Cytoplasmic p21 super(WAF1/CIP1) correlates with Akt activation and poor response to tamoxifen in breast cancer

P21 super(WAF1/Cip1) (p21) translocates to the cytoplasm inducing cell cycle progression and survival upon Akt/PKB activation. We studied whether heregulin ss1 (HRGss1), that activates the P13K/Akt and MAPK pathways, also misallocates p21. We also explored whether HRGss1 interferes with the effects of tamoxifen. The clinical material studied helped us to clarify whether p21 was associated with phosphorylated Akt, recurrence-free survival and response to tamoxifen. MCF-7 cells treated with HRGss1 -/+ E2 were analyzed by flow cytometry to observe how the different compounds affected tamoxifen-induced cell cycle arrest and apoptosis. Total cell lysate and nuclear and cytoplasmic fractions were used to detect p21, phospho-Akt and other proteins by Western blotting. Immunofluorescence was used to visualize p21 super(+) cells upon HRGss1 and E2 stimulation. The localization of p21 in breast cancer was studied by immunohistochemistry in frozen tumor sections from 280 patients. In MCF-7 we found that HRGss1 counteracted the inhibition of p21 expression by tamoxifen and caused p21 cytoplasmic accumulation. HRGss1 partially counteracted the cytostatic effect of tamoxifen but abrogated its cytotoxic effect. The clinical material revealed that nuclear p21 (P=0.022) and cytoplasmic p21 (P=0.00001) were associated with phospho-Akt. Based on p21 cell location, we identified 3 subgroups of ER super(+) patients: the p21N super(+)/C super(-) group for whom tamoxifen was needed otherwise the survival was poor (P=0.0082), the p21N super(+)/C super(+) or p21N super(-)/C super(-) group, that responded to tamoxifen (P=0.034), and the p21C super(+)/N super(-) group, that might not benefit from this treatment (P=0.7). In conclusion, HRGss1 inhibits tamoxifen-induced apoptosis, contributes to p21 cytoplasmic expression while the cellular localization of p21 interacts with the benefit from tamoxifen treatment.