All ETDs from UAB

Advisory Committee Chair

Yuhua Song

Advisory Committee Members

Donald J Buchsbaum

Aaron Lucius

Selvarangan Ponnazhagan

Jack Rogers

Document Type

Dissertation

Date of Award

2017

Degree Name by School

Doctor of Philosophy (PhD) School of Engineering

Abstract

Drug resistant estrogen receptor positive (ER-positive) and triple-negative (TN) breast cancers, are prolific breast cancers with poor clinical outcome. TRA-8 is a death receptor-5 (DR5) specific agonistic antibody with tumoricidal activity in vitro and in vivo, without inducing normal hepatocyte apoptosis. ER-positive and TN breast cancers have a range of sensitivity to TRA-8 mediated cytotoxicity. TRA-8 activation of DR5 leads to DR5 oligomerization that is critical for formation of the death inducing signaling complex (DISC) at the death domain of DR5 and initiation of apoptotic signaling. Calmodulin (CaM) is overexpressed in breast cancer and is involved in regulating breast cancer growth, proliferation, and transformation to malignancy. The role of CaM in regulation of DR5-mediated apoptotic signaling has yet to be fully revealed. The goal of this study is to characterize CaM and DR5 interaction, and to determine the role of CaM binding to DR5 in DR5-mediated DISC formation for DR5-mediated apoptotic signaling in breast cancer cells. Results demonstrated CaM directly binds to DR5 death domain (DD) in a calcium dependent manner in breast cancer cells. The CaM binding site in DR5 is 354WEPLMRKLGL363 located in DR5 DD. Residues Trp354, Agr359, Glu355, Leu363, and Glu367 in DR5 are important for DR5-mediated DISC formation to signal apoptosis and are also required for CaM-DR5 binding. The changed electrostatic potential distribution in the CaM binding site in DR5 DD by W354A, E355K, R359A, L363N, or E367K mutations of DR5 could directly contribute to the experimentally observed decreased CaM-DR5 binding. In TRA-8 sensitive breast cancer cells, CaM was recruited into DR5-mediated DISC in a calcium dependent manner. CaM antagonist, trifluoperazine (TFP), inhibited CaM recruitment into the DISC and attenuated DR5 oligomerization and thus DISC formation. TFP and Ca2+ chelator, EGTA, impeded TRA-8 activated caspase-dependent apoptotic signaling, and TFP reduced TRA-8 induced cell cytotoxicity. For TRA-8 resistant TN breast cancer cells, CaM antagonist(s) enhanced TRA-8 induced cytotoxicity for TRA-8 resistant TNBC cells. CaM knock-down promoted DR5-mediated DISC formation in TRA-8 resistant TNBC cells. TFP enhanced TRA-8 activated DR5 oligomerization, DR5-mediated DISC formation and TRA-8 activated caspase cleavage for apoptosis in TRA-8 resistant TNBC cells. For TRA-8 sensitive breast cancer cells, CaM could mediate phosphatase SHP-1 recruitment to DR5, thus dephosphorylating DR5 and facilitating DR5 oligomerization and DR5-mediated DISC formation for apoptosis. For TRA-8 resistant TN breast cancer cells, CaM could mediate the formation of DR5-mediated anti-apoptotic complex that would impede DR5-mediated DISC formation. CaM antagonists would inhibit CaM binding to DR5 and disrupt the DR5-mediated anti-apoptotic complex formation. Leading to promoting DR5-medaiated DISC for apoptosis in TRA-8 resistant TN breast cancer cells. Understanding the role CaM in DR5-mediated apoptosis could help the identification of therapeutic strategies for breast cancer treatment to overcome the drug resistance in ER-positive breast cancer and TN breast cancer.

Included in

Engineering Commons

Share

COinS