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

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 G₁ cell cycle arrest followed by apoptosis. The initial G₁ 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 within the central nervous system. Second, NDRG4 is more than 60% identical in amino acid sequence to NDRG2. This sequence similarity is likely behind the overlapping functions of these two proteins in certain cell types within the brain. For example, in PC12 neuronal cells, both NDRG4 and NDRG2 promote neurite extension (16–18). In combination with the brain-specific expression pattern of NDRG4, these functional and sequence similarities suggest that NDRG4 may recapitulate the tumor suppressive function of NDRG2 in primary brain neoplasms.To determine if the similarities between NDRG2 and NDRG4 extend to the context of GBM, we investigated the role of NDRG4 in GBM cell lines and primary human astrocytes. In contrast to the established roles of NDRG2 and other NDRG family members, we found that the role of NDRG4 in GBM is not tumor suppressive. On the contrary, both astrocytes and GBM cells require the presence of NDRG4 for cell cycle progression and survival.