Crosstalk between PKC alpha and Notch-4 in endocrine-resistant breast cancer cells

The Notch pathway is functionally important in breast cancer. Notch-1 has been reported to maintain an estrogen-independent phenotype in estrogen receptor a (ER alpha) + breast cancer cells. Notch-4 expression correlates with Ki67. Notch-4 also plays a key role in breast cancer stem-like cells. Estrogen-independent breast cancer cell lines have higher Notch activity than estrogen-dependent lines. Protein kinase C alpha (PKC alpha) overexpression is common in endocrine-resistant breast cancers and promotes tamoxifen (TAM)-resistant growth in breast cancer cell lines. We tested whether PKC alpha overexpression affects Notch activity and whether Notch signaling contributes to endocrine resistance in PKC alpha-overexpressing breast cancer cells. Analysis of published microarray data from ER alpha + breast carcinomas shows that PKC alpha expression correlates strongly with Notch-4. Real-time reverse transcription PCR and immunohistochemistry on archival specimens confirmed this finding. In a PKC alpha-overexpressing, TAM-resistant T47D model, PKC alpha selectively increases Notch-4, but not Notch-1, expression in vitro and in vivo. This effect is mediated by activator protein-1 (AP-1) occupancy of the Notch-4 promoter. Notch-4 knockdown inhibits estrogen-independent growth of PKC alpha-overexpressing T47D cells, whereas Notch-4IC expression stimulates it. Gene expression profiling shows that multiple genes and pathways associated with endocrine resistance are induced in Notch-4IC- and PKC alpha-expressing T47D cells. In PKC alpha-overexpressing T47D xenografts, an orally active g-secretase inhibitor at clinically relevant doses significantly decreased estrogen-independent tumor growth, alone and in combination with TAM. In conclusion, PKC alpha overexpression induces Notch-4 through AP-1. Notch-4 promotes estrogen-independent, TAM-resistant growth and activates multiple pathways connected with endocrine resistance and chemoresistance. Notch inhibitors should be clinically evaluated in PKC alpha- and Notch-4-overexpressing, endocrine-resistant breast cancers.