein-1 (VILIP-1), a member of the visininrecoverin neuronal calcium-sensor protein family, has an important role in regulating cAMP levels, cell signaling and differentiation in central nervous system
Except for a few tumor cell lines from central nervous system and colon, VILIP-1 protein was commonly absent in human cancer cell lines, including those derived from prostate, lung, ovarian and renal tumors as well as those from melanoma and leukemia (Figure 1)
We focused on lung-derived cells to further examine VILIP-1 protein expression in normal human bronchial epithelial cells (NHBE) and a total of 12 non-small cell lung cancer (NCSLC) cell lines (Figure 2A)
Summary
Visinin-like protein-1 (VILIP-1), a member of the visininrecoverin neuronal calcium-sensor protein family, has an important role in regulating cAMP levels, cell signaling and differentiation in central nervous system. Our group identified VILIP-1 to be differentially expressed in chemically-induced murine skin cancer cells of high and low invasive ability by differential display, indicating a new function of VILIP-1 in cancer [3,4]. VILIP-1 was expressed in normal basal epidermal keratinocytes, while its expression was markedly decreased or undetectable in aggressive and invasive squamous cell carcinoma (SCC). Less aggressive SCCs showed expression of VILIP-1 protein. Ectopic overexpression of VILIP-1 resulted in a cAMP-mediated decrease of in vivo and in vitro growth and invasiveness of SCC cells [3]. Reduced invasiveness and elevated cAMP levels were accompanied by decreased MMP-9 as well as lowered RhoA activity [4]
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