Heregulin-triggered Her-2/neu signaling enhances nuclear accumulation of p21WAF1/CIP1 and protects breast cancer cells from cisplatin-induced genotoxic damage

Elevated levels of p21 WAF1/CIP1 , an important mediator of DNA repair, have been observed in various aggressive tumors as well as linked to chemoresistance. We examined whether heregulin (HRG), a member of the EGF-like growth factor family closely related to breast cancer tumurigencsis and metastasis, modulates p21 WAF1/CIP1 expression and cellular localization. We used a model system that consisted of MCF-7 cells and MCF-7 cells engineered to overexpress the full-length cDNA of the human HRG gene (MCF-7/HRG). MCF-7/HRG cells demonstrate constitutive hyperactivation of Her-2/neu receptor as well as activation of down-stream PI-3'K/AKT and MAPK signaling cascades. Immunoblotting analyses showed that MCF-7/HRG cells significantly up-regulate p21 WAF1/CIP1 expression relative to control MCF-7/pBABE cells, while a strong nuclear accumulation of p21 WAF1/CIP1 in MCF-7/HRG cells was revealed by immunofluorescence microscopy studies. Protein degradation analyses demonstrated that the half-life of p21 WAF1/CIP1 protein was increased from -35 min in control MCF-7/pBABE cells to ≥3 h in MCF-7/HRG cells. Pharmacological inactivation of the PI-3'K/AKT and MAPK completely prevented HRG-induced accumulation of p21 WAF1/CIP1 . A structural deletion mutant of HRG (HRG-M4) lacking the N-terminus sequence and the cytoplasmic-transmembrane region of HRG was generated to investigate whether secretion of HRG and transactivation of Her-2/neu actively contributed to HRG-regulated p21 WAF1/CIP1 expression and cellular localization. MCF-7 cells engineered to overexpress HRG-M4 did not demonstrate either activation of Her-2/neu, PI-3'K/AKT, or MAPK. Remarkably, HRG-M4 overexpression completely abolished the ability of HRG to promote nuclear accumulation of p21 WAF1/CIP1 and concomitantly enhanced the apoptotic effects of cisplatin towards breast cancer cells. This novel interplay between HRG and p21 WAF1/CIP1 strongly suggests that one mechanism of HRG-rcgulmcd breast cancer cell proliferation, survival, and/or sensitivity to genotoxic damage is to stabilize and promote a nuclear accumulation of p21 WAF1/CIP1