Regulation of the oncogenic phenotype by the nuclear body protein ZC3H8

John A Schmidt,Keith G Danielson,Tianjiao Wang,Janice E Knepper,Amber Shelton,Gerard T Walker,Sara G Radecki,Emily R Duffner

doi:10.1186/s12885-018-4674-1

Abstract

BackgroundThe Zc3h8 gene encodes a protein with three zinc finger motifs in the C-terminal region. The protein has been identified as a component of the Little Elongation Complex, involved in transcription of small nuclear RNAs. ZC3H8 is overexpressed in a number of human and mouse breast cancer cell lines, and elevated mRNA levels are associated with a poorer prognosis for women with breast cancer.MethodsWe used RNA silencing to decrease levels of expression in mouse mammary tumor cells and overexpression of ZC3H8 in cells derived from the normal mouse mammary gland. We measured characteristics of cell behavior in vitro, including proliferation, migration, invasion, growth in soft agar, and spheroid growth. We assessed the ability of these cells to form tumors in syngeneic BALB/c mice. ZC3H8 protein was visualized in cells using confocal microscopy.ResultsTumor cells with lower ZC3H8 expression exhibited decreased proliferation rates, slower migration, reduced ability to invade through a basement membrane, and decreased anchorage independent growth in vitro. Cells with lower ZC3H8 levels formed fewer and smaller tumors in animals. Overexpression of ZC3H8 in non-tumorigenic COMMA-D cells led to an opposite effect. ZC3H8 protein localized to both PML bodies and Cajal bodies within the nucleus. ZC3H8 has a casein kinase 2 (CK2) phosphorylation site near the N-terminus, and a CK2 inhibitor caused the numerous PML bodies and ZC3H8 to coalesce to a few larger bodies. Removal of the inhibitor restored PML bodies to their original state. A mutant ZC3H8 lacking the predicted CK2 phosphorylation site showed localization and numbers of ZC3H8/PML bodies similar to wild type. In contrast, a mutant constructed with a glutamic acid in place of the phosphorylatable threonine showed dramatically increased numbers of smaller nuclear foci.ConclusionsThese experiments demonstrate that Zc3h8 expression contributes to aggressive tumor cell behavior in vitro and in vivo. Our studies show that ZC3H8 integrity is key to maintenance of PML bodies. The work provides a link between the Little Elongation Complex, PML bodies, and the cancer cell phenotype.

Highlights

The Zc3h8 gene encodes a protein with three zinc finger motifs in the C-terminal region
Zc3h8 expression in mouse mammary carcinoma cells We identified a potential role for Zinc Finger CCCHType containing 8 (Zc3h8) in tumorigenesis using PCR to locate integration sites of the mouse mammary tumor virus (MMTV) in tumors of BALB/cV mice
We show that the level of Zc3h8 expression in murine mammary cells influences aggressive behavior in vitro and tumor formation in vivo

Summary

Introduction

The Zc3h8 gene encodes a protein with three zinc finger motifs in the C-terminal region. The protein has been identified as a component of the Little Elongation Complex, involved in transcription of small nuclear RNAs. ZC3H8 is overexpressed in a number of human and mouse breast cancer cell lines, and elevated mRNA levels are associated with a poorer prognosis for women with breast cancer. The zinc finger protein ZC3H8 was first identified as expressed in fetal liver [1], but moderate levels can be detected in a variety of tissues, and the gene is amplified in 2–6% of solid tumors of the breast [2]. The Zc3h8 gene encodes a protein of predicted molecular weight 34 kDa of unknown function. Recent work has demonstrated that the zinc finger domains of some of the family members from disparate species are functionally interchangeable, suggesting a common strategy for binding RNA [13]

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