Novel CARP-1-H2AX interaction regulates apoptosis in breast cancer cells

CCAR1/CARP-1 is a peri-nuclear phosphoprotein that regulates apoptosis signaling by breast cancer therapeutic Doxorubicin and a novel class of CARP-1 Functional Mimetic (CFM) compounds. Prior reports have established that Doxorubicin exposure causes DNA damage in cells. Phosphorylation of histone 2AX (often referred as γH2AX) protein is involved in regulating DNA damage repair and apoptosis signaling. Although, currently it is unclear whether CFMs also induce DNA damage, we found that Doxorubicin or CFM-4.16 treatments caused elevated CARP-1 and γH2AX in HBC cells. In fact, a robust nuclear or peri-nuclear co-localization of CARP-1 and γH2AX occurred in HBC cells undergoing apoptosis. Knock-down of CARP-1 abolished increase of CARP-1 and γH2AX, their co-localization, and apoptosis in CFM-4.16 or Doxorubicin-treated HBC cells. We found that CARP-1 directly binds with H2AX, and the H2AX-interacting epitope is located within CARP-1 amino acids 600 to 652. We generated HBC cells that have stable expression of myc-tagged, wild-type CARP-1 (1-1150) or CARP-1 (1-1150; Δ600-652) mutant. Further analyses revealed that CARP-1 (1-1150), but not CARP-1 (1-1150; Δ600-652) mutant, interacted with H2AX. Moreover, HBC cells expressing CARP-1 (1-1150; Δ600-652) mutant were resistant to apoptosis by Doxorubicin or CFM-4.16, and had diminished levels of γH2AX, when compared with their counterparts expressing wild-type CARP-1. We have earlier reported involvement of the Stress-Activated Protein Kinases (SAPKs) such as p38 and JNKs in apoptosis by Doxorubicin or CFMs. Since the PI3K family of serine-threonine kinases ATM/ATR, as well as JNK SAPKs, phosphorylate serine-139 of H2AX following DNA damage, our current studies are investigating whether H2AX binding with CARP-1 serves to facilitate phosphorylation of CARP-1 and H2AX proteins for transduction of apoptosis signaling. Citation Format: Sreeja C. Sekhar, Vino T. Cheriyan, Arun K. Rishi. Novel CARP-1- H2AX interaction regulates apoptosis in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4378.