The Role Of Notch Signaling Pathway In The Pathogenesis Of Pancreatic Adenocarcinoma Induced By Chronic Pancreatitis

IntroductionPancreatic cancer is one of the most malignant gastrointestinal tumors and the fourth leading cause of cancer-related mortality worldwide due to its difficulty for early diagnosis, rapid progression and poor prognosis. Therefore, it is necessary to reveal the development mechanism of pancreatic cancer for early prevention and treatment. PK mouse model is the first transgenic mouse model recapitulating the full spectrum of human PanINs to PDAC. The studies found that patients suffering from chronic pancreatitis have a16-fold increased risk of developing pancreatic cancer. The Notch signaling pathway, playing an important role in human embryonic development, was reactivated in chronic pancreatitis and pancreatic cancer. Chronic pancreatitis significantly accelerated the development of PDAC in mouse expressing KrasG12v. However, the mechanism of chronic pancreatitis accelerating PDAC development is not clear. Therefore, in advantage of PK mouse model, this research aimed to study the action mechanism of Notch signaling pathway in the process of chronic pancreatitis inducing PDAC.MethodsAfter hybridization of LSL-KrasG12D and PDX1-Cre mice, the hybrid offspring genotypes were detected by PCR to obtain LSL-KrasG12D;PDX1-Cre mice which were then divided into three groups, one for vehicle treatment, one for caerulein treatment inducing chronic pancreatitis, and the other for caerulein in combination of Notch signaling pathway blocker DBZ treatment. After that, each group was further divided into two subgroups according to intervention time, namely5and12days. The mice were sacrificed and dissected the day after the end of drug intervention. The pancreatic tissue is divided into three parts. One part is for paraffin block used in subsequent HE staining evaluating lesions and immunohistochemistry testing the protein expression. One part is used for qRT-PCR testing the mRNA activity of Notch signaling pathway. The other is used for western blotting testing the protein activity of Notch signaling pathway. Results(1) After hybridization of LSL-KrasG12D mice and PDX1-Cre mice, about800offspring were obtained, in which there were about140LSL-KrasG12D; PDX1-Cre mice. The heterozygous positive rate is about1/5.(2) After drug intervention for5days, the mice in caerulein group and in caerulein in combination with DBZ group both appeared chronic pancreatitis, while the lesion in caerulein group was more severe than that in the latter group. The pancreatic tissue in vehicle group appeared normal. After drug intervention for12days, the mice in caerulein group and in caerulein in combination with DBZ group both appeared neoplasia, while the infiltration degree of PanINs and PDAC in caerulein group was severe than that in the latter group microscopically. The pancreatic tissue in vehicle group appeared normal in gross anatomy, and showed local PanIN1lesion microscopically.(3) The infiltration degree of chronic inflammation cells, T lymphocytes (CD3+) and macrophages (F4/80+) in caerulein in combination with DBZ group was significantly lower than that in caerulein treatment group. And the serum inflammatory cytokines TNF-α and IL-6levels in the former group were significantly lower than that in the latter group mainly in chronic pancreatitis lesion. After caerulein in combination with DBZ treatment for12days, the activity of Notch signaling pathway was partly inhibited. The results of qRT-PCR showed that Notch4, Dll4, Hes1, Hey1mRNA expression were significantly reduced. Western blotting results showed that Notch2, Notch4and D114protein expression were significantly reduced, that Notch3and Jagged2protein expression were mild reduced while the expression of Hes1without significant change. The results of immunochemistry revealed that the protein expression of Notchl, Notch4, Jaggedl, Dll1, D114and Hesl were significantly reduced. In addition, after DBZ treatment for5days, the protein expression of Notch1, Notch2, Notch3, Jagged1and Hes1were also significantly reduced.(4) PanIN1A has a mucus-rich characteristic, wherein residual mature acinar cell markers chymotrypsin and amylase positive acinar cells could be seen, reflecting a transitional state of transformation from acinar cells to ductal cells. The positivity of PanIN1lesion in immature acinar cell markers clusterin and PDX1reflected its characteristics of immature acinar cell. And CK19positive expression of PanINs and PDAC reflected their ductal characteristic. In addition, the percentage of Ki67positive PanINs epithelial cells in caerulein treatment group for12days was significantly higher than that in caerulein in combination with DBZ treatment group, while the percentage of TUNEL positive cells in the former group was significantly lower than that in the latter group.ConclusionsBlocking the Notch signaling pathway in the process of chronic pancreatitis inducing pancreatic cancer could alleviate the inflammation, inhibit the epithelial proliferation of PanINs and induce their apoptosis, resulting in the delay of pancreatic cancer progression. Mature acinar cells could dedifferentiate and then transdifferentiate into PanINs and PDAC cells.