Normal Human Pancreatic Ductal Cells Research Articles

Necroptosis-Related Prognostic Model for Pancreatic Carcinoma Reveals Its Invasion and Metastasis Potential through Hybrid EMT and Immune Escape.

Necroptosis, pro-inflammatory programmed necrosis, has been reported to exert momentous roles in pancreatic cancer (PC). Herein, the objective of this study is to construct a necroptosis-related prognostic model for detecting pancreatic cancer. In this study, the intersection between necroptosis-related genes and differentially expressed genes (DEGs) of pancreatic ductal adenocarcinoma (PDAC) was obtained based on GeneCards database, GEO database (GSE28735 and GSE15471), and verified using The Cancer Genome Atlas (TCGA). Next, a prognostic model with Cox and LASSO regression analysis, and divided the patients into high-risk and low-risk groups. Subsequently, the Kaplan-Meier (KM) survival curve and the receiver operating characteristic (ROC) curves were generated to assess the predictive ability of overall survival (OS) of PC patients. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to predict the potential biofunction and possible mechanical pathways. The EMTome database and an immune analysis were applied to further explore underlying mechanism. Finally, clinical samples of PDAC patients were utilized to verify the expression of model genes via immunohistochemistry (IHC), and the normal human pancreatic ductal cell line, hTERT-HPNE as well as human pancreatic ductal carcinoma cell lines, PANC-1 and PL45, were used to identify the levels of model genes by Western blot (WB) and immunofluorescence (IF) in vitro. The results showed that 13 necroptosis-related DEGs (NRDEGs) were screened based on GEO database, and finally four of five prognostic genes, including KRT7, KRT19, IGF2BP3, CXCL5, were further identified by TCGA to successfully construct a prognostic model. Univariate and multivariate Cox analysis ultimately confirmed that this prognostic model has independent prognostic significance, KM curve suggested that the OS of low-risk group was longer than high-risk group, and the area under receiver (AUC) of ROC for 1, 3, 5 years was 0.733, 0.749 and 0.667, respectively. A GO analysis illustrated that model genes may participate in cell-cell junction, cadherin binding, cell adhesion molecule binding, and neutrophil migration and chemotaxis, while KEGG showed involvement in PI3K-Akt signaling pathway, ECMreceptor interaction, IL-17 signaling pathway, TNF signaling pathway, etc. Moreover, our results showed KRT7 and KRT19 were closely related to EMT markers, and EMTome database manifested that KRT7 and KRT19 are highly expressed in both primary and metastatic pancreatic cancer, declaring that model genes promoted invasion and metastasis potential through EMT. In addition, four model genes were positively correlated with Th2, which has been reported to take part in promoting immune escape, while model genes except CXCL5 were negatively correlated with TFH cells, indicating that model genes may participate in immunity. Additionally, IHC results showed that model genes were higher expressed in PC tissues than that in adjacent tumor tissues, and WB and IF also suggested that model genes were more highly expressed in PANC-1 and PL45 than in hTERT-HPNE. Tracing of a necroptosis-related prognostic model for pancreatic carcinoma reveals its invasion and metastasis potential through EMT and immunity. The construction of this model and the possible mechanism of necroptosis in PDAC was preliminarily explored to provide reliable new biomarkers for the early diagnosis, treatment, and prognosis for pancreatic cancer patients.

Islet Neogenesis Associated Protein (INGAP) induces the differentiation of an adult human pancreatic ductal cell line into insulin-expressing cells through stepwise activation of key transcription factors for embryonic beta cell development

Regeneration of β-cells in diabetic patients is an important goal of diabetes research. Islet Neogenesis Associated Protein (INGAP) was discovered in the partially duct-obstructed hamster pancreas. Its bioactive fragment, pentadecapeptide 104–118 (INGAP-P), has been shown to reverse diabetes in animal models and to improve glucose homeostasis in patients with diabetes in clinical trials. Further development of INGAP as a therapy for diabetes requires identification of target cells in the pancreas and characterization of the mechanisms of action. We hypothesized that adult human pancreatic ductal cells retain morphogenetic plasticity and can be induced by INGAP to undergo endocrine differentiation. To test this hypothesis, we treated the normal human pancreatic ductal cell line (HPDE) with either INGAP-P or full-length recombinant protein (rINGAP) for short-term periods. Our data show that this single drug treatment induces both proliferation and transdifferentiation of HPDE cells, the latter being characterized by the rapid sequential activation of endocrine developmental transcription factors Pdx-1, Ngn3, NeuroD, IA-1, and MafA and subsequently the expression of insulin at both the mRNA and the protein levels. After 7 days, C-peptide was detected in the supernatant of INGAP-treated cells, reflecting their ability to secrete insulin. The magnitude of differentiation was enhanced by embedding the cells in Matrigel, which led to islet-like cluster formation. The islet-like clusters cells stained positive for nuclear Pdx-1 and Glut 2 proteins, and were expressing Insulin mRNA.These new data suggest that human adult pancreatic ductal cells retain morphogenetic plasticity and demonstrate that a short exposure to INGAP triggers their differentiation into insulin-expressing cells in vitro. In the context of the urgent search for a regenerative and/or cellular therapy for diabetes, these results make INGAP a promising therapeutic candidate.

