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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 interacts with DNA methyltransferase 1 associated protein 1 (DMAP1) in gastric cancer. (A) Co-immunoprecipitation (Co-IP) of MDGA2 binding proteins followed by mass spectrometry identified DMAP1 to be a MDGA2-binding protein. (B) Co-IP of MDGA2 and DMAP1 with each other from proteins of MDGA2-transfected AGS and BGC823 cells. The presence of MDGA2 and DMAP1 in the Co-IP products was confirmed by western blot analysis using specific antibodies. (C) Direct interaction between MDGA2 and DMAP1 was shown by glutathione S-transferase (GST) pull-down assay. (D) Western blot analysis for DMAP1 expression. (E) MDGA2 increased the DMAP1 level by inhibiting its ubiquitin-mediated degradation. (F1) Confocal immunofluorescence analysis of MDGA2 and DMAP1 expression in MDGA2- and empty vector-transfected cells. The scale bar of 50 M is for unframed images of both cell groups. The yellow-frame fields were enlarged arbitrarily. (F2) Western blot detection of MDGA2 and DMAP1 in the membrane, cytoplasmic and nuclear fractions of AGS and BGC823 cells transfected with MDGA2 expression vector. (G) Western blot analysis showed increased levels of ataxia telangiectasia mutated (ATM) and p53 by MDGA2 over-expression in AGS and BGC823 cells and xenograft tumours.
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GUTJNL2015309276F5: MDGA2 interacts with DNA methyltransferase 1 associated protein 1 (DMAP1) in gastric cancer. (A) Co-immunoprecipitation (Co-IP) of MDGA2 binding proteins followed by mass spectrometry identified DMAP1 to be a MDGA2-binding protein. (B) Co-IP of MDGA2 and DMAP1 with each other from proteins of MDGA2-transfected AGS and BGC823 cells. The presence of MDGA2 and DMAP1 in the Co-IP products was confirmed by western blot analysis using specific antibodies. (C) Direct interaction between MDGA2 and DMAP1 was shown by glutathione S-transferase (GST) pull-down assay. (D) Western blot analysis for DMAP1 expression. (E) MDGA2 increased the DMAP1 level by inhibiting its ubiquitin-mediated degradation. (F1) Confocal immunofluorescence analysis of MDGA2 and DMAP1 expression in MDGA2- and empty vector-transfected cells. The scale bar of 50 M is for unframed images of both cell groups. The yellow-frame fields were enlarged arbitrarily. (F2) Western blot detection of MDGA2 and DMAP1 in the membrane, cytoplasmic and nuclear fractions of AGS and BGC823 cells transfected with MDGA2 expression vector. (G) Western blot analysis showed increased levels of ataxia telangiectasia mutated (ATM) and p53 by MDGA2 over-expression in AGS and BGC823 cells and xenograft tumours.

Mentions: To gain insight into the molecular mechanistic basis of the tumour suppressive effect of MDGA2 in gastric cancer, we sought to identify its interacting partners by Co-IP followed by identification of associated proteins using mass spectrometry (see online supplementary table S3). DMAP1 was found to interact with MDGA2 by comparing the anti-His IP product of AGS cells overexpressing MDGA2-His with AGS cells transfected with control vector (pcDNA3.1/His) or with anti-IgG IP product (figure 5A). To validate the interaction between MDGA2 and DMAP1, Co-IP using DMAP1 antibody was also performed in AGS and BGC823 cells. MDGA2 could also be co-precipitated by DMAP1 antibody in both cell lines, as shown by western blot analysis (figure 5B), confirming the physical interaction between MDGA2 and DMAP1. We then performed a GST pull-down assay using purified recombinant GST-DMAP1 and MDGA2-His proteins to verify the specificity of the interaction between DMAP1 and MDGA2 under cell-free conditions. Coomassie blots showed that MDGA2 existed in a complex with GST-DMAP1 but not with GST (figure 5C), demonstrating specific binding between MDGA2 and DMAP1.


