Identification Of The Molecular Mechanisms And Target Proteins Of APOBEC3G In Pancreatic Cancer

Purpose:Pancreatic cancer is a common gastrointestinal malignancy cancers, which is the fourth cause of cancer-related deaths, with occult onset, rapid progression, early metastasis, and limited treatment. To clarify the molecular mechanisms of pancreatic cancer is the basis and premise for the treatment. Recent research suggests that many gastrointestinal cancers are related with virus infection, but the role of virus infection in the biological behaviors of cancer cells remains unclear. As a virus-reactive protein, APOBEC enzyme can cause hypermutation in the viral DNA, presenting extensive anti-virus capability. Recent studies have found that APOBEC protein not only has antiviral activity, but also has an important function in cancers. The purpose of this study is to screen and identify APOBEC family members involved in the malignant behavior of tumor cells; to detect the A3G expression in pancreatic cancer; to observe the molecular mechanisms of APOBEC3G(A3G) in pancreatic cancer, and to identify the target proteins, thereby clarifying the effect of A3G on malignant behavior of pancreatic cancer.Methods:The whole genome cDNA expression microarray was used to screen virus-reactive proteins involved in the malignant behavior of cancer cells. The mRNA level of A3G was examined using TaqMan real-time PCR in3kinds of pancreatic cancer cells,11matched human pancreatic cancer and para-cancerous tissues. Immunohistochemistry and immunofluorescence were used to validate the A3G expression in54matched human pancreatic cancer pathological slides. To evaluate the role of A3G in pancreatic cancer in vivo, pancreatic cancer cell line stably expressing A3G was established and injected subcutaneously into nude mice. To investigate the molecular mechanisms of A3G early promoting the tumor formation, the cloning formation assay, anoikis assay and flow cytometry analysis were used. The expression of apoptosis-related proteins were detected by western blot. To clarify the molecular mechanism of A3G-induced anoikis resistance, the Akt kinase activity and PTEN expression were detected. Furthermore, the interaction between A3G and PTEN was examined by immunoprecipitation. The Wnt pathway activity was detected by luciferase dual reporter gene assay, and the target protein in Wnt pathway was revealed by immunoprecipitation. The domain mutants were established to analyze the binding sites of A3G and target proteins.Results:Microarray screening found that virus-reactive protein A3G might be involved in the malignant biological behavior of cancer; A3G was significantly up-regulated in pancreatic cancer cells and tissues. Up-regulation of A3G promoted xenograft tumor formation at early stage, and inhibited the tumor sizes at late stage. We further found that A3G could inhibit anoikis in pancreatic cancer cells, with the elevated expression of anti-apoptotic proteins Bcl-2, Mcl-1, and phospho-Bcl-2. Overexpression of A3G participated in anoikis resistance through the activation of Akt kinase activity, Akt pathway was activated by A3G-induced inactivation of PTEN. Furthermore, The combination of A3G and PTEN required the the CD2domain of A3G, the C2tensin-type and PDZ domain of PTEN. We also found that A3G was involved in the regulation of Wnt pathway by binding to FZD1. The combination of A3G and FZD1depended on the phosphorylation level, with no significant correlation with the domain of A3G, but was associated with the conserved and PDZ domain of FZD1. Conclusions:The virus-reactive protein A3G is up-regulated in pancreatic cancer cells. Overexpression of A3G activates Akt pathway through inactivation of PTEN, which leads to the anoikis resistance in pancreatic cancer cells, meanwhile A3G combines with FZD to disrupt the activity of Wnt pathway, suggesting that A3G affects the malignant behaviors of pancreatic cancer cells besides antiviral ability.