GNB2L1 and its O-GlcNAcylation regulates metastasis via modulating epithelial-mesenchymal transition in the chemoresistance of gastric cancer

<div><p>GNB2L1 and its O-GlcNAcylation has been reported to play roles in gastric cancer metastasis. However, the roles of GNB2L1 in chemoresistance of gastric cancer has never been determined. In the present study, we found that GNB2L1 was downregulated in chemoresistant patients of gastric cancer, and observed the decrease of GNB2L1 in protein levels instead of mRNA levels in different chemoresistant gastric cancer cell lines. Further we proved that this downregulation of GNB2L1 was resulted from its elevated O-GlcNAcylation catalyzed by OGT in both cell lines and patients. Next, we investigate the function of GNB2L1 and its O-GlcNAcylation on gastric cancer metastasis during chemoresistance, and confirmed Ser124 as the major O-GlcNAcylation site on GNB2L1 that regulated its function on metastasis. Furthermore, our data demonstrated that GNB2L1 modulated EMT via regulating the translation of EMT-related proteins in the process of chemoresistance. In summary, this study indicated that GNB2L1 and its O-GlcNAcylation regulated metastasis via modulating the translation of EMT-related proteins in the chemoresistance of gastric cancer.</p></div

Chemoresistant gastric cancer is associated with decreased protein level of GNB2L1.

<p>(A-C) Protein levels of GNB2L1 in tumor tissues from chemosensitive patients and chemoresistant patients were determined by WB (A) and IHC (B-C). Representative images (A-B) and statistical data (C) were shown. (D) Transcriptional levels of GNB2L1 in tumor tissues were determined by qPCR. (E-F) Protein levels and mRNA levels of GNB2L1 were further assessed in SGC-7901 cells and several chemoresistant cells derived from SGC-7901 cells. Progressive disease (PD) was considered as chemoresistant; complete remission (CR), partial remission (PR) and stable disease (SD) as chemosensitive. GAPDH was used as loading control. N.S., not significant; ***, P <0.001.</p

OGT elevated O-GlcNAcylation on GNB2L1 in chemoresistant gastric cancer.

<p>(A-B) The O-GlcNAcylation levels of GNB2L1 in different chemoresistant cells (A) and different tissue samples (B) were assessed via IP analysis, and protein levels of OGT and OGA were also determined via WB. Progressive disease (PD) was considered as chemoresistant; complete remission (CR), partial remission (PR) and stable disease (SD) as chemosensitive. GAPDH was used as loading control.(C-E) Correlation analysis of GNB2L1 levels with OGT (D) or OGA (E) in chemoresistant gastric cancer patients. Representative images (C) and statistical data (D-E) were shown. *, P<0.05.</p

GNB2L1 and its O-GlcNAcylation regulated EMT in chemoresistance.

<p>(A) The protein levels of EMT-related markers, E-cadherin and N-cadherin were determined by WB in SGC-7901/DDP cells. (B-D) The changes of E-cadherin and N-cadherin in protein level were further confirmed in tumor tissues by IHC (B). The correlation between GNB2L1 and E-cadherin or N-cadherin in clinical chemoresistant gastric cancer cases was determined by IHC (C-D). Representative images (B) and statistical data (C-D) were shown. (E) Transcriptional alternation of E-cadherin was assessed in qPCR analysis. (F) The translation alternation of EMT-related proteins was determined in SGC-7901/DDP transfected with or without GNB2L1 via WB. And some cells were treated with CHX (50 μM) for 30 min. GAPDH was used as loading control. All experiments were repeated more than 3 times. PD, progressive disease (considered as chemoresistance); n.s., not significant.</p