A novel function for the tumor suppressor p16(INK4a): Induction of anoikisvia upregulation of the alpha(5)beta(1) fibronectin receptor

The tumor suppressor gene p16(INK4a) inhibits the kinase activity of the cy clin-dependent kinase 4-6/cyclin D complexes and subsequent phosphorylation of critical substrates necessary for transit through the G1 phase of the c ell cycle. Recent studies suggested that control of the G1/S boundary might not be the sole biological function of p16(INK4a). We hypothesized that p1 6(INK4a) might influence hitherto unknown critical features of a malignant epithelial phenotype, such as anchorage dependence, Here we provide evidenc e that stable transfection of p16(INK4a) restitutes apoptosis induction upo n loss of anchorage (anoikis) in a variety of human cancer cells. Anoikis i n p16(INK4a)-transfected cells was evidenced by DNA fragmentation and poly( ADP-ribose) polymerase cleavage upon cultivation on polyhydroxyethylmethacr ylate-coated dishes and was associated with suppression of anchorage-indepe ndent growth as well as complete loss of tumorigenicity. p16(INK4a)-mediate d anoikis was due to selective transcriptional upregulation of the alpha(5) integrin chain of the alpha(5)beta(1) fibronectin receptor as detected by FACS(R) analysis, immunoprecipitation, Northern blotting, and nuclear run-o n assays. Addition of soluble fibronectin and inhibitory alpha(5) antibodie s to nonadherent cells completely abolished p16(INK4a)-mediated anoikis, wh ereas laminin was ineffective. Furthermore, antisense-induced downregulatio n of the alpha(5) integrin chain in p16(INK4a)-transfected cells restored r esistance to anoikis, These data suggest a novel functional interference be tween a cell cycle-regulating tumor suppressor gene and membrane-bound inte grins, thus regulating a hallmark feature of an epithelial transformed phen otype: susceptibility to anoikis.