ATP1A1 Integrates AKT and ERK Signaling via Potential Interaction With Src to Promote Growth and Survival in Glioma Stem Cells

Abstract

Glioma stem cells (GSCs) have been considered to be responsible for treatment failure due to their self-renewal and limitless proliferative property. Recently, the Na⁺/K⁺-ATPase a1 (ATP1A1) subunit was described as a novel therapeutic target for gliomas. Interestingly, our previous proteomics study revealed that ATP1A1 is remarkably overexpressed in GSCs. In the current study, we investigated the role of ATP1A1 in regulating growth, survival, and tumorigenicity of primary human GSCs and the underlying molecular mechanism. We tested RNA and protein expression of ATP1A1 in glioma tissues and GSCs. In addition, we knocked down ATP1A1 in GSCs and assessed the effects thereof on growth, survival, and apoptosis. The role of ATP1A1 in signaling pathways was investigated in vitro. We found that the ATP1A1 expression level was associated with the grade of glioma. Knockdown of ATP1A1 in GSCs in vitro inhibited cell proliferation and survival, increased apoptosis, and halted cell-cycle progression at the G1 phase. Cell proliferation and survival were resumed upon rescue of ATP1A1 expression in ATP1A1-knockdown GSCs. The ERK1/2 and AKT pathways were inhibited through suppression of Src phosphorylation by ATP1A1 knockdown. Collectively, our findings suggest that ATP1A1 overexpression promotes GSC growth and proliferation by affecting Src phosphorylation to activate the ERK1/2 and AKT signaling pathways.

Keywords: ATP1A1; ERK/AKT; GSCs; Src; proliferation; survival.

Figure 1

ATP1A1 is overexpressed in high-grade glioma tissues. (A) ATP1A1 expression was analyzed by immunoblotting in three GBM samples, three WHO grade III astrocytomas, three WHO grade II gliomas, three WHO grade I gliomas, and normal human brain tissue. (B) ATP1A1 expression was analyzed by immunohistochemical staining in gliomas of different grades in tissue arrays. ATP1A1 was slightly stained in normal brain tissue (a), WHO grade I astrocytomas (b), and WHO grade II astrocytomas (c). In contrast, moderate ATP1A1 staining was observed in WHO grade III astrocytomas (d) and GBM WHO grade IV samples (e). Hematoxylin counterstaining was used to visualize nuclei. Scale bar, 100 μm. (f) Bars represent the numbers of tumor samples that fall into each staining category for each tumor grade.

Figure 2

ATP1A1 is overexpressed in primary GSCs, which express stemness markers nestin and SOX2. (A) ATP1A1 protein expression was assessed by immunoblotting for seven pairs of patient-matched GSCs and differentiated glioma cells. (B) Representative images of immunocytochemical staining for nestin (red) and SOX2 (green) in GSCs, for GFAP (green) in differentiated GBM cells (C), and for ATP1A1 (red) in GSCs and differentiated GBM cells (D). Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (blue). Scale bars, 200 μm. Error bars represent the SD, ^*P < 0.05, ^**P < 0.01 (B).

Figure 3

ATP1A1 knockdown decreases viability in GSC and tumor growth and proliferation. (A) CCK-8 and (B) BrdU incorporation assays were used to detect cell viability and proliferation in sh-NC- and sh-ATP1A1-transfected compared to non-transfected GSCs1 and GSCs2 cells. BrdU incorporation into DNA was measured by a colorimetric assay and absorbance was measured at 450 nm. (C) sh-ATP1A1- and sh-NC-transfected cells were injected subcutaneously into the flank of athymic nude mice (n = 5 mice/group). Tumor volume was monitored at the indicated times to plot tumor growth curves. The experiment was stopped at day 25, and the xenograft tumors were weighed. Data represent the mean ± SD, ^*P < 0.05, ^**P < 0.01.

Figure 4

ATP1A1 knockdown in GSCs induces cell-cycle arrest and increases apoptosis. (A) The cellular DNA content was analyzed by flow cytometry. The histogram shows the percentage of cells in each phase of the cell cycle (right panel). The data represent means of five experiments. (B) Immunoblot analysis of key proteins in the G1, S, and G2–M cell-cycle phases in sh-ATP1A1- and sh-NC-transfected GSCs. (C) FACS analysis of cell apoptosis in sh-ATP1A1- and sh-NC-transfected GSCs. The cells were double stained with Annexin V and PI. Early apoptotic cells are in the lower right quadrant, late apoptotic cells are in the upper right quadrant. The histogram in the right panel shows average percentages of apoptotic cells which contain the early apoptotic cells and late apoptotic cells. (D) Immunoblot analysis of apoptosis-related proteins in sh-ATP1A1- and sh-NC-transfected cells. Experiments were repeated independently at least three times (A–D). ^*P < 0.05, ^**P < 0.01.

Figure 5

ATP1A1 enhances PI3K and MAPK/ERK signaling potentially via interacting with Src. (A) Effects of ATP1A1 on the phosphorylation status of ERK, JNK, p38, and AKT in GBM GSCs. (B) ATP1A1 cDNA was re-introduced into ATP1A1-knockdown cells and effectively restored signaling activation. (C) Effects of FTS on the phosphorylation status of ERK and AKT in GBM GSCs/pCMV-ATP1A1. (D) Effects of PP2 on the phosphorylation status of ERK and AKT in GBM GSCs/pCMV-ATP1A1. (E) ATP1A1/Src interaction was assayed by immunoprecipitation in GBM GSCs. (F) Changes in SRC expression in ATP1A1-silenced cells compared with control cells by immunoprecipitation with an anti-ATP1A1 antibody and IgG. To confirm that equal amounts of each cell extract were used for immunoprecipitation, whole-cell extracts were immunoblotted with anti-ATP1A1 and IgG (input). (G) Effects of ATP1A1 on the phosphorylation status of Src in sh-NC- and sh-ATP1A1-transfected GBM GSCs.

Figure 6

ATP1A1 promotes GSC proliferation and survival in a Src-dependent manner. (A) Src knockdown efficiency in GBM GSCs/pCMV6-ATP1A1 was confirmed by western blotting. (B) Cell growth curves of BrdU incorporation assays and (C) CCK-8 assays in GBM GSCs/pCMV6-ATP1A1 cells with Src knockdown. Data were collected at 24, 48, and 72 h. (D) Flow-cytometric analysis of GBM GSCs/pCMV6-ATP1A1 cells after Src knockdown. The cells were double stained with Annexin V and PI. Early apoptotic cells are in the lower right quadrant, late apoptotic cells are in the upper right quadrant. The histogram in the right panel shows average percentages of apoptotic cells which contain the early apoptotic cells and late apoptotic cells. Error bars represent the SD, ^*P < 0.05, ^**P < 0.01.