Akt and ERK control the proliferative response of mammary epithelial cells to the growth factors IGF-1 and EGF through the cell cycle inhibitor p57 ^Kip2

Epithelial cells respond to growth factors including epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), and insulin. Using high-content immunofluorescence microscopy, we quantitated differences in signaling networks downstream of EGF, which stimulated proliferation of mammary epithelial cells, and insulin or IGF-1, which enhanced the proliferative response to EGF but did not stimulate proliferation independently. We found that the abundance of the cyclin-dependent kinase inhibitors p21 ^Cip1 and p57 ^Kip2 increased in response to IGF-1 or insulin but decreased in response to EGF. Depletion of p57 ^Kip2, but not p21 ^Cip1, rendered IGF-1 or insulin sufficient to induce cellular proliferation in the absence of EGF. Signaling through the PI3K (phosphatidylinositol 3-kinase)-Akt-mTOR (mammalian target of rapamycin) pathway was necessary and sufficient for the increase in p57 ^Kip2, whereas MEK [mitogen-activated or extracellular signal-regulated protein kinase (ERK) kinase]-ERK activity suppressed this increase, forming a regulatory circuit that limited proliferation in response to unaccompanied Akt activity. Knockdown of p57 ^Kip2 enhanced the proliferative phenotype induced by tumor-associated PI3K mutant variants and released mammary epithelial acini from growth arrest during morphogenesis in three-dimensional culture. These results provide a potential explanation for the context-dependent proliferative activities of insulin and IGF-1 and for the finding that the CDKN1C locus encoding p57 ^Kip2 is silenced in many breast cancers, which frequently show hyperactivation of the PI3K pathway. The status of p57 ^Kip2 may thus be an important factor to assess when considering targeted therapy against the ERK or PI3K pathways.