BRCA2Silencing Associated Aberrant Pitx2Expression Promotes PanIN Malignant Transformation

Background﹠ObjectivePancreatic ductal adenocarcinoma (PDAC) is a lethal disease and early diagnosis andtreatment are of paramount importance. One of the most crucial thing is to understand themechanism of pancreatic intraepithelial neoplasia (PanIN) malignant transformation toPDAC. The mutation of KrasG12D initiates PanIN and the inactivated tumor suppressivegenes, including p16, Tp53or Smad4promote KrasG12D-driven carcinogenesis,respectively. However it is unclear whether BRCA2silencing promotes PanIN malignanttransformation. In the present study, we aim to investigate the biological function andmolecular mechanism of BRCA2in the process of malignant transformation from PanINto PDAC.Methods1Lentivirus-mediated BRCA2silencing vector was transduction into PanIN cell whichnamed PanIN-BR cell (PanIN-C cell was used as negative control). MTT assay, soft agarassay, Matrigel migration and invasion assay, FACS analysis, xenografted modelexperiment were used to investigate the biological function between PanIN-BR cell andPanIN-C cell.2Gene expression profiling was employed to examine the differentially expressed genesin PanIN BR cell and PanIN-C cell. The genes were identified and subjected to Real-timePCR and Western blot analysis. After silencing Pitx2expression in PanIN cell or inductionof Pitx2expression into PanIN-BR cell, soft agar assay and Matrigel invasion assay were used to investigate the biological function of the cell compared with the negative one.3Immunohistochemistry assay was used to detect the Pitx2protein level in pancreaticcancer and matched peritumoral pancreatic tissues. Also, soft agar assay and Matrigelinvasion assay were used to investigate the biological function of human pancreatic cancercells.4Methylation-specific real-time PCR and Real-time PCR were used to detect Pitx2methylation level and Pitx2expression respectively. After Induction of Pitx2expressioninto mouse PDAC cells(Tp53mutation) and PanIN-SR cell(PanIN cell with Smad4silencing), soft agar assay and Matrigel invasion assay were used to investigate thebiological function. Real-time PCR and Western blot assay were performed to investigatethe relevant of Pitx2and Smad4.Results1BRCA2silencing promoted PanIN cell anchorage-dependent growth, colony formationability, migration, invasion and xenograft tumor growth. But apoptosis detected by FACSanalysis showed there was no significant difference.2Microarray results showed that Wisp1, Ccna2, Cxcr4, Twist1, Ccnb2, Pitx2, Wisp2,Cdkn2a and Cdkn2b were the most differentially expressed genes and the magnitude ofexpression changes detected by Real-time PCR and Western blot were comparable withthe fold changes detected by the microarray. Moreover, silencing Pitx2expression inPanIN cell promoted the colony formation and invasion ability，and induction of Pitx2expression into PanIN-BR cell, the colony formation ability and invasion ability of the cellwere attenuated compared with the negative one.3Pitx2protein level was detected moderate nuclear staining in normal tissues, PanIN-Itissues, PanIN-II tisses, and weak nuclear staining in PanIN-III tissues. However, Pitx2protein level was detected weak or lack nuclear staining in PDAC tissues. The overallsurvival rate of patients with Pitx2positive-expression was higher than patients with Pitx2negative-expression. Moreover, Pitx2attenuates colony formation ability and invasion ability of pancreatic cancer cells.4Pitx2DNA methylation level in PDAC tissues and matched normal peritumoralpancreatic tissues were extremely low, and the DNA methylation level and mRNAexpression level of Pitx2in36fresh human PDAC tissues did not show any relationship.Also, After Induction of Pitx2expression into mouse PDAC cells(Tp53mutation) andPanIN-SR cell(PanIN cell with Smad4silencing), the colony formation ability andinvasion ability were attenuated compared with the negative one, respectively. Furthermore, Smad4restoration increased the expression of Pitx2in human pancreatic cancercells.ConclusionBRCA2silencing promoted PanIN cell malignant transformation. Induction of Pitx2expression may attenuated the acquired malignant behavior because of BRCA2silencing,Tp53mutation and Smad4silencing. Pitx2expression was sharply reduced in pancreaticcancer tissues and decreased Pitx2expression was probably associated with a poorprognosis in patients with pancreatic cancer. Pitx2attenuates colony formation ability andinvasion ability of PanIN cell and human pancreatic cancer cells. Pitx2expression was notassociated with the methylation level in pancreatic cancer, while was associated withSmad4. In summary, our findings show that Pitx2may be act as a tumor suppressor inpancreatic carcinogenesis, and may serve a potential marker for the diagnosis, treatmentand prognosis of pancreatic cancer.