Transcriptional Repression by Rb-E2F and Regulation of Anchorage-Independent Survival

Abstract

Mutations that lead to anchorage-independent survival are a hallmark of tumor cells. Adhesion of integrin receptors to extracellular matrix activates a survival signaling pathway in epithelial cells where Akt phosphorylates and blocks the activity of proapoptotic proteins such as the BCL2 family member Bad, the forkhead transcription factor FKHRL-1, and caspase 9. Insulin-like growth factor 1 (IGF-1) is a well-established epithelial cell survival factor that also triggers activation of Akt and can maintain Akt activity after cells lose matrix contact. It is not until IGF-1 expression diminishes (∼16 h after loss of matrix contact) that epithelial cells deprived of matrix contact undergo apoptosis. This suggests that IGF-1 expression is linked to cell adhesion and that it is the loss of IGF-1 which dictates the onset of apoptosis after cells lose matrix contact. Here, we examine the linkage between cell adhesion and IGF-1 expression. While IGF-1 is able to maintain Akt activity and phosphorylation of proapoptotic proteins in cells that have lost matrix contact, Akt is not able to phosphorylate and inactivate another of its substrates, glycogen synthase kinase 3β (GSK-3β), under these conditions. The reason for this appears to be a rapid translocation of active Akt away from GSK-3β when cells lose matrix contact. One target of GSK-3β is cyclin D, which is turned over in response to this phosphorylation. Therefore, cyclin D is rapidly lost when cells are deprived of matrix contact, leading to a loss of cyclin-dependent kinase 4 activity and accumulation of hypophosphorylated, active Rb. This facilitates assembly of a repressor complex containing histone deacetylase (HDAC), Rb, and E2F that blocks transcription of the gene for IGF-1, leading to loss of Akt activity, accumulation of active proapoptotic proteins, and apoptosis. This feedback loop containing GSK-3β, cyclin D, HDAC-Rb-E2F, and IGF-1 then determines how long Akt will remain active after cells lose matrix contact, and thus it serves to regulate the onset of apoptosis in such cells.

ACKNOWLEDGMENTS

We thank C. J. Sherr for helpful comments during the course of these studies; K. L. Guan, R. A. Weinberg, R. A. Roth, J. Massague, D. A. Cantrell, K. Helin, R. Baserga, and S. J. Korsmeyer for reagents; and M. J. Holtzman and D. C. Look for primary human tracheal epithelial cell cultures.

R.G.F. was supported by a postdoctoral fellowship from the American Lung Association. J.T.Y. was supported by NIH training grant HL07873. These studies were supported by grants from the National Institutes of Health to D.C.D.