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MDGA2 is a novel tumour suppressor cooperating with DMAP1 in gastric cancer and is associated with disease outcome

View Article: PubMed Central - PubMed

ABSTRACT

Background: Using the promoter methylation assay, we have shown that MDGA2 (MAM domain containing glycosylphosphatidylinositol anchor 2) is preferentially methylated in gastric cancer. We analysed its biological effects and prognostic significance in gastric cancer.

Methods: MDGA2 methylation status was evaluated by combined bisulfite restriction analysis and bisulfite genomic sequencing. The effects of MDGA2 re-expression or knockdown on cell proliferation, apoptosis and the cell cycle were determined. MDGA2 interacting protein was identified by mass spectrometry and MDGA2-related cancer pathways by reporter activity and PCR array analyses. The clinical impact of MDGA2 was assessed in 218 patients with gastric cancer.

Results: MDGA2 was commonly silenced in gastric cancer cells (10/11) and primary gastric cancers due to promoter hypermethylation. MDGA2 significantly inhibited cell proliferation by causing G1–S cell cycle arrest and inducing cell apoptosis in vitro, and suppressed xenograft tumour growth in both subcutaneous and orthotopic xenograft mouse models (both p<0.001). The anti-tumorigenic effect of MDGA2 was mediated through direct stabilising of DNA methyltransferase 1 associated protein 1 (DMAP1), which played a tumour suppressive role in gastric cancer. This interaction activated their downstream key elements of p53/p21 signalling cascades. Moreover, promoter methylation of MDGA2 was detected in 62.4% (136/218) of gastric cancers. Multivariate analysis showed that patients with MDGA2 hypermethylation had a significantly decreased survival (p=0.005). Kaplan–Meier survival curves showed that MDGA2 hypermethylation was significantly associated with shortened survival in patients with early gastric cancer.

Conclusions: MDGA2 is a critical tumour suppressor in gastric carcinogenesis; its hypermethylation is an independent prognostic factor in patients with gastric cancer.

No MeSH data available.


Related in: MedlinePlus

MDGA2 induces p53/p21 signalling pathway. (A) Luciferase reporter assays showed that p53 and p21 were significantly activated by MDGA2 expression. Date are mean±SD. *p<0.05, **p<0.001. (B) p53 knockdown by short interference RNA (siRNA) in cells with stable MDGA2 transfection was examined by western blot analysis. (C) p53 knockdown in AGS and BGC823 cells transfected with MDGA2 partially blunts the effects of MDGA2 overexpression on cell growth, as evidenced by cell viability and (D) colony formation assays. Data shown are mean±SD. *p<0.05, **p<0.01, ***p<0.005. (E) Downstream targets of MDGA2 identified by p53 signalling pathway PCR array. (F) Schematic illustration of the molecular mechanism of MDGA2 in gastric cancer. MDGA2 binds to DNA methyltransferase 1 associated protein 1 (DMAP1) to stabilise DMAP1 by inhibiting its ubiquitin-mediated degradation, which subsequently activates the ataxia telangiectasia mutated (ATM)/p53/p21 signalling pathway to mediate the tumour suppressive function by inhibiting cell cycle progression and promoting apoptosis. Besides activation of p21 and p53, upregulation of p21 (figure 3C), ATM and p53 (figure 5G) by MDGA2 was well confirmed at the protein level by western blot analysis.
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GUTJNL2015309276F7: MDGA2 induces p53/p21 signalling pathway. (A) Luciferase reporter assays showed that p53 and p21 were significantly activated by MDGA2 expression. Date are mean±SD. *p<0.05, **p<0.001. (B) p53 knockdown by short interference RNA (siRNA) in cells with stable MDGA2 transfection was examined by western blot analysis. (C) p53 knockdown in AGS and BGC823 cells transfected with MDGA2 partially blunts the effects of MDGA2 overexpression on cell growth, as evidenced by cell viability and (D) colony formation assays. Data shown are mean±SD. *p<0.05, **p<0.01, ***p<0.005. (E) Downstream targets of MDGA2 identified by p53 signalling pathway PCR array. (F) Schematic illustration of the molecular mechanism of MDGA2 in gastric cancer. MDGA2 binds to DNA methyltransferase 1 associated protein 1 (DMAP1) to stabilise DMAP1 by inhibiting its ubiquitin-mediated degradation, which subsequently activates the ataxia telangiectasia mutated (ATM)/p53/p21 signalling pathway to mediate the tumour suppressive function by inhibiting cell cycle progression and promoting apoptosis. Besides activation of p21 and p53, upregulation of p21 (figure 3C), ATM and p53 (figure 5G) by MDGA2 was well confirmed at the protein level by western blot analysis.

