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Introduction. Anoikis is a form of apoptosis triggered by the lack of attachment to the extracellular matrix (ECM). Anoikis resistant transformed cells have increased survival times in the absence of attachment facilitating their migration and colonization of distant sites. Clonal lines derived from the human osteosarcoma cell line SAOS-2 were established and clones sensitive to anoikis were chosen. Culturing these clones under cycles of adhered and suspended conditions resulted in the induction of an anoikis resistant phenotype (SAOSar). The purpose of this study was to identify the signal transduction pathway involved during anoikis resistance of SAOSar cells. Methods. Anoikis was assayed after culture on poly-HEMA treated culture wells followed by propidium iodide staining and flow cytometry analyses. Src, PI-3K and Akt activity were pharmacologically inhibited using commercially available specific inhibitors. Activated states of FAK, Src, and Akt were determined by western immunoblot using phosphospecific antibodies Results. Akt is known as an important mediator that promotes cell survival. Therefore, its role during anoikis of SAOS cells was examined. Increased phosphorylation of Akt at Ser473 was found in SAOSar cells regardless of their culture conditions, suggesting a constitutively active state. Pharmacological inhibition of Akt or its upstream mediator, PI3-K, resulted in anoikis sensitivity. Since FAK, through the formation of focal adhesions, is central in coordinating the signals that arise from the ECM, it was expected that in anoikis resistant SAOSar cells FAK would remain phosphorylated despite suspended conditions, and thus maintaining the survival signals. No difference was found among the phosphorylation patterns of FAK between apoptotic (SAOSp) and non-apoptotic (SAOSar) cells after culture under anoikis-inducing conditions. These results indicate that the major component of the focal adhesion complex is functioning normally and is not aberrantly upregulated in anoikis resistant SAOSar cells. Moreover, this suggests that anoikis resistance in SAOSar cells is achieved through a mechanism independent of FAK. Since Src is known to be involved in anchorage-dependent survival, its role was then analyzed. Src activity was found to be upregulated in SAOSar cells. Moreover, pharmacological inhibition of Src activity correlated with a decrease in PKB/Akt activity and restored sensitivity to anoikis. Conclusions. The mechanism of anoikis resistance in SAOSar cells involves the activation of PKB/Akt by Src, independently of FAK tyrosine phosphorylation. In this way, Src activation in suspended anoikis resistant SAOSar cells is able to maintain survival by activating PKB/Akt, therefore circumventing the requirement for attachment-mediated integrin signaling.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]