GATA6 is an astrocytoma tumor suppressor gene identified by gene trapping of mouse glioma model

Abstract

Malignant astrocytomas are the most common and lethal adult primary brain tumor. Retroviral gene trapping of nontransformed neonatal astrocytes from a glial fibrillary acidic protein (GFAP): V12 HaRas murine astrocytoma model led to isolation of the transcription factor Gata6. Loss of Gata6 resulted in enhanced proliferation and transformation of astrocytes. Human malignant astrocytoma cell lines, explant xenografts, and operative specimens demonstrated loss of GATA6 expression. Loss-of-function GATA6 mutations with loss of heterozygosity of the GATA6 locus were found in human malignant astrocytoma specimens but not in lower-grade astrocytomas or normal adult astrocytes. Knockdown of Gata6 expression in V12 Ha-Ras or p53؊/؊ astrocytes, but not in parental murine or human astrocytes, led to acceleration of tumorgenesis. Knockin GATA6 expression in human malignant astrocytoma cells reduced their tumorgenic growth with decreased VEGF expression. Collectively, these data demonstrate that GATA6, isolated from a murine astrocytoma model, is a novel tumor suppressor gene that is a direct target of mutations during malignant progression of murine and human astrocytomas. This work also demonstrates the utility of random mutagenesis strategies, such as gene trapping, on murine cancer models toward discovery of novel genetic alterations in corresponding human cancers.

GATA4 is a regulator of astrocyte cell proliferation and apoptosis in the human and murine central nervous system

“…GATA4 is expressed ubiquitously in post-natal and adult human CNS Our interest in GATA4 commenced with the result that a panel of six well-characterized human GBM cell lines had complete loss GATA4, similar to our prior report on GATA6 (Kamnasaran et al, 2007), compared with a non-transformed but immortalized normal human astrocyte (NHA) cell line (Sonoda et al, 2001) (Figure 1a). However, compared with GATA6 expression in the CNS, which we had previously characterized (Kamnasaran and Guha, 2005), the CNS characterization of GATA4 at the cellular and developmental level was lacking.…”

“…Gata4 transcript was detected in murine astrocytes (Supplementary Figure 1b), with IF analysis showing Gata4 staining positively in the nuclei of primary murine adult astrocytes (Figure 2c, i-iv) marked with GFAP. Collectively, our human and murine data show ubiquitous Gata4 Functional analysis of GATA4 in cell cycle control of human and murine astrocytes We focused our functional analysis of GATA4 to astrocytes, as our prior study implicates GATA6 to be a tumor suppressor in human GBMs (Kamnasaran and Guha, 2005;Kamnasaran et al, 2007) and our current findings show GATA4 expression to be lost in a majority of human GBM cell lines, Figure 1a. We used loss and gain of function studies of GATA4 using GATA4 small interfering RNA (siRNA)-mediated gene silencing and overexpressing GATA4 using the fulllength human and murine GATA4 cDNA.…”

“…Using a gene-trap mutagenesis strategy on a spontaneous transgenic murine glioma model developed in our laboratory, we identified GATA6 as a novel tumor suppressor gene. Loss of GATA6 was involved in glioma progression not only in the murine model, but also in the human glioblastoma (glioblastoma multiforme (GBM)), the most common and malignant primary CNS tumor (Kamnasaran et al, 2007). These observations on GATA6 furthered our interest in other GATA members in the CNS, particularly GATA4, since both GATA6 and GATA4 are embryonic lethal and have similar tissue expression patterns in the CNS.…”

“…NMA cultures were established from 1-month-old adult CD1 mice (Jackson Labs, Bar Harbor, ME, USA) as previously described (Luo et al, 2000;Kamnasaran et al, 2007). p53 À/À murine astrocytes were a gift from Dr Tak W Mak (University of Toronto).…”

“…Flow cytometry was done as previously (Kamnasaran et al, 2007) described and detail in Supplementary Materials and Methods.…”