Elevation of MMP-9 and IDO induced by pancreatic cancer cells mediates natural killer cell dysfunction.

BackgroundNatural killer (NK) cells play a key role in non-specific immune response in different cancers, including pancreatic cancer. However the anti-tumor effect of NK cells decreases during pancreatic cancer progression. The regulatory pathways by which NK cells facilitate tumor immune escape are unclear, therefore our purpose was to investigate the roles of the contributory factors.MethodsNK cells isolated from fresh healthy peripheral blood were co-cultured with normal human pancreatic ductal cells hTERT-HPNE and human pancreatic cancer cell lines SW1990 and BxPc-3 in vitro. Then NK cell function was determined by Flow cytometric analysis of surface receptors and cytotoxic granules in NK cells, NK cell apoptosis and cytotoxicity, and Enzyme-linked immunosorbent assay of cytokines. Expression level of MMP-9, IDO and COX-2 in hTERT-HPNE and SW1990 cells were detected by quantitative RT-PCR. Statistical differences between data groups were determined by independent t-tests using SPSS 19.0 software.ResultsOur results showed that NK cell function was significantly downregulated following exposure to pancreatic cancer cells compared to normal pancreatic cells, as demonstrated by lower expressions of activating surface receptors (NKG2D, DNAM-1, NKp30 and NKp46) and cytotoxic granules (Perforin and Granzyme B); decreased secretion of cytokines (TNF-α and IFN-γ); and reduced cytotoxicity against myelogenous leukemia K562 cells. Further investigations revealed that MMP-9 and IDO may be implicated in SW1990 cell-induced NK cell dysfunction by facilitating tumor immune evasion. Blockade by TIMP-1 and/or 1-MT could partially restore NK function.ConclusionsTaken together, elevation of MMP-9 and IDO induced by pancreatic cancer cells mediates NK cell dysfunction. Our findings could contribute to the development of NK cell-based immunotherapy in patients with pancreatic cancer.

Abstract 5207: Targeting prion protein as a potential oncogene in pancreatic cancer.

Abstract Background: Human prion protein (PrP) has been found to be up-regulated in gastric, breast, pancreatic cancers and its expression in cancer cells contributes to cancer progression and resistance to various cancer therapies. Current literature suggests that overexpression of PrP induces inherent resistance to cancer therapeutics, while repression of PrP induces sensitization to cancer therapeutics. Our previous studies using expression microarray have revealed that PrP is not detected in normal human pancreatic ductal cells but is expressed in human pancreatic ductal adenocarcinoma (PDAC) cell lines and 40% of pancreatic cancer specimens. Further, expression of PrP confers a worse prognosis in PDAC patients. The poor prognosis of pancreatic cancer demonstrates needs for better target and drug development. Our recent studies also found that PrP is universally expressed in fetal pancreatic precursor ductal cells, and co-expresses with pancreatic stem cell markers, such as Pdx1 and Notch1, suggesting that PrP is a marker of pancreatic stem cells and may cross-talk with Notch1 during development and pancreatic tumorigenesis. However, the role for PrP in the establishment or progression of cancer is unknown, and whether PrP cross-talks with oncogenes such as Notch in these processes need to be elucidated. Methods: We applied immunoprecipitation (Abcam, Anti-Prion protein antibody 8H4) and western blotting to investigate the binding partners for PrP. Knockdown and over-expression of PRNP gene were carried out on various pancreatic cell lines, followed by in vitro proliferation assay and apoptotic activity analysis of these cells. Results: Pancreatic cancer cell lines express different levels of PrP, e.g. BxPC3 and Pan02.03 have higher PrP expression when compared to Capan-1. Western blot analysis revealed that PrP can bind with Notch1 protein in both BxPC-3 and CAPAN-1 cell lines. Moreover, down-regulation of PrP led to decreased cell proliferation and accelerated apoptosis, accompanied by down-regulation of cleaved/activated Notch1 (ICN1) and Notch2 (ICN2) expression in BxPC3 cells, but had lesser effect on CAPAN-1 cells. In comparison, over-expression of PrP significantly increased the proliferation rate of CAPAN-1 cells and dramatically upregulated the expression of ICN1. In conjunction with our previous findings that PrP exists as a pro-prion that binds filamin A in pancreatic cancer cells, these findings suggest that PrP may mediate pancreatic cancer cell progression through regulation of Notch activity and other cell mechanic and signaling integrators. This hypothesis is being further tested by xenograft analysis. Elucidating the mechanisms for PrP mediated Notch activation in PDAC will further our long-term goal of understanding the role of oncogenic PrP in conferring pancreatic cancer aggressiveness, and potentially will lead to novel therapeutic options for treatment of pancreatic cancer. Citation Format: Yiwei Wang, Lan Zhou, Wei Xin. Targeting prion protein as a potential oncogene in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5207. doi:10.1158/1538-7445.AM2013-5207

