TAZ is a WW domain containing a transcription coactivator that modulates mesenchymal differentiation and development of multiple organs. In this study, we show that TAZ is phosphorylated by the Lats tumor suppressor kinase, a key component of the Hippo pathway, whose alterations result in organ and tissue hypertrophy in Drosophila and contribute to tumorigenesis in humans. Lats phosphorylates TAZ on several serine residues in the conserved HXRXXS motif and creates 14-3-3 binding sites, leading to cytoplasmic retention and functional inactivation of TAZ. Ectopic expression of TAZ stimulates cell proliferation, reduces cell contact inhibition, and promotes epithelial-mesenchymal transition (EMT). Elimination of the Lats phosphorylation sites results in a constitutively active TAZ, enhancing the activity of TAZ in promoting cell proliferation and EMT. Our results elucidate a molecular mechanism for TAZ regulation and indicate a potential function of TAZ as an important target of the Hippo pathway in regulating cell proliferation tumorigenesis.

SUMMARY Mammary epithelia are composed of luminal and myoepithelial/basal cells whose neoplastic transformations lead to distinct types of breast cancers with diverse clinical features. We report that mice deficient for the CDK4/6 inhibitor p18Ink4c spontaneously develop ER-positive luminal tumors at a high penetrance. Ink4c deletion stimulates luminal progenitor cell proliferation at pubertal age and maintains an expanded luminal progenitor cell population throughout life. We demonstrate that GATA3 binds to and represses INK4C transcription. In human breast cancers, low INK4C and high GATA3 expressions are simultaneously observed in luminal A type tumors and predict a favorable patient outcome. Hence, p18INK4C is a downstream target of GATA3, constrains luminal progenitor cell expansion and suppresses luminal tumorigenesis in the mammary gland. SIGNIFICANCE Breast cancer is heterogenous with tumors pathologically distinct and diverse in their responsiveness to treatment. We show that the CDK inhibitor p18INK4c gene is repressed by GATA3, a transcription factor specifying mammary luminal cell fate, and that low INK4C and high GATA3 expressions are associated with human luminal A type tumors and with better patient survival. p18Ink4c null mice have an expanded luminal progenitors at a young age and throughout life and develop ER+ luminal tumors at high penetrance. These results identify the escape of luminal progenitors from quiescence as a rate-limiting step for initiation of mammary luminal tumors. The Ink4c-null mouse represents a unique model for study and for developing therapeutic strategies to treat luminal tumors.

BRCA1 mutation carriers are predisposed to developing basal-like breast cancers with high metastasis and poor prognosis. Yet, how BRCA1 suppresses formation of basal-like breast cancers is still obscure. Deletion of p18 Ink4c (p18), an inhibitor of CDK4 and CDK6, functionally inactivates the RB pathway, stimulates mammary luminal stem cell (LSC) proliferation, and leads to spontaneous luminal tumor development. Alternately, germline mutation of Brca1 shifts the fate of luminal cells to cause luminal-to-basal mammary tumor transformation. Here, we report that disrupting Brca1 by either germline or epithelium-specific mutation in p18-deficient mice activates epithelial-to-mesenchymal transition (EMT) and induces dedifferentiation of LSCs, which associate closely with expansion of basal and cancer stem cells and formation of basal-like tumors. Mechanistically, BRCA1 bound to the TWIST promoter, suppressing its activity and inhibiting EMT in mammary tumor cells. In human luminal cancer cells, BRCA1 silencing was sufficient to activate TWIST and EMT and increase tumor formation. In parallel, TWIST expression and EMT features correlated inversely with BRCA1 expression in human breast cancers. Together, our findings showed that BRCA1 suppressed TWIST and EMT, inhibited LSC dedifferentiation, and repressed expansion of basal stem cells and basal-like tumors. Thus, our work offers the first genetic evidence that Brca1 directly suppresses EMT and LSC dedifferentiation during breast tumorigenesis. Cancer Res; 74(21); 6161-72. Ó2014 AACR.

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