Molecular signaling regulating anchorage-independent cell growth and survival

The question remains open whether the signaling pathways shown to be important for growth and transformation in adherent cultures proceed similarly and play similar roles for cells grown under anchorage-independent conditions. We have used the methylcellulose (MC) cell culture system, which permits the growth and colony formation of cells in non-adherent conditions, and subsequent recovery of the cells, and characterized the signal transduction pathways promoting anchorage-independent growth of transformed cells in two experimental systems.; The signaling functions essential for Ros-induced anchorage-independent growth were analyzed in chicken embryo fibroblasts (CEF) infected with the avian sarcoma virus UR2, encoding the oncogenic receptor protein tyrosine kinase (RPTK) v-Ros, or with two of its transformation-impaired mutants, F419 and DI. The overall tyrosine phosphorylation of cellular proteins in CEF transformed by v-Ros or by two oncogenic non-receptor PTKs, v-Src and v-Yes, was dramatically reduced in non-adherent conditions compared to that in adherent conditions. The UR2 transformation-defective mutants were differentially impaired compared to UR2 in the activation of PI3 kinase and Stat3, and in the induction of PI3 kinase-dependent cyclin A-associated Cdk2 activity, in non-adherent conditions. Consistently, the constitutively activated mutants of PI3 kinase and Stat3 rescued the ability of the UR2 mutants, F419 and DI, to promote anchorage-independent growth and dominant negative mutants of P13 kinase and Stat3, and the Cdk2 inhibitor roscovitine, inhibited UR2-induced anchorage-independent growth.; We analyzed the signaling pathways critical for promoting the enhanced anoikis resistance and potentiation of anchorage-independent growth of rat intestinal epithelial (RIE) cells treated with TGF-β1 and EGF, as compared to those treated with EGF alone. In anchorage-independent conditions, the TGF-β1- and EGF-treated cells showed sustained and enhanced activation of Erk/MAPK and Akt/PKB up to 24 hours, and enhanced phosphorylation and inactivation of the Akt substrates GSK3β and FKHRL1, compared to control cells or cells treated with TGF-β1 or EGF alone. EGF-mediated protection from anoikis was inhibited by pharmacological and dominant negative inhibitors of both MAPK kinase and PI3 kinase, whereas TGF-β1 plus EGF-mediated protection from anoikis was inhibited only by inhibitors of PI3 kinase. TGF-β1 plus EGF-induced anchorage-independent growth was more refractile to the inhibition of MAP kinase and PI3 kinase, compared to that induced by EGF alone. TGF-β1 plus EGF-treated RIE cells showed attenuation of the growth suppressive effects of TGF-β1 signaling including reduced nuclear translocation of Smad3 and decreased activation of p38 MAPK compared to treatment with either growth factor alone. Treatment with the p38 MAPK inhibitor SB202190 enhanced the EGF-induced anchorage-independent growth.; In summary, we have analyzed the signaling pathways activated by oncogenes and growth factors in fibroblasts and epithelial cells, the two major cell types in human cancers. Our studies provide an insight into the molecular mechanisms promoting anchorage-independent growth of transformed cells, which contributes to better understanding of tumorigenesis.