PH Regulatory Transporters in Pancreatic Ductal Adenocarcinoma (PDAC)

PDAC is among the most aggressive human malignacies, with a 5‐ year survival rate of 1–4%. Elucidation of specific molecular alterations in PDAC is urgently needed to devise new treatment strategies. Malignant tumors with rapid progression such as PDAC often undergo extensive metabolic changes resulting in increased acid production. We and others have shown in other cancers that this leads to the upregulation of acid extruding transporters, which in turn favors further cancer progression. The aim of our studies is to elucidate the regulation and roles of pH regulatory ion transporters in PDAC. We used qPCR to screen a panel of 4 human PDAC cell lines (AsPC‐1, BxPC‐3, Panc‐1, MIAPaCa‐2) against a normal human pancreatic ductal cell line (HPDE). The Na+/H+ exchanger 2 (NHE2) was upregulated in 3 of 4 cell lines, whereas NHE1 was upregulated only in BxPC3 cells; the V‐type H+‐ATPase subunit a3 was upregulated in all PDAC cell lines; all cell lines except BxPC3 exhibited upregulation of either AE2 or AE3 Cl−/HCO3−exchangers, and also the Na+‐HCO3− transporters SLC4A7 and –A8 tended to be upregulated in PDAC; surprisingly, the H+/lactate cotransporter MCT1 was downregulated in all PDAC cell lines and a similar trend was seen for MCT4. In conclusion, PDAC cell lines exhibit dramatic changes in the mRNA expression level of pH‐regulatory transporters. Ongoing experiments are addressing the functional relevance of this in PDAC.Funding: Marie Curie Actions (ITN IonTraC)

Prosurvival role of heat shock factor 1 in the pathogenesis of pancreatobiliary tumors.

Several mechanisms have evolved to ensure the survival of cells under adverse conditions. The heat shock response is one such evolutionarily conserved survival mechanism. Heat shock factor-1 (HSF1) is a transcriptional regulator of the heat shock response. By the very nature of its prosurvival function, HSF1 may contribute to the pathogenesis of cancer. The current study investigates the role of HSF1 in the pathogenesis of pancreatobiliary tumors. HSF1 was downregulated in pancreatic cancer (MIA PaCa-2 and S2-013) and cholangiocarcinoma (KMBC and KMCH) cell lines by HSF1-specific small interfering RNA (siRNA). Nonsilencing siRNA was used as control. The effect of HSF1 downregulation on viability and apoptosis parameters, i.e., annexin V, terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL), and caspase-3, was measured. To evaluate the cancer-specific effects of HSF1, the effect of HSF1 downregulation on normal human pancreatic ductal cells was also evaluated. HSF1 is abundantly expressed in human pancreatobiliary cancer cell lines, as well as in pancreatic cancer tissue, as demonstrated by Western blot and immunohistochemistry, respectively. Inhibition of HSF1 expression by the HSF1 siRNA sequences leads to time-dependent death in pancreatic and cholangiocarcinoma cell lines. Downregulation of HSF1 expression induces annexin V and TUNEL positivity and caspase-3 activation, suggesting activation of a caspase-dependent apoptotic pathway. Although caspase-3 inhibition protects against cell death induced by HSF1 expression, it does not completely prevent it, suggesting a role for caspase-independent cell death. HSF1 plays a prosurvival role in the pathogenesis of pancreatobiliary tumors. Modulation of HSF1 activity could therefore emerge as a novel therapeutic strategy for cancer treatment.