Mentions: To understand the molecular basis of the tumour suppressive property of MDGA2, we performed luciferase reporter assays to assess the effect of MDGA2 on the activities of p53, p21, NF-κB, MAPK and Wnt signalling pathways. MDGA2 increased p53 and p21 luciferase reporter activities in both AGS and BGC823 cells, but not other pathways (figure 7A). We thus tested whether p53 knockdown could blunt the effect of MDGA2 overexpression. Our results show that p53 knockdown in AGS and BGC823 cells transfected with MDGA2 could partially block the effects of MDGA2 on cell growth, as evidenced by cell viability and colony formation assays (figure 7B–D). To further determine the downstream mediators of p53 signalling derived by MDGA2, gene expression profiles in MDGA2 stably transfected AGS and BGC823 cells were analysed by p53 signalling pathway PCR array. When compared with empty vector-transfected cells, MDGA2 modulated the expression of p53 signalling-related genes involved in apoptosis, proliferation, cell cycle and tumour suppressive function (figure 7E). In addition, knockdown DMAP1 by siDMAP1 transfection in AGS and BGC823 cells with stable MDGA2 overexpression partially decreased the protein expression of p53 and p21 as determined by western blot analysis (see online supplementary figure S2), suggesting that MDGA2 induces p53/p21 signalling cascade partly depending on DMAP1.


