Alcohol consumption in women has been associated with an increased risk of breast cancer (~10-50%/1-3 drinks a day), particular in estrogen receptor positive (ER+) disease;however, its mechanisms remain to be elucidated. To date, there have been no reports on the role of MSK1 (mitogen-stress activated protein kinase), Runx2 (Runt-related gene 2), H3ph (histone H3 phosphorylation) and Brf1 (TFIIIB-related factor 1) in alcohol-associated mammary tumor formation. Our overall objective is to explore the molecular mechanism of alcohol-associated breast cancer by determining the role of alcohol-induced deregulation of Pol III genes (RNA polymerase III-dependent genes) in ER+ breast cancer. Deregulation of Pol III genes would serve to enhance the protein synthetic capacity of cells, which is required to promote cellular growth, proliferation, transformation and tumor development. Brf1 is a subunit of TFIIIB complex and specifically regulates Pol III gene transcription. Brf1 and products of Pol III genes, such as 5S rRNAs and tRNAs, are elevated in both transformed and tumor cells suggesting that they play a crucial role in tumorigenesis. Our studies indicate that enhancement of Brf1 and Pol III gene expression is correlated with tumor formation in alcohol-fed mice. We established that ethanol dramatically induces Pol III gene transcription in ER+ breast carcinoma cells. Alcohol increases ER? expression to enhance Brf1 and Pol III gene expression, where this process is mediated by alcohol-activated JNK1. Alcohol increases c-Jun expression and induces H3ph. Runx2 is regulated by ER? and JNK1 and is associated with mammary gland development and ER+ breast cancer. Importantly, our preliminary results indicate that alcohol activates MSK1 and that alcohol increases the activity of Runx2-dependent reporter and enhances expression of Runx2. Based on these studies, we propose a ground-breaking hypothesis that ethanol activates JNK1 and MSK1 to mediate H3ph. H3ph increases ER? expression, which modulates Runx2 activity. In turn, Runx2 upregulates Brf1 expression and Pol III gene transcription, thereby contributing to cell transformation and mammary tumor development. This application is a logical extension of our work to better define the role of MSK1, H3ph, Runx2 and Brf1 in alcohol-promoted mammary tumor development. To test this hypothesis, we will characterize alcohol-induced signaling events of deregulation of Brf1 and Pol III gene by inhibiting JNK1, MSK1 and Aurora B pathway. We will identify the epigenetic regulation of Brf1 by modified histone H3 by determine alcohol-induced H3 modifications, such as H3ph, H3ac (histone H3 acetylation) and H3me (histone H3 methylation). We will determine whether Runx2 modulates Brf1 expression and Pol III gene transcription by increasing and decreasing Runx2 expression and will identify whether MSK1 and H3ph mediate Runx2 expression. We will evaluate the role of Brf1 and MSK1 in alcohol-promoted mammary tumor formation by using Brf1 conditional KO mice and MSK KO mice. These studies using cell culture and animal models will characterize the roles of JNK1, MSK1, Aurora B and H3ph as important signaling molecules to modulate Brf1 expression and Pol III gene transcription in alcohol-associated mammary tumor development. The results from this project will provide a potential treatment for patients with breast cancer.
The proposed project seeks to characterize function of Runx2 in alcohol-promoted mammary tumor development and to establish the roles of JNK1, c-Jun, phosphorylated histone H3 in this process. We will feed MMTV-neu mice with ethanol to observe mammary tumor formation. We will elucidate the molecular mechanism of alcohol-associated breast cancer and determine the role of Runx2 in alcohol- induced deregulation of Brf1 and Pol III genes, cell transformation and mammary tumor formation. We will characterize the specific pathway, JNK1-H3ph-ERa-Runx2-Brf1, which mediates alcohol-induced mammary tumor development. Results from this project may provide valuable information, which could lead to the development of an early detection approach and a new therapeutic strategies for human breast carcinoma.
