BackgroundEpithelial-mesenchymal transition (EMT) is a dedifferentiation process that mainly involves in mesenchymal marker upregulation, epithelial maker downregulation and cell polarity loss. Related hypoxia factors play a crucial role in EMT, however, it remains few evidence to clarify the role of HIF-2α in EMT in pancreatic cancer.MethodIn this study, we investigated the expression of HIF-2α and E-cadherin by immunohistochemistry in 70 pancreatic cancer patients, as well as the correlation to the clinicopathologic characteristics. Then we regulated the expression of HIF-2α in pancreatic cancer cells to examine the role of HIF-2α on invasion and migration in vitro. Finally, we tested the relation of HIF-2α and EMT related proteins by Western blot and determined whether HIF-2α regulated EMT through Twist regulating the expression of E-cadherin by Chromatin immunoprecipitation (ChIP) assay.ResultsWe found that HIF-2α protein was expressed positively in 67.1 % (47/70) of pancreatic cancer tissues and 11.4 % (8/70) of adjacent non-tumor pancreatic tissues, and there was a significant difference in the positive rate of HIF-2α protein between two groups (χ2 = 45.549, P < 0.05). In addition, the staining for HIF-2α was correlated with tumor differentiation (P < 0.05), clinical stage (P < 0.05) and lymph node metastasis (P < 0.05), while E-cadherin expression was only correlated with lymph node metastasis (P < 0.05). HIF-2α promoted cell migration, invasion in vitro, and regulated the expression of E-cadherin and MMPs, which are critical to EMT. Our further ChIP assay suggested that only Twist2 could bind to the promoter of E-cadherin in -714 bp region site, but there is no positive binding capacity in -295 bp promoter region site of E-cadherin. Clinical tissues IHC staining showed that Twist2 and E-cadherin expression had an obviously negative correlation in pancreatic cancer. Nevertheless, it had no obvious correlation between Twist1 and E-cadherin.ConclusionThese findings indicated that HIF-2α promotes EMT in pancreatic cancer by regulating Twist2 binding to the promoter of E-cadherin, which meant that HIF-2α and this pathway may be effective therapeutic targets for pancreatic cancer.

AimWe investigated the expression of the inhibitory costimulatory molecules B7-H1, B7-H3, and B7-H4 in human pancreatic cancer to define their clinical significance and mechanism in a tumor microenvironment.Patients and methodsSixty-three pancreatic cancer tissues and 12 normal pancreatic tissues were examined in our research. Patients were enrolled in the study between December 2000 and August 2010. Expression levels of the B7 family of molecules and densities of tumor-infiltrating lymphocytes in the tissues were characterized with immunohistochemical assays.ResultsMore than 50% of the patients expressed B7-H1 and B7-H4, and nearly 100% of the patients expressed B7-H3. B7-H1 expression was correlated with tumor size, B7-H3 expression was correlated with lymph-node metastasis and differentiation grade, and B7-H4 expression was correlated with tumor size, lymph-node metastasis, and invasion depth. High B7-H4 expression was also correlated with poor survival in pancreatic cancer. We determined the value of these three B7 family molecules in the postoperative survival prognosis for patients with pancreatic cancer, and pancreatic cancer patients with less coexpression of the B7 family of molecules had a significantly higher survival rate. B7-H1 expression was found to be negatively related to the intensity of both CD3+ T cells and CD8+ T cells, and B7-H4 expression was negatively related to CD3+ T-cell infiltration intensity, but not to CD8+ T cells.ConclusionB7-H1, B7-H3, and B7-H4 are involved in pancreatic cancer progression, and their coexpression could be a valuable prognostic indicator. Negative regulation of T-cell infiltration might be the main mechanism of action of the B7 family of molecules in pancreatic cancer.

Our data indicate that Tim-3 expression on NK cells is regulated by T-bet, and that Tim-3 levels correlate with advanced stages of gastric cancer.

In numerous types of cancer, the expression of a novel member of the B7 ligand family, the B7-H3 immunoregulatory protein, has been correlated with a poor prognosis. In the present study, we investigated the role of B7-H3 in chemoresistance in pancreatic carcinoma. Silencing of B7-H3, through lentivirus-mediated delivery of stable short hairpin RNA, was observed to increase the sensitivity of the human pancreatic carcinoma cell line Patu8988 to gemcitabine as a result of enhanced drug-induced apoptosis. Overexpression of B7-H3 caused the cancer cells to be more resistant to the drug. Subsequently, we investigated the underlying mechanisms of B7-H3-mediated gemcitabine resistance, and found that the levels of survivin decreased in cells in which B7-H3 had been knocked down. In vivo animal experiments demonstrated that tumors in which B7-H3 had been knocked down displayed a slower growth rate compared with the control xenografts. Notably, gemcitabine treatment led to a strong antitumor activity in mice with tumors in which B7-H3 had been knocked down; however, this effect was only marginal in the control group. Furthermore, survivin expression was weak in gemcitabine-treated tumors in which B7-H3 had been knocked down and apoptosis was increased, as revealed by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining. In summary, the present study demonstrated that B7-H3 induces gemcitabine resistance in pancreatic carcinoma cells, at least partially by downregulating survivin expression. These results provide novel insights into the function of B7-H3 and encourage the design and investigation of approaches targeting this protein in treating pancreatic carcinoma.