MDGA2 is a novel tumour suppressor cooperating with DMAP1 in gastric cancer and is associated with disease outcome
MDGA2 interacts with DNA methyltransferase 1 associated protein 1 (DMAP1) in gastric cancer. (A) Co-immunoprecipitation (Co-IP) of MDGA2 binding proteins followed by mass spectrometry identified DMAP1 to be a MDGA2-binding protein. (B) Co-IP of MDGA2 and DMAP1 with each other from proteins of MDGA2-transfected AGS and BGC823 cells. The presence of MDGA2 and DMAP1 in the Co-IP products was confirmed by western blot analysis using specific antibodies. (C) Direct interaction between MDGA2 and DMAP1 was shown by glutathione S-transferase (GST) pull-down assay. (D) Western blot analysis for DMAP1 expression. (E) MDGA2 increased the DMAP1 level by inhibiting its ubiquitin-mediated degradation. (F1) Confocal immunofluorescence analysis of MDGA2 and DMAP1 expression in MDGA2- and empty vector-transfected cells. The scale bar of 50 M is for unframed images of both cell groups. The yellow-frame fields were enlarged arbitrarily. (F2) Western blot detection of MDGA2 and DMAP1 in the membrane, cytoplasmic and nuclear fractions of AGS and BGC823 cells transfected with MDGA2 expression vector. (G) Western blot analysis showed increased levels of ataxia telangiectasia mutated (ATM) and p53 by MDGA2 over-expression in AGS and BGC823 cells and xenograft tumours.
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Related In: Results  -  Collection

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GUTJNL2015309276F5: MDGA2 interacts with DNA methyltransferase 1 associated protein 1 (DMAP1) in gastric cancer. (A) Co-immunoprecipitation (Co-IP) of MDGA2 binding proteins followed by mass spectrometry identified DMAP1 to be a MDGA2-binding protein. (B) Co-IP of MDGA2 and DMAP1 with each other from proteins of MDGA2-transfected AGS and BGC823 cells. The presence of MDGA2 and DMAP1 in the Co-IP products was confirmed by western blot analysis using specific antibodies. (C) Direct interaction between MDGA2 and DMAP1 was shown by glutathione S-transferase (GST) pull-down assay. (D) Western blot analysis for DMAP1 expression. (E) MDGA2 increased the DMAP1 level by inhibiting its ubiquitin-mediated degradation. (F1) Confocal immunofluorescence analysis of MDGA2 and DMAP1 expression in MDGA2- and empty vector-transfected cells. The scale bar of 50 M is for unframed images of both cell groups. The yellow-frame fields were enlarged arbitrarily. (F2) Western blot detection of MDGA2 and DMAP1 in the membrane, cytoplasmic and nuclear fractions of AGS and BGC823 cells transfected with MDGA2 expression vector. (G) Western blot analysis showed increased levels of ataxia telangiectasia mutated (ATM) and p53 by MDGA2 over-expression in AGS and BGC823 cells and xenograft tumours.
Mentions: To gain insight into the molecular mechanistic basis of the tumour suppressive effect of MDGA2 in gastric cancer, we sought to identify its interacting partners by Co-IP followed by identification of associated proteins using mass spectrometry (see online supplementary table S3). DMAP1 was found to interact with MDGA2 by comparing the anti-His IP product of AGS cells overexpressing MDGA2-His with AGS cells transfected with control vector (pcDNA3.1/His) or with anti-IgG IP product (figure 5A). To validate the interaction between MDGA2 and DMAP1, Co-IP using DMAP1 antibody was also performed in AGS and BGC823 cells. MDGA2 could also be co-precipitated by DMAP1 antibody in both cell lines, as shown by western blot analysis (figure 5B), confirming the physical interaction between MDGA2 and DMAP1. We then performed a GST pull-down assay using purified recombinant GST-DMAP1 and MDGA2-His proteins to verify the specificity of the interaction between DMAP1 and MDGA2 under cell-free conditions. Coomassie blots showed that MDGA2 existed in a complex with GST-DMAP1 but not with GST (figure 5C), demonstrating specific binding between MDGA2 and 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–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