PROJECT SUMMARY/ABSTRACT Overview: There is an urgent need to understand the molecular basis of HER2+ breast cancer, given that the majority of patients eventually become refractory to treatment including with anti-HER2 therapy. Herein, we provide preliminary data linking poor clinical outcome of human HER2+ breast cancers with the presence of high cytoplasmic levels of the transcription factor ThPOK (cytoThPOK). Further, using a novel mouse model of cytoplasmically restricted ThPOK (ThPOK?NLS mice), we establish a causal relationship between cytoThPOK and development of highly penetrant Her2+ breast cancer. Proteomic analysis of mouse breast cancer cells indicates that cytoThPOK interacts with multiple cytosolic proteins implicated in Her2 signaling, including several SH3 proteins that probably bind to a conserved proline-rich motif in ThPOK. Given the central role of HER2 signaling in HER2+ breast cancer biology, we hypothesize that cytoThPOK interaction with these factors enhances HER2-mediated signaling in some way, and that this represents an important driver of breast cancer development/progression, that has so far been overlooked. Research Focus: POK transcription factors have been implicated in diverse human cancers, which was presumed to reflect direct effects on transcription. In contrast, we now implicate cytosolic localization of ThPOK in breast cancer in humans and mice, demonstrating a novel mode of POK-mediated oncogenesis not based on nuclear function. These findings provide an innovative and compelling premise for the proposed studies. Specific Aims: We will elucidate the role of cytoThPOK in breast cancer according to 3 aims: SA-1: Elucidating molecular basis of ThPOK?NLS?mediated oncogenesis - to test effect of cytoThPOK on HER2/EGFR expression and signaling in cell lines, and test the requirement for the ThPOK SH3-interaction domain for cytoThPOK-mediated breast cancer in mice. SA-2: Dissecting prognostic value and molecular basis for cytoplasmic localization of ThPOK in human BC - to evaluate correlation between high cytoThPOK and survival in different human HER2+ breast cancer subtypes, and determine the molecular basis for cytoplasmic localization of ThPOK in human breast cancer cells. SA-3: Elucidating effect of cytoThPOK on BC tumor maintenance - to elucidate whether cytoThPOK is required for tumor maintenance/progression of established cancers from ThPOK?NLS mice, and characterize a new humanized ThPOK mouse model that expresses a variant hThPOKp.H22pTfs*6 allele found in some human breast cancer patients. Impact: Given the high incidence of cancer observed in mice with enforced cytoplasmic ThPOK localization and the high frequency of cytoplasmic ThPOK localization in human HER2+ breast cancer patients, elucidating the molecular basis by which cytoThPOK promotes breast cancer is likely to have high impact on human health. Here we combine novel animal models and molecular approaches to elucidate these mechanisms. These studies have the capacity to shift the basic conceptual framework by which POK factors are presumed to promote oncogenesis. Potentially, elucidating these mechanisms may lead to novel therapeutic approaches to target HER2+ breast cancer and other human malignancies in which cytoThPOK may be implicated.