MDGA2 is a novel tumour suppressor cooperating with DMAP1 in gastric cancer and is associated with disease outcome
MDGA2 induces p53/p21 signalling pathway. (A) Luciferase reporter assays showed that p53 and p21 were significantly activated by MDGA2 expression. Date are mean±SD. *p<0.05, **p<0.001. (B) p53 knockdown by short interference RNA (siRNA) in cells with stable MDGA2 transfection was examined by western blot analysis. (C) p53 knockdown in AGS and BGC823 cells transfected with MDGA2 partially blunts the effects of MDGA2 overexpression on cell growth, as evidenced by cell viability and (D) colony formation assays. Data shown are mean±SD. *p<0.05, **p<0.01, ***p<0.005. (E) Downstream targets of MDGA2 identified by p53 signalling pathway PCR array. (F) Schematic illustration of the molecular mechanism of MDGA2 in gastric cancer. MDGA2 binds to DNA methyltransferase 1 associated protein 1 (DMAP1) to stabilise DMAP1 by inhibiting its ubiquitin-mediated degradation, which subsequently activates the ataxia telangiectasia mutated (ATM)/p53/p21 signalling pathway to mediate the tumour suppressive function by inhibiting cell cycle progression and promoting apoptosis. Besides activation of p21 and p53, upregulation of p21 (figure 3C), ATM and p53 (figure 5G) by MDGA2 was well confirmed at the protein level by western blot analysis.
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GUTJNL2015309276F7: MDGA2 induces p53/p21 signalling pathway. (A) Luciferase reporter assays showed that p53 and p21 were significantly activated by MDGA2 expression. Date are mean±SD. *p<0.05, **p<0.001. (B) p53 knockdown by short interference RNA (siRNA) in cells with stable MDGA2 transfection was examined by western blot analysis. (C) p53 knockdown in AGS and BGC823 cells transfected with MDGA2 partially blunts the effects of MDGA2 overexpression on cell growth, as evidenced by cell viability and (D) colony formation assays. Data shown are mean±SD. *p<0.05, **p<0.01, ***p<0.005. (E) Downstream targets of MDGA2 identified by p53 signalling pathway PCR array. (F) Schematic illustration of the molecular mechanism of MDGA2 in gastric cancer. MDGA2 binds to DNA methyltransferase 1 associated protein 1 (DMAP1) to stabilise DMAP1 by inhibiting its ubiquitin-mediated degradation, which subsequently activates the ataxia telangiectasia mutated (ATM)/p53/p21 signalling pathway to mediate the tumour suppressive function by inhibiting cell cycle progression and promoting apoptosis. Besides activation of p21 and p53, upregulation of p21 (figure 3C), ATM and p53 (figure 5G) by MDGA2 was well confirmed at the protein level by western blot analysis.
Mentions: To understand the molecular basis of the tumour suppressive property of MDGA2, we performed luciferase reporter assays to assess the effect of MDGA2 on the activities of p53, p21, NF-κB, MAPK and Wnt signalling pathways. MDGA2 increased p53 and p21 luciferase reporter activities in both AGS and BGC823 cells, but not other pathways (figure 7A). We thus tested whether p53 knockdown could blunt the effect of MDGA2 overexpression. Our results show that p53 knockdown in AGS and BGC823 cells transfected with MDGA2 could partially block the effects of MDGA2 on cell growth, as evidenced by cell viability and colony formation assays (figure 7B–D). To further determine the downstream mediators of p53 signalling derived by MDGA2, gene expression profiles in MDGA2 stably transfected AGS and BGC823 cells were analysed by p53 signalling pathway PCR array. When compared with empty vector-transfected cells, MDGA2 modulated the expression of p53 signalling-related genes involved in apoptosis, proliferation, cell cycle and tumour suppressive function (figure 7E). In addition, knockdown DMAP1 by siDMAP1 transfection in AGS and BGC823 cells with stable MDGA2 overexpression partially decreased the protein expression of p53 and p21 as determined by western blot analysis (see online supplementary figure S2), suggesting that MDGA2 induces p53/p21 signalling cascade partly depending on DMAP1.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Using the promoter methylation assay, we have shown that MDGA2 (MAM domain containing glycosylphosphatidylinositol anchor 2) is preferentially methylated in gastric cancer. We analysed its biological effects and prognostic significance in gastric cancer.

Methods: MDGA2 methylation status was evaluated by combined bisulfite restriction analysis and bisulfite genomic sequencing. The effects of MDGA2 re-expression or knockdown on cell proliferation, apoptosis and the cell cycle were determined. MDGA2 interacting protein was identified by mass spectrometry and MDGA2-related cancer pathways by reporter activity and PCR array analyses. The clinical impact of MDGA2 was assessed in 218 patients with gastric cancer.

Results: MDGA2 was commonly silenced in gastric cancer cells (10/11) and primary gastric cancers due to promoter hypermethylation. MDGA2 significantly inhibited cell proliferation by causing G1&ndash;S cell cycle arrest and inducing cell apoptosis in vitro, and suppressed xenograft tumour growth in both subcutaneous and orthotopic xenograft mouse models (both p&lt;0.001). The anti-tumorigenic effect of MDGA2 was mediated through direct stabilising of DNA methyltransferase 1 associated protein 1 (DMAP1), which played a tumour suppressive role in gastric cancer. This interaction activated their downstream key elements of p53/p21 signalling cascades. Moreover, promoter methylation of MDGA2 was detected in 62.4% (136/218) of gastric cancers. Multivariate analysis showed that patients with MDGA2 hypermethylation had a significantly decreased survival (p=0.005). Kaplan&ndash;Meier survival curves showed that MDGA2 hypermethylation was significantly associated with shortened survival in patients with early gastric cancer.

Conclusions: MDGA2 is a critical tumour suppressor in gastric carcinogenesis; its hypermethylation is an independent prognostic factor in patients with gastric cancer.

No MeSH data available.


Related in: MedlinePlus