NDRG4 Is Required for Cell Cycle Progression and Survival in Glioblastoma Cells

Abstract

NDRG4 is a largely unstudied member of the predominantly tumor suppressive N-Myc downstream-regulated gene (NDRG) family. Unlike its family members NDRG1-3, which are ubiquitously expressed, NDRG4 is expressed almost exclusively in the heart and brain. Given this tissue-specific expression pattern and the established tumor suppressive roles of the NDRG family in regulating cellular proliferation, we investigated the cellular and biochemical functions of NDRG4 in the context of astrocytes and glioblastoma multiforme (GBM) cells. We show that, in contrast to NDRG2, NDRG4 expression is elevated in GBM and NDRG4 is required for the viability of primary astrocytes, established GBM cell lines, and both CD133 ؉ (cancer stem cell (CSC)-enriched) and CD133 ؊ primary GBM xenograft cells.While NDRG4 overexpression has no effect on cell viability, NDRG4 knockdown causes G 1 cell cycle arrest followed by apoptosis. The initial G 1 arrest is associated with a decrease in cyclin D1 expression and an increase in p27 Kip1 expression, and the subsequent apoptosis is associated with a decrease in the expression of XIAP and survivin. As a result of these effects on cell cycle progression and survival, NDRG4 knockdown decreases the tumorigenic capacity of established GBM cell lines and GBM CSC-enriched cells that have been implanted intracranially into immunocompromised mice. Collectively, these data indicate that NDRG4 is required for cell cycle progression and survival, thereby diverging in function from its tumor suppressive family member NDRG2 in astrocytes and GBM cells.The N-Myc downstream-regulated gene (NDRG) 5 family consists of four genes (NDRG1-4) that can be divided into two subfamilies based on sequence homology: NDRG1 and NDRG3 are in the first subfamily, and NDRG2 and NDRG4 make up the second subfamily. Although the four NDRG family members show distinct spatiotemporal expression patterns during embryonic development and in adult tissues (1-10), all four are highly expressed in the brain (4). To date, however, NDRG2 is the only NDRG family member that has been studied in the context of GBM cells and astrocytes. NDRG2 mRNA and protein levels are lower in GBM than in normal brain tissue, normal glial cells, and low grade astrocytomas (11-14), suggesting a tumor suppressive function. Data from experimental and clinical studies support this hypothesis: NDRG2 overexpression inhibits GBM cell proliferation (15), and decreased NDRG2 expression correlates with decreased GBM patient survival (13).In contrast to its subfamily member NDRG2, NDRG4 has not been studied in GBM cells or astrocytes. Nevertheless, available evidence supports the hypothesis that NDRG4 has an important role in this context that is similar to the role of NDRG2. First, unlike the relatively ubiquitous expression patterns of NDRG1-3, NDRG4 expression is restricted to a small number of tissues including the brain, where it is expressed at particularly high levels (7, 10). This restricted expression pattern suggests that NDRG4 plays an important role withi...

NDRG4 is Downregulated in Glioblastoma and Inhibits Cell Proliferation

“…However, NDRG3 could serve as a tumor promoter for prostate cancer PC3 cell line because overexpression of NDRG3 increased cell growth and migration capability of PC3 cells ). We showed that NDRG4's expression was downregulated at both RNA and protein levels in GBM tissues compared to normal brain tissues, which is in conflict with the findings by Schilling et al (2009), who showed that NDRG4 is upregulated in GBM compared to human cortex tissues and that knocking down of NDRG4 reduced the cell viability of GBM cells. The discrepancies could be due to several possible reasons.…”

“…The discrepancies could be due to several possible reasons. In the report by Schilling et al (2009), the realtime PCR of NDRG4 was performed by comparing normal primary human astrocytes (NHA 1 and 2) and cultured cells derived from three human GBM xenograft samples (Schilling et al, 2009), whereas in the TCGA as well as in our data (Fig 1 and Supplementary Table 1), the analysis was performed by comparing human GBM tissues with normal brain tissues. The sample size (three cases of GBM) was also very small in Schilling's case but quite large (410 TCGA GBM samples and 49 GBM samples from our laboratory).…”

“…However, Schilling et al (2009) recently reported that NDRG4 is upregulated in GBM compared to human cortex tissues, and knock down of NDRG4 reduced the cell viability of GBM cells. To resolve this discrepancy, we performed additional experiments, and found that NDRG4 is indeed downregulated, at both RNA and protein levels by quantitative RT-PCR and Western blot analysis, in GBM compared to normal tissues.…”

NDRG4 Protein-deficient Mice Exhibit Spatial Learning Deficits and Vulnerabilities to Cerebral Ischemia

“…Fax: 81-6-6835-1176; E-mail: kame@ri.ncvc.go.jp. 2 To whom correspondence may be addressed: 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. Fax: 81-6-6835-6894; E-mail: hyanamot@ res.ncvc.go.jp.…”

“…Furthermore, accumulating evidence implicates their roles in development, cancer metastasis, and the immune system (1)(2)(3)(4)(5).…”