Lofgren, Kristopher Andrew2011-07-192011-07-192010-12https://hdl.handle.net/11299/109443University of Minnesota Ph.D. dissertation. December 2010. Major: Microbiology, Immunology and Cancer Biology. Advisor: Carol Ann Lange. 1 computer file (PDF); vii, 100 pages.Protein tyrosine kinases (PTKs) regulate cellular proliferation and differentiation during development and homeostasis of normal tissues. Additionally, PTKs are frequently overexpressed in cancers. As such, they have become promising targets for new therapies. Breast tumor kinase (Brk/PTK6) is a non-receptor tyrosine kinase initially cloned from a metastatic breast tumor, and is overexpressed in ~86% of human breast tumors and several breast cancer cell lines. While expressed in differentiating cells in the skin and intestine, Brk is notably absent from normal mammary tissue and non-transformed mammary epithelial cell lines. The role of Brk/PTK6 in breast pathology is unclear. We hypothesized that Brk expression in non-transformed mammary epithelium would promote tumorigenesis. To determine the effects Brk expression in the normal mammary gland, we sought to create a mouse model for tissue-specific Brk expression. We expressed a WAP-driven Brk/PTK6 transgene in FVB/n mice, and analyzed wild type and transgenic mammary glands after forced weaning. Brk-transgenic dams exhibited delayed mammary gland involution and infrequent tumors in aged mice. Transgenic mammary glands displayed decreased STAT3 phosphorylation, a marker of early stage involution. Signaling mediators downstream of Brk, including STAT5 and p38 MAPK were activated relative to wild-type mice. Brk-mediated signaling to STAT5 and p38 MAPK was recapitulated with Brk expression in non-transformed HC11 murine mammary epithelial cells. Additionally, non-transformed HMEC and HC11 cells expressing Brk exhibited increased anchorage-independent survival when cultured on PolyHEMA coated dishes. Samples from breast tumor biopsies were subjected to IHC analysis for co-expression of Brk, phospho-STAT5, and phospho-p38 MAPK. Ductal and lobular carcinomas expressing Brk exhibited elevated phospho-STAT5 and phospho-p38 MAPK, respectively, whereas non-malignant tissues were Brk-null with dramatically less phospho-STAT5 and phospho-p38 expression. HMEC cells expressing Brk underwent luminal hollowing (reliant on cell death) similar to normal mammary alveologenesis when cultured on Matrigel, but are partially resistant to doxorubicin treatment, suggesting a context dependent component to Brk-mediated survival. HGF, EGF, or IGF treatment in cells that endogenously express Brk (non-transformed HaCaT keratinocytes and MDA-MB-231 breast cancer cells), resulted in growth factor specific changes in Brk localization, but only in MDA-MB-231 cells. Brk-knockdown in breast cancer cells (T47D and SKBR3) identified differential phosphorylation of Erk5, and notably, p38 MAPK in response to EGF and heregulin-β1 treatment. These studies illustrate that forced expression of Brk/PTK6 in non-transformed mammary epithelial cells in vivo mediates STAT5 and p38 MAPK phosphorylation and promotes increased cellular survival, delayed involution, and latent tumor formation. Brk expression in human breast tumors may contribute to progression by inducing these same pathways, as evidenced in clinical samples of invasive breast carcinoma. Models of Brk expression in non-transformed mammary epithelial cells in vitro promote cellular survival in a context dependent manner, however, remain a strong tool for identifying molecules contributing to Brk mediated breast tumor progression. Inhibition of STAT5 and/or p38 MAPK may provide strong therapeutic potential in Brk-positive breast cancer.en-USBreast cancerBrk/PTK6Involutionp38 MAPKMicrobiology, Immunology and Cancer BiologyThesis or Dissertation