Experimental Study Of Integrin αυβ6 In Promoting Malignant Progression Of Pancreatic Cancer

Background and significanceIntegrins are a family of cell surface adhesion molecules which mediate cell-cell and cell-extracellular matrix (ECM) interactions, influence the expression of cellular gene, and trigger intracellular signaling pathways to modulate cellular biological behavior. Among the various members of integrin family,αvβ6 appears to be directly implicated in the cancer progression. As an epithelial-specific integrin,αvβ6 usually is not constitutively expressed in normal epithelium and benign epithelial tumor tissues. Induced expression of avP6 has been observed in a variety of epithelial malignancies including breast, colorectum, stomach, pancreas, ovary, cervical cancers as well as oral and skin squamous cell carcinomas. There is increasing evidence to suggest that integrinαvβ6 plays important roles in tumor proliferation, invasion, and metastasis, especially in the field of colon cancer. Our previous study discovered that transfection ofαvβ6 gene could remarkably increase the MMP-9 secretion of colon cancer cells, promote the degradation of ECM, and mediate the potentials of colon cancer cells to metastasize to the liver. There is a direct binding between avP6 and ERK, and this novel binding defines a direct signal pathway for avP6 to regulate the progression of the malignant tumors. High avP6 expression was detected at the invading edges of colon cancer specimens, and low avP6 expression within the interior of tumor masses. Previous study has confirmed that high avP6 expression in human colon carcinoma is a prognostic indicator of poor survival. However, the effects of avP6 expression on biological behavior of pancreatic carcinoma cells, chemoresistance, and prognosis have not been studied up to now.Pancreatic cancer is one of the malignancies with the worst prognosis because of aggressive invasion, early metastasis, and almost complete resistance to existing chemotherapeutic agents, radiation therapy, and immunotherapy. Most patients can't receive curative surgical resection at diagnosis and the five-year survival rate of patients remains less than 5%in the past 20 years. The aim of this study is to investigate the roles of integrinαvβ6 in cell proliferation, invasion, metastasis, apoptosis, chemoresistance and prognostic significance in pancreatic carcinoma, which would help us to further understand the molecular biologic mechanisms of the malignant progression of pancreatic carcinoma. Targeting therapy directed againstαvβ6 has the potential to enhance therapeutic efficacy for aggressive pancreatic carcinoma and might ultimately improve the patient's clinical outcome.Part IThe expression and prognostic significance of integrinαvβ6 in pancreatic ductal adenocarcinoma tissuesObjective To investigate clinicopathologic significance and prognostic value ofαvβ6 and TGF-β1 expression in pancreatic ductal adenocarcinoma tissues.Methods We generated the microarray of 92 surgically resected pancreatic ductal adenocarcinoma specimens,10 normal autopsy pancreatic tissues were used as controls. The expression status ofαvβ6 and TGF-β1 was examined by immunohistochemical staining. Clinical and pathological data were retrospectively analyzed. We compared the staining results with clinicopathologic characteristics and patients survival. Univariate and multivariate survival analyses were performed to assess their prognostic value. The correlation between avP6 and TGF-β1 expression were also analyzed.Results Normal pancreatic epithelium did not exhibit any staining forαvβ6. In pancreatic carcinomas, positive staining of avP6 was detected in 52 patients (56.5%) and highαvβ6 expression was associated with larger tumor size (P=0.041), advanced TNM stage (P =0.027) and lymph node metastasis (P=0.030). TGF-β1 stained the cytoplasms of tumor cells in 61 cases (66.3%) and high TGF-β1 expression was correlated with poor tumor differentiation and advanced TNM stage. Furthermore, the results of Spearman correlation test showed that avP6 expression correlated positively with TGF-β1 expression. The patients with lowαvβ6 expression had much longer median survival time than those with highαvβ6 expression (P=0.001). The results of univariate and multivariate survival analyses indicated that highαvβ6 expression was an independent unfavorable prognostic factor in pancreatic ductal adenocarcinoma tissues.Conclusions The concomitant expression ofαvβ6 and TGF-β1 is likely to play an important role in the progression of pancreatic carcinoma. High expression of avP6 is an independent unfavorable prognostic indicator in pancreatic carcinoma tissues.Significance We revealed the expression status of integrinαvβ6 and TGF-β1 and their correlation in pancreatic carcinoma tissues, compared the results with various clinicopathologic parameters and patients'survival. High expression ofαvβ6 was defined as an independent unfavorable prognostic indicator in pancreatic carcinoma for the first time. These findings might have potential value for the judgment of patient's prognosis and targeted therapy directed against avP6.PartⅡThe effects of integrinαvβ6 on the proliferation, invasion and metastasis of pancreatic carcinoma cellsObjective To study the important roles of integrinαvβ6 on the proliferation, invasion and metastasis of pancreatic carcinoma cells.Methods we examined the expression status of avP6 in three pancreatic adenocarcinoma cell lines by RT-PCR and Flow cytometry. PANC-1 and Capan-2 cells were selected to determine the effects of function-blocking mAb againstαvβ6 (10D5) on the cellular malignant behavior. MTT assays were performed to test the changes of cell proliferation. Transwell