Abstract 1713: Molecular dissection of Zc3h8 functional domains and the implications for the oncogenic phenotype

Abstract

Abstract Zc3h8 encodes a zinc finger protein that is amplified in 2-6% of human breast tumors. Higher expression of Zc3h8 also results in lower rates of disease-free survival. Amplification of Zc3h8 expression has been shown to increase cell proliferation, migration, invasion and tumor growth in mice. Conversely, decreased expression alters cell behavior by decreasing cell proliferation, migration, invasion, and tumor growth in mice. Zc3h8 comprises three CCCH non-classical zinc finger domains at the C-terminus of the protein that we hypothesize bind to RNA. The interactions between Zc3h8 and nucleic acids may be important for the localization and/or function of Zc3h8 and therefore may be important for the cellular phenotypes observed when the gene is over or under expressed in cells. We used in vitro and in vivo assays to demonstrate that mutations in any of the three zinc finger domains results in a protein that fails to rescue aggressive cellular phenotypes in cells with reduced expression of Zc3h8, while the wild type protein can rescue these phenotypes. This implies that all three zinc finger domains are required for Zc3h8 function in promoting rapid and aggressive cell proliferation. In addition to zinc finger domains, Zc3h8 also contains a casein kinase-2 (CK2) phosphorylation site that may be important for protein localization and/or activation. We have previously shown that CK2 inhibitors alter Zc3h8 localization to nuclear PML Bodies. Mouse cells overexpressing Zc3h8 with a Thr to Ala (T32A) change that prevents CK2 phosphorylation grow very slowly in culture and cannot grow in more challenging 3D or soft agar growth chambers. However, Zc3h8 with a Thr to Glu (T32E) change that mimics constitutive CK2 phosphorylation results in rapidly growing cells that surpass overexpression of Zc3h8 wild type. Zc3h8 phospho-mutants also have altered localization with Zc3h8 T32E becoming diffuse in the nucleus and T32A localizing to nuclear foci. These data suggest that the phosphorylation state of Zc3h8 and its ability to bind nucleic acids via zinc finger domains are critical for the oncogenic behavior that results from amplified expression. We also hypothesize that Zc3h8 has an important role in establishing or maintaining PML Bodies involved in genome maintenance, transcription, and/or post-transcription regulation. Citation Format: John A. Schmidt, Tyler Doan, Emanuel Irizarry, Emily Harris, Keith G. Danielson, Janice E. Knepper. Molecular dissection of Zc3h8 functional domains and the implications for the oncogenic phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1713.