Bcl-2 interacting protein 3 (BNIP3) is involved in various cellular processes and is considered a key regulator of hypoxia-induced apoptosis. In the present study, the expression of BNIP3 in pancreatic cancer tissues, the correlation with clinicopathological characteristics and prognosis and the regulation of this protein in pancreatic cancer cell lines with regard to the induction of apoptosis were investigated. BNIP3 expression was significantly lower in pancreatic cancer tissues compared with normal epithelia and was associated with tumor size, clinical stage, and lymph node metastasis. The expression of BNIP3 correlated positively to the proapoptotic protein Bax and negatively to the antiapoptotic protein Bcl-2, whereas the induction of apoptosis by BNIP3 was independent of caspase 3 and 9 activation. The restoration of BNIP3 expression in pancreatic cancer cells in vitro, caused loss of ΔΨm, increase in ROS production, and apoptosis induction. The opposite effect was observed in pancreatic cancer cells, following BNIP3 silencing by RNAi. The absence of BNIP3 expression in pancreatic cancer cells was related to gene methylation that suppressed binding of HIF-1α to the BNIP3 promoter, whereas 5-Aza-2′-deoxycytidine (Aza-dC) treatment restored BNIP3 expression and sensitized pancreatic cancer cells to BNIP3-induced apoptosis. The findings indicated that BNIP3 was significantly downregulated in pancreatic cancer resulting in reduced apoptosis induction. Silencing of BNIP3 expression was associated with methylation of the hypoxia-responsive element (HRE) site that in turn inhibited the binding of HIF-1α to the BNIP3 promoter. The data suggest that BNIP3 reactivation is a potential target for therapeutic intervention against pancreatic cancer.

Vasculogenic mimicry (VM) is a blood supply modality that occurs independently of endothelial cell angiogenesis. Hypoxia and the epithelial-mesenchymal transition (EMT) induce VM formation by remodeling the extracellular matrix. Our previous study demonstrated that hypoxia-inducible factor-2 alpha (HIF-2α) promotes the progress of EMT in pancreatic cancer; however, whether HIF-2α promotes VM formation in pancreatic cancer remains unknown. In this study, we investigated HIF-2α expression and VM by immunohistochemistry in 70 pancreatic cancer patients as well as the role of Twist1and Twist2 in HIF-2α-induced VM in vitro and in vivo. We found that the overexpression of HIF-2α and VM were correlated with poor tumor differentiation, late clinical stage and lymph node metastasis, and a poor prognosis in pancreatic cancer. Moreover, the upregulation of HIF-2α in SW1990 cells induced VM formation, whereas the opposite results were found after silencing HIF-2α in AsPC-1 cells. A mechanistic study indicated that HIF-2α might regulate the binding of twist1 to vascular endothelial cadherin (VE-cadherin) to promote VM formation in pancreatic cancer cells, and that the P1 (-421bp) and P4 (-2110bp) regions of the Twist1 binding sequences are positive regulatory elements for VE-cadherin. In addition, we confirmed that the overexpression of HIF-2α increased Twist1 expression and promoted tumor growth and VM formation in pancreatic cancer xenografts in nude mice. These findings indicated that HIF-2α might play a critical role in VM and that HIF-2α and the pathway of HIF-2α inducing VM formation are potential therapeutic targets for pancreatic cancer.

MicroRNAs (miRNAs) have emerged as important regulators in the development of pancreatic cancer and may be a valuable therapeutic application. Aberrant expression of microRNA-375 (miR-375) has been reported to be involved in development and progression in various types of cancers, but few studies have been conducted to determine its relationship with pancreatic cancer. Quantitative RT-PCR was used to detect the levels of miR-375 expression in pancreatic cancer tissue samples and cells. The cell growth rate of pancreatic cancer cells transfected with pre-miR-375 was examined by CCK8 assay. The effects of miR-375 on cell cycle and apoptosis were assessed by flow cytometry analyses. In this study, we found that the expression levels of miR-375 was significantly lower in pancreatic cancer tissues compared with nontumorous tissues. We found that miR-375 level in pancreatic cancer was associated with lymph nodes metastasis and clinical stage, and did not correlated with any other factors such as sex, age, position, tumor size, or histological grading. The CCK8 assay showed that that cells transfected with pre-miR-375 inhibited cell proliferation in Panc-1 and SW1990 cells. Flow cytometry analysis indicated that upregulation of miR-375 led to an increase in the percentage of cells in G0/G1 phase in the cell cycle distribution and induced cell apoptosis. Our research suggested that miR-375 has potential as a novel suppressor gene in pancreatic cancer and its downregulation may promote the progression of pancreatic cancer. Overexpression of miR-375 impacts cell proliferation, cell cycle distribution, and apoptosis of pancreatic cancer cells, miR-375 may play an important role in the novel therapeutic strategy for pancreatic cancer.

Abstract.Increasing evidence has demonstrated that malignant cells exhibit increased glucose uptake, which facilitates survival and growth in a hypoxic environment. The glucose transporter-1 (GLUT-1) is overexpressed in a variety of malignant tumors. However, the association between GLUT-1 expression and clinicopathological factors, 18 F-fluorodeoxyglucose uptake and tumor proliferation in pancreatic cancer has not been investigated to date. In the present study, the expression of GLUT-1 in 53 pancreatic cancer tissues was analyzed, which revealed that GLUT-1 was overexpressed in pancreatic tissue and correlated with poor prognosis and clinicopathological characteristics, including increased tumor size, clinical stage and lymph node metastasis, maximum standardized uptake value (SUV max ) and Ki-67 expression. The receiver operating characteristic curve analysis indicated that a cut-off SUV max value of 4.830 was associated with optimal sensitivity (88%) and specificity (71.4%) for the detection of strong positive GLUT-1 expression. In addition, as the expression of GLUT-1 was found to correlate with Ki-67 expression, GLUT-1 may exhibit a significant effect on cell proliferation in pancreatic cancer. Overall, these findings indicate that GLUT-1 may represent a prognostic indicator, and a potential therapeutic target for pancreatic cancer.