invasion assays were performed to test the changes of invasive capability. To determine whether blockingαvβ6 function would affect MMP-9 secretion, we performed gelatin zymography and Western blotting analysis. The function of integrinαvβ6 was analyzed via comparing the differences between the data of treated cells and control cells.Results High expression of avP6 at both mRNA and protein levels was observed in PANC-1 cells. Capan-2 cells showed relatively high expression ofαvβ6, while CFPAC-1 cells showed lowαvβ6 expression. Therefore, PANC-1 and Capan-2 cells were selected for subsequent applications. After incubation with 10D5 for 24h, the proliferation rate of PANC-1 and Capan-2 cells decreased to (61.94±3.62)%and (67.35±5.02)%respectively compared to the control cells. The number of invasive PANC-1 and Capan-2 cells dramatically decreased after blockingαvβ6 function with 10D5 (71±9/HP versus 19±5/HP, 36±6/HP versus 13±4/HP, respectively). Furthermore, pretreatment with anti-MMP-9 antibody Ab-1 greatly reduced the number of invasive cells in both cell lines (20±4/HP, 12±5/HP, respectively) at similar levels in the absence and presence of 10D5 or control IgG2a, indicating that the invasion ability of pancreatic carcinoma cells through reconstituted basement membrane was mediated through integrinαvβ6, and the MMP-9 activity is essential for cell invasion. The results of gelatin zymography and Western blotting showed that after suppressingαvβ6 function with 10D5, the MMP-9 secretion levels in PANC-1 and Capan-2 cells decreased to (9.28±3.11)%and (6.35±2.46)%respectively when compared to those of control cells.Conclusions Integrinαvβ6 plays important roles in promoting the proliferation, invasion, and MMP-9 secretion of pancreatic carcinoma cells. Blocking the function ofαvβ6 could inhibit cellular proliferation, invasive, and metastatic capabilities. Significance We tested the expression status ofαvβ6 in three pancreatic carcinoma cell lines, revealed the important roles of avP6 on the proliferation, invasion and metastasis of pancreatic carcinoma cells, and provided the exact experimental evidence for the targeted therapy againstαvβ6 for pancreatic carcinoma.PartⅢThe effects of integrin avP6 on cell apoptosis and chemosensitivity of pancreatic carcinoma cellsObjective To study the important roles of integrin avP6 on cell apoptosis and chemosensitivity to gemcitabine in pancreatic carcinoma cells.Methods We designed and constructed three small interfering RNA (siRNA) against the different positions ofαvβ6 open reading frame. After testing for the most effective targeting sequence, theαvβ6 siRNA was transfected into PANC-1 pancreatic carcinoma cells using the Lipofectamine TM 2000 reagent. A scrambled version ofαvβ6 siRNA was used as a control siRNA. RT-PCR and Western blotting analyses were performed to determine the silencing effect ofαvβ6 expression. MTT assays were performed to detect the changes of cell proliferation. The effects ofαvβ6 siRNA transfection on cell cycle distribution and cell apoptosis were determined by flow cytometry. To quantify apoptosis, cells were stained with an Annexin V-FITC apoptosis assay kit. Gemcitabine was used as chemotherapeutics, and we detected the changes of chemosensitivity and gemcitabine-induced apoptosis after silencingαvβ6 expression by siRNA. Caspase 3 activity was measured using a fluorometric protease assay kit. The effects of avP6 on tumorigenicity and tumor growth in vivo were assessed by subcutaneous implantation of avP6 siRNA and control siRNA transfected PANC-1 cells in athymic nude mice.Results 48 hours following siRNA transfection, the expression ofαvβ6 in PANC-1 cells was markedly suppressed at both mRNA and protein levels byαvβ6 siRNA but not control siRNA, and inhibitive effect persisted 96h after transfection. Silencing of avP6 expression inhibited the cell proliferation in a time-dependent manner. At 96h post-transfection,41%ofαvβ6 siRNA-transfected cells were arrested in G2/M phase, and the apoptotic rate of avP6 siRNA-treated cells was increased to 13.5±2.3%when compared with control siRNA-treated cells. Gemcitabine treatment could induce the apoptosis of PANC-1 cells. Silencing avP6 expression with siRNA significantly enhanced gemcitabine sensitivity and increased gemcitabine-induced caspase-mediated apoptosis. Tumorigenicity assay showed that the average time for the macroscopic tumor formed from theαvβ6 siRNA-transfected cells was about (15±4.6) days, while it was about (9±2.7) days from the control siRNA-transfected cells. At 6 weeks after implantation, the average volume of tumor formed from the avP6 siRNA-transfected cells was significantly decreased compared with that formed from control siRNA transfected cells (170±32mm3 vs 760±48mm3, P<0.01).Conclusions Integrin avP6 plays important roles in inhibiting cell apoptosis and enhancing chemoresistance to gemcitabine in pancreatic carcinoma cells. Silencing ofαvβ6 expression by siRNA significantly inhibited cell growth in vitro and in vivo, caused cell cycle arrest at G2/M phase, induced cell apoptosis, and enhanced gemcitabine sensitivity. Integrinαvβ6 is a potential therapeutic target for highly-resistant pancreatic carcinoma and has promising perspective for clinical application.Significance The siRNA against integrin avP6 was successfully constructed and transfected into PANC-1 pancreatic carcinoma cells for the first time. Silencing ofαvβ6 expression by siRNA significantly inhibited cell growth in vitro and in vivo, resulted in cell cycle G2/M arrest, induced cell apoptosis, and enhanced chemosensitivity to gemcitabine. These findings might provide a new approach for the gene therapy directed against integrinαvβ6 for pancreatic carcinoma.