Aberrant expression of the epidermal growth factor receptor (EGFR) is an indicator of poor prognosis in breast cancer. However, the mechanism by which EGFR becomes elevated is unclear. In a genetic screen to identify genes that mediate EGF-independent proliferation of breast cancer cells, we isolated YB-1 (Y-box binding protein 1), a multifunctional regulator of gene transcription and translation. We found that YB-1 overexpression in immortal human mammary epithelial cells (HMECs) conferred EGF independence and increased EGFR levels. YB 1 binds to the EGFR gene control regions, and elevated YB-1 levels in breast cancer samples correlate with EGFR expression and with poor patient outcome. Moreover, YB 1 transgenic mice develop mammary tumors. Interestingly, Akt phosphorylates YB-1 on Ser102, a residue necessary for YB-1 nuclear localization, EGFR induction, and growth stimulation. We hypothesize that YB-1 and EGFR are part of a positive feedback loop whereby EGFR signaling increases YB-1 nuclear localization; nuclear YB-1 binds to promoter regions to regulate transcription of EGFR and other genes controlling proliferation, which contributes to breast cancer formation. We propose the following Specific Aims: 1) Investigate the transcriptional regulation of EGFR by YB-1 in HMECs. We will analyze functional YB-1 binding elements in the EGFR gene that mediate induction by YB-1 using chromatin immunoprecipitation, reporter assays, and electrophoretic mobility assays. 2) Investigate the mechanism by which YB-1 stimulates EGF-independent cell growth. We will determine if (a) YB-1-induced EGF independence of breast epithelial cells requires YB-1 Ser102 phosphorylation and nuclear translocation; (b) YB-1 activates EGFR signaling pathways; (c) knockdown of YB-1 in HMECs reverses EGF independence and triggers cell cycle arrest, senescence or apoptosis; (d) YB-1-induced EGF independence requires both induction of EGFR and repression of negative growth regulators. 3) Examine the role of YB-1 and EGFR in tumor formation. We will determine if targeting YB-1 and/or EGFR in human breast cancer cells reduces their anchorage-independent growth in vitro and their tumorigenic and metastatic potential in an orthotopic nude mouse model. The proposed studies will deepen our understanding of the way breast cancer develops. Most importantly, this work will examine whether targeting YB-1 and EGFR in tumors represents a viable strategy for therapy in a subset of breast cancer where treatment options are currently limited. ? ? ?
