AND-34-induced Rac activation and antiestrogen resistance in breast cancer requires phosphatidylinositol 3-kinase

AND-34 is a novel signaling protein with an amino-terminus Src homology 2 (SH2) domain and a C-terminus with modest homology to the Cdc25 domain of Ras-subfamily GDP exchange factors (GEF). In prior studies, AND-34 was found on overexpression to induce antiestrogen resistance in human breast cancer cell lines. The current study explored the mechanism by which this occurs. AND-34 is constitutively expressed in primary splenic B cells but not in primary splenic T cells or thymocytes. B cell AND-34 associates with the docking molecules p130Cas and Hef-1 through its C-terminus GEF domain. Overexpression of AND-34 in murine B cell lines and NIH 3T3 cells induces morphologic changes characterized by the formation of long filamentous actin-containing cellular extensions and polymerized actin (F-actin) redistribution. Consistent with these morphologic alterations, overexpression of AND-34 in murine B cell lines activates the Rho family GTPase Cdc42. AND-34 also augments both autophosphorylation and kinase activity of the Cdc42 effector protein PAK1 and inhibits SDF-1alpha-induced B cell polarization. Overexpression of AND-34 in the estrogen-dependent breast cancer cell line MCF-7 markedly augments levels of activated Rac and, to a lesser degree, Cdc42, in a SH2 and GEF domain-dependent manner. Similarly, both the SH2 and GEF-like domains are required for AND-34-mediated antiestrogen resistance. As phosphatidylinositol 3-kinase (PI3K) is known to lead to Rac and Cdc42 activation, the effects of AND-34 on this enzyme were examined. AND-34 stimulates the activity of PI3K and Akt, a downstream target of PI3K. AND-34 overexpression augments levels of Akt phosphorylated at serine 473. Inhibition of PI3K by the pharmacologic inhibitor LY294002 and by overexpression of a dominant-negative PI3K (pDelta85) abolishes AND-34-induced Rac activation. Furthermore, inhibition of PI3K activity blocks AND-34-mediated antiestrogen resistance in estrogen-dependent breast cancer cell lines. Although R-Ras, which is known to activate PI3K, is a potential substrate for AND-34's GEF domain, GEF assays and studies with constitutively active R-Ras mutants do not support a role for this GTPase in AND-34 mediated Rac activation. This study demonstrates a role for PI3K in AND-34 induced Rac activation and antiestrogen resistance in breast cancer cell lines.