The Role Of AREG/EGFR Signaling Pathway In Pancreatic Cancer Epithelial-mesenchymal Transition And Immortalization Of Human Pancreatic Stellate Cell Line

Background & Objective:The majority of pancreatic cancer patients are diagnosed at an advanced stage and thus not candidate for treatment with curative intent. Because pancreatic cancer patients usually show partial responses to traditional radiotherapy and cytotoxic chemotherapy, finding potential therapeutic target and new diagnostic, prognostic biomarkers have important implication for pancreatic cancer patients. To study the the mechanism of the interaction between pancreatic stellate cell (PSC) and pancreatic cancer cell is a hot topic in pancreatic cancer pathogenesis. To understand the interaction between these two cells provides a much more realistic model for complex intrapancreatic microenvironment in pancreatic cancer.This study includes two main parts.Part one, the role of AREG/EGFR in pancreatic cancer was preliminarily studied. (1) investigate the expression of AREG, EGFR, and EGFRvIII in resected pancreatic cancer tissues and explore the clinicopathological and prognostic significance of their expression in pancreatic cancer.(2) investigate the role of AREG/EGFR/ERK/NF- κB signaling pathway in pancreatic cancer epithelial-mesenchymal transition.Part two, summarize the process of human pancreatic stellate cell line immortalization, which provides the experimental material basis for the further study of the role of AREG in PSC.Materials and Methods:Part one. (1) Immunohistochemical staining was performed to detect the expression of AREG, EGFR, and EGFRvⅢ in 92 pancreatic cancer patients. The categorical variables were compared using the x2 test. The correlation between AREG and EGFR expression was analyzed. Survival curves were calculated using the Kaplan-Meier method and compared using the log-rank test. A Cox proportional-hazards regression model was used to perform multivariate survival analysis using a forward variable selection procedure.(2) Knock down of AREG expression by RNAi and overexpression of AREG by recombinant protein was performed to investigate the biological behavior changes in pancreatic cancer. Cell movement ability was tested by cell scratch assay, cell migration assay and cell invasion assay. Western blot was performed to analyze the effect of AREG/EGFR on the expression of EMT proteins and transcription factors. EGFR、MEK1/2 and NF- κ B inhibitors were used to verify the downstream signaling pathway expression by AREG/EGFR activation. MMP/TIMP protein chip was used to screen for MMP/TIMP proteins whose expression altered by AREG knockdown. Candidate proteins were further validated by immunoblotting. Meanwhile, the expression of AREG in 4 cases of primary culture of PSCs was verified, the influence of PSC on migration and invasion of pancreatic cancer cell was studied.Part two. The immortalization of human pancreatic stellate cells. The PSC was transfected with SV40LT and hTERT genes, colonies were isolated by pruomycin selection and expanded to immortalized cell lines. The morphology of the cells was observed by microscope. Immunohistochemistry, karyotype analysis, growth curve and nude mice transplantation were performed to investigate its biological characteristics.Results:Part one. (1) EGFR, EGFRvⅢ and AREG expression and AREG/EGFR co-expression were detected in 43 (46.7%),12 (13%),49 (53.3%) and 22 (23.9%) cases, respectively. EGFR and EGFRvⅢ were predominantly localized at the cellular membrane and cytoplasm, AREG was mainly detected in the cytoplasm. There was no significant association between AREG and EGFR expression (0=0.039, P=0.709). A significant association between EGFR expression and tumor differentiation was observed in our study. AREG/EGFR co-expression was significantly associated with tumor differentiation. In the univariate survival analysis, AREG expression(P=0.021, P=0.003), high TNM stage(P=0.003, P=0.001), and poor tumor differentiation(P=0.009, P=0.002) were significant adverse prognostic factors for DFS and OS. Poor tumor differentiation(DFS HR=1.785, P=0.021; OS HR=2.125, P=0.004) was an independent unfavorable prognostic factor for DFS and OS. AREG expression (HR=1.822, P=0.03), high TNM stage (HR=2.25, P=0.03) and positive resection margins (HR=1.84, P=0.045) were all independent prognostic indicators for poor OS.(2) Cell scratch assay, cell migration assay and cell invasion assay results showed that knocking down AREG expression decreased AsPC-1 cells migration and invasion ability. Western blot showed that, knocking down AREG expression reduced the expression level of Vimentin, β-catenin, Snail, Slug and ZEB1. Knocking down AREG also increased the expression of E-cadherin, ZO-1. Meanwhile, the phosphorylation level of AREG receptor EGFR was significantly reduced. In AREG knock-down AsPC-1 cells, the activation levels of several signaling pathways, including AKT, ERK and STAT3 were down regulated, among which the ERK signaling pathway was the most obvious. The level of nuclear factor kappa B decreased significantly at the same time. According to the results of MMP/TIMP protein chip, Knocking down AREG also decreased MMP-9 expression. In PANC-1 cells, exogenous overexpression of AREG enhanced migration, invasion ability of PANC-1 cells. Meanwhile, western blot showed that, E-cadherin and ZO-1 expression level were decreased, Vimentin, β-catenin and MMP-9 expression level were up-regulated. The expression level of transcription factor Snail and Slug were increased; the expression level of ZEB1 did not change significantly. The signaling pathways of AKT, ERK and STAT3 showed different degrees of activation, the most obvious effects were observed in the ERK and AKT signaling pathways. The level of nuclear factor kappa B increased significantly at the same time. The enhancement effect was blocked by PD153035 and/or U1026. AREG/EGFR signal axis enhanced the migration, invasion and EMT capability of PANC-1 cells, this effects could be blocked by QZN. AREG in 4 cases of primary culture of PSCs can be expressed in different degrees, PSC can significantly enhanced the migration and invasion capability of PANC-1 cells, the chemotactic effect of pancreatic cancer cells could not be antagonized by AREG neutralizing antibody.Part two. Primary PSCs were cultured by outgrowth method. After SV40LT and hTERT virus transfection and puromycin selection, an immortalized human pancreatic stellate cell line imPSC was established. RT-qPCR and Western blot showed that im PSC cell expressed high level of SV40LT and hTERT. a-SMA and Vimentin expression were detected by IHC on imPSC cell line. In addition, a small amount of GFAP expression was detected. Karyotype analysis of im PSC cells revealed a mean number of 60 chromosomes. The growth curve of im PSC cells showed typical S-shaped. The nude mice transplantation revealed that im PSC cells had no tumorigenicity ability.Conclusion:AREG expression is an important prognostic biomarker in PDAC. AREG expression is positively associated with the migration and invasion capability. The mechanisms include the followings:AREG binds to EGFR, then activates the downstream signal through the activation of EGFR intracellular region (pY1068 phosphorylation), and results in ERK, AKT and STAT3 signaling pathway activation in pancreatic cancer cells. The binding of AREG to EGFR raises the nuclear NF- κ B level, which increases the EMT related transcription factor Snail and Slug expression. Up regulation of Snail and Slug expression reduces the expression of E-cadherin. The binding of AREG to EGFR also increases Vimentin and MMP-9 expression and results in the epithelial-mesenchymal transitions in pancreatic cancer cells. These results suggest that AREG/EGFR signaling axis may serve as an attractive therapeutic target for pancreatic cancer. Meanwhile, we have established an immortalized human pancreatic stellate cell line. This will provide a reliable and ideal experimental model for the study of pancreatic stellate cell function, the interaction mechanism between pancreatic stellate cell and pancreatic cancer cell and pancreatic cancer tumor microenvironment.