A Study On The Expression,Antigen Identification,Functions And Mechanisms Of MG5 In Gstric Cancer

Background: Gastric cancer(GC) is a major health problem in China. Biomarkers, especially those for pre-warning and early detection of GC are of great importance to improve the survival chance of patients. But there is no such an ideal biomarker in practice. Our lab has produced a batch of monoclonal antibodies with high sensitivity and specificity for GC detection, which warrants large-scale retrospective study and basic researches such as antigen identification. Objectives: To evaluate the clinical value of MG5 in pre-warning, early detection and prognosis in GC, to identify the antigen specific to MG5 and to explore the biological functions and related mechanism in GC.Methods: Immunohistochemistry(IHC) was applied to the expression of MG5 in numerous gastric mucosal precancerosis(GMP, N=509 and 467) and various common malignancies(n=747), and the correlation between their expression and clinical features including precancerous progression, GC development and prognosis; methods including WB, IP, MS, IF, lentivirus infection were performed to clarify the antigen specific to MG5; assays including MTT, FACS, lentivirus infection, WB, IF, Co-IP, MS, in vivo tumor growth, lysosome binding and uptake were used to investigate the biological functions and related molecular mechanisms of MG5 in GC. Results: Pre-warning function of MG5 in numerous patients with gastric mucosal precancerosis(GMP)We graded the expression of MG5 into four levels:-(negative), +(positive), ++(strong positive) and +++(extreme positive) and set the risk of precancerous progression(PP) and GC occurrence as “1” for the negative expression group. As compared with the negative expression group, we found that: 1. PP risk of GMP with MG5++ and MG5+++ were 3.35 and 5.21. 2. GC risk of GMP with MG5+ 2.51, with MG5++ 4.12. 3. GC risk of IM/DYS with MG5+ was 2.71, with MG5++ 6.01. 4. The GC risk of GMP with MG5+HP+ was 2.80, with MG5++HP+ 3.48 and with MG5++ 14.68. P value for all the positive results were less than 0.05 and were considered as significant. These results reveal that MG5 is a useful pre-warning marker for GC alone or in conjunction with HP infection and GMP subtypes.Expression and clinical significance of MG5 in diverse tumorsMG5 positivity was significantly elevated in eight kinds of tumors(n=747) as compared with adjacent normal tissues(P all <0.05). Clinicopathologically, MG5 expression was found correlated with female gender in esophageal squamous cell carcinoma(P=0.028) and with high pathologic grade in breast ductal carcinoma(P=0.003). Noticeably, the medium survival time of female patients with gastric adenocarcinoma(GA) or with esophageal squamous cell carcinoma(ESCC) was significantly shortened when MG5 expression was positive(P=0.028 and 0.003, respectively); and the medium survival time of patients with lung squamous cell carcinoma(LSCC) was also shortened when MG5 was positive and simultaneously the tumor had invaded the lymph node(P=0.040). In multivariate analysis, MG5 was proven to be an independent prognostic predictor for female GA patients(P=0.015) and for LSCC patients with node metastasis(P=0.030).Antigen identification of MG5 as LAMP2AIP based MS analysis indicated that LAMP2 was possibly the identity of MG5-Ag. Co-focal immunofluorescence demonstrated that MG5-Ag was located in the lysosome and was co-localized with LAMP2. Western blot showed that both MG5 antibody and anti-LAMP2 could recognize a band at ~120Kd in LAMP2-positive cell line Hep G2 and in LAMP2-null cell line MCF-7 when infected with LAMP2-lentivirus. MG5 antibody can only recognize over-expressed LAMP2 A isoform but not LAMP2 B, whereas the commercial LAMP antibody(Santa) can recognize both. LAMP2 A silencing and overexpression via lentivirus infection further conformed that MG5 antibody is specific for LMAP2 A. Co-IF revealed that MG5 was primarily located in lysosome. All these data proved that MG5 is LAMP2 A.Functions and mechanis ms of LAMP2A/MG5 in prolife ration and survival of gastric cancer cellLAMP2A knockdown significantly slowed down the proliferation of GC cells, accompanied with increased Rho E level. Rho E up-regulation can directly suppress GC cell proliferation. Decreasing Rho E level can partly reverse the compromised proliferation caused by LAMP2 A knockdown. Rho E was demonstrated to be a substrate of CMA because it can interact with LAMP2 A and HSC70, and be bond and engulfed by lysosomes. Beside, when CMA was compromised by silencing LAMP2 A, GC cells became susceptible to H2O2 induced apoptosis, and O verexpress ion of DJ-1 can partly reverse this phenomenon. Furthermore we found that oxidation stress can promote the interaction between DJ-1 and CMA components(LAMP2A and HSC70), and enhance its binging and uptake by lysosomes. Conclusions:MG5 is a promising biomarker for GC pre-warning. MG5 expression was a useful prognostic biomarker for female GC patients and also for lung cancer patients with node invasion. Rho E and oxidized DJ-1 are two novel substrates of CMA, and their accumulation are responsible for the compromised proliferation and increased apoptosis in GC cells.