Date of Graduation
5-2014
Document Type
Dissertation (PhD)
Program Affiliation
Cancer Biology
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Khandan Keyomarsi
Committee Member
Jeffrey Rosen
Committee Member
Elsa Flores
Committee Member
Pierre McCrea
Committee Member
Jinsong Liu
Abstract
Elafin, an endogenous serine protease inhibitor, is a critical component of the epithelial barrier against neutrophil elastase (NE) activity. The central hypothesis examined in this dissertation was that elafin has tumor suppressive properties in breast cancer. In support of this hypothesis, immunohistochemical (IHC) analysis revealed that elafin was downregulated in the majority of invasive breast tumors and a subset of pre-invasive ductal carcinoma in situ (DCIS) compared to elafin expression in the normal mammary epithelium. To understand the role of elafin in the mammary epithelium and the impetus for its downregulation during breast tumorigenesis, primary and immortalized human mammary epithelial cells (HMECs) were utilized as a model system. Elafin was highly expressed in G0-arrested HMECs, suggesting a previously unrecognized role for elafin in growth control. Stable knockdown (KD) of elafin compromised the ability of HMECs to maintain G0-arrest during long-term growth factor deprivation. This effect was reversed by re-expression of wild-type elafin but not elafin-M25G lacking protease inhibitory function, suggesting a role for deregulated protease activity. Elafin KD HMECs demonstrated enhanced sensitivity to NE-induced proliferation. Mechanistically, activation of the ERK signaling pathway downstream of toll-like receptor 4 (TLR4) was essential to the mitogenic effect of NE in this system. Compared to HMECs, the majority of breast cancer cell lines lack endogenous elafin expression. Adenoviral-mediated expression of elafin was utilized to evaluate the tumor suppressive properties of elafin in breast cancer cell lines. Rb-status was identified as the critical factor governing the anti-tumor effect of elafin in this system. In breast cancer cell lines expressing functional Rb, the expression of elafin triggered Rb-dependent cell cycle arrest. However, in breast cancer cell lines lacking functional Rb, elafin expression induced caspase-3 dependent apoptotic cell death. Elafin is a critical counterbalance against NE-activity. IHC analysis revealed that high levels of NE-expressing tumor-associated neutrophils (TAN) were associated with reduced recurrence-free survival, high tumor grade, and triple-receptor negative breast cancer (TNBC). ERK-catalyzed phosphorylation of p90RSK (T359/S363) and Rb phosphorylation (S807/811) were significantly enriched in NE-positive breast tumors, suggesting that NE-induced ERK signaling and proliferation may be relevant to human breast cancer. The in vivo role of deregulated NE in breast tumorigenesis was examined in the C3(1)TAg mouse model of TNBC. Knockout of NE in C3(1)TAg mice significantly reduced tumor growth and proliferation. Elafin has tumor suppressive properties in the context of breast cancer and is a critical counterbalance against the growth promoting effect of NE in vitro and in vivo. Deregulated NE-activity is a viable therapeutic target in breast cancer.
Keywords
Elafin, Neutrophil Elastase, Breast Cancer, human mammary epithelial cells, serine protease, C3(1)TAg