Abstract 1134: Association of ZC3H8 with nuclear bodies and its role in promoting tumor cell behavior in vitro and in vivo

Abstract

Abstract The Zinc Finger CCCH-Type Containing 8 (zc3h8 or Fliz1) gene is overexpressed in many mouse and human breast cancer cell lines that we examined, although the exact function of this gene and the ZC3H8 protein are largely unknown. To characterize ZC3H8 protein, we localized it to distinct sub-nuclear domains that also contain markers for promyelocytic leukemia (PML) nuclear bodies and Cajal bodies. Furthermore, when cells are treated with a casein kinase-2 (CK2) inhibitor, ZC3H8 mislocalizes out of the nuclear bodies and diffuses into the nucleoplasm. CK2 is a known activator of transcription and a potential drug target. ZC3H8 contains a putative CK2 phosphorylation site at position T32, and may be regulated by this kinase. Based on this localization and other preliminary data, we hypothesize that ZC3H8 has a role in transcriptional regulation or RNA modification at these nuclear bodies. This hypothesis is supported by published studies that have found a role for ZC3H8 in the Little Elongation Complex (LEC), which transcribes snRNA involved in RNA splicing in nuclear bodies. Since many tumors and cancer cell lines overexpress zc3h8, we used stably expressed shRNA to knock down expression in two independent mouse breast cancer cell lines for in vitro and in vivo studies. Cells with reduced zc3h8 expression have decreased rates of proliferation and migration compared to overexpressing cells when tested using MTS and wound healing assays. Decreased rates of migration in wound healing assays are reversed by overexpression of the known migration-associated gene MEMO1. Additionally, cells with reduced expression of zc3h8 failed to form colonies in soft agar or pass through an extracellular matrix coating a transwell chamber- a phenotype that can be rescued using recombinant human zc3h8. Conversely, untransformed mouse mammary COMMA-1D cells gain the ability to form colonies in soft agar when transfected to stably overexpress zc3h8. Our in vitro studies suggest that zc3h8 expression has a role in tumorigenesis. These observations have been supported with in vivo studies in mice. We implanted cells knocked down for zc3h8 and control cells into the mammary glands of syngeneic female mice. Mice injected with cells that have reduced expression of zc3h8 form substantially smaller tumors by weight compared to control. When taken together, these data show that zc3h8 has a role in the formation and progression of cancer through its association with nuclear bodies involved in transcriptional regulation and RNA modification. Citation Format: John A. Schmidt, Keith G. Danielson, Jani L. Swiatek, Emily R. Duffner, Janice E. Knepper. Association of ZC3H8 with nuclear bodies and its role in promoting tumor cell behavior in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1134.