MicroRNA miR-17-5p is overexpressed in pancreatic cancer, associated with a poor prognosis, and involved in cancer cell proliferation and invasion

The microRNA-17-92 cluster is an oncogene in human B cell lymphomas and lung cancers. Previous microRNA microarray data revealed that miR-17-5p, a member of the miR-17-92 cluster, is upregulated in pancreatic cancer. However, the involvement of miR-17-5p expression in pancreatic carcinogenesis has not well been studied. In the present study, we measured the miR-17-5p expression levels in pancreatic cancer cell lines, primary cultures of normal human pancreatic ductal cells, formalin-fixed paraffin-embedded (FFPE) tissue samples derived from 80 patients who underwent pancreatectomy for pancreatic cancer and microdissected cells (including normal ductal epithelial, pancreatic intraepithelial neoplasia-1B and invasive ductal carcinoma cells) by qRT-PCR. Furthermore, we investigated the effects of upregulation of miR-17-5p expression on the proliferation and invasion of pancreatic cancer cells. We found that pancreatic cancer cells expressed higher levels of miR-17-5p than primary cultured normal ductal cells. miR-17-5p was also overexpressed in pancreatic cancer in FFPE and microdissected samples. Furthermore, analysis of macrodissected FFPE samples revealed that high miR-17-5p expression was associated with a poor prognosis (p = 0.03). In addition, in vitro experiments revealed that SUIT-2 and KP-2 pancreatic cancer cells transfected with the miR-17-5p precursor showed significantly higher cell growth ratios than the corresponding control cells (p < 0.001 and p = 0.012, respectively), as well as significantly higher numbers of invading cells (p < 0.0001 for both). The present findings suggest that miR-17-5p plays important roles in pancreatic carcinogenesis and cancer progression, and is associated with a poor prognosis in pancreatic cancer.

Evidence for a membrane carbonic anhydrase IV anchored by its C-terminal peptide in normal human pancreatic ductal cells.

The high concentration of HCO(3)(-) ions (150 mM) in the human pancreatic ducts raises the question of the membrane proteins responsible for their secretion in addition to the Cl(-)/HCO(3)(-) exchanger. In this study, we investigated the expression of carbonic anhydrase IV (CA IV), a possible candidate. Experiments were carried out on specimens of normal human pancreas obtained from brain-dead donors ( n=9) as well as on isolated human ductal cells. Two antibodies were generated: CA IV NH(2) antibody directed against the NH(2) terminal of human glycosyl phosphatidylinositol (GPI)-anchored CA IV and CA IV COOH antibody directed against the COOH terminal of the same protein before its association with a GPI in the rough endoplasmic reticulum. A 35-kDa CA IV was detected in the homogenates of human pancreas. Immunocytochemistry demonstrated the expression of CA IV in centroacinar cells and in intercalated, intralobular, and interlobular ductal cells. The immunoreactivity observed with the CA IV COOH antibody was mainly localized on luminal membranes of ductal cells. Treatment of purified plasma membranes with phosphatidylinositol-phospholipase C indicated that the CA IV expressed in pancreatic ducts was not GPI-anchored. Its detection in the same extracts by the CA IV COOH antibody indicated that it was anchored by a hydrophobic segment at the carboxy terminal. Taken together, these results suggest that normal human pancreatic ductal cells express a 35-kDa CA IV anchored in their luminal plasma membrane by a hydrophobic segment of the COOH terminus. In view of its localization and its mode of anchorage in luminal plasma membranes, this CA IV may participate in the maintenance of luminal pH.

Lipoxygenase Inhibitors Abolish Proliferation of Human Pancreatic Cancer Cells

Epidemiologic and animal studies have linked pancreatic cancer growth with fat intake, especially unsaturated fats. Arachidonic acid release from membrane phospholipids is essential for tumor cell proliferation. Lipoxygenases (LOX) constitute one pathway for arachidonate metabolism, but their role in pancreatic cancer growth is unknown. The expression of 5-LOX and 12-LOX as well as their effects on cell proliferation was investigated in four human pancreatic cancer cell lines (PANC-1, MiaPaca2, Capan2, and ASPC-1). Expression of 5-LOX and 12-LOX mRNA was measured by nested RT-PCR. Effects of LOX inhibitors and specific LOX antisense oligonucleotides on pancreatic cancer cell proliferation were measured by 3H-thymidine incorporation. Our results showed that (1) 5-LOX and 12-LOX were expressed in all pancreatic cancer cell lines tested, while they were not detectable in normal human pancreatic ductal cells; (2) both LOX inhibitors and LOX antisense markedly inhibited cell proliferation in a concentration-dependent and time-dependent manner; (3) the 5-LOX and 12-LOX metabolites 5-HETE and 12-HETE as well as arachidonic and linoleic acids directly stimulated pancreatic cancer cell proliferation; (4) LOX inhibitor-induced growth inhibition was reversed by 5-HETE and 12-HETE. The current studies indicate that both 5-LOX and 12-LOX expression is upregulated in human pancreatic cancer cells and LOX plays a critical role in pancreatic cancer cell proliferation. LOX inhibitors may be valuable for the treatment of pancreatic cancer.

The expression of carbonic anhydrases II and IV in the human pancreatic cancer cell line (Capan 1) is associated with bicarbonate ion channels

Human pancreatic ductal cells of the Capan 1 cell line differentiate progressively during growth. After the exponential growth phase, the cells elongate and become polarized with their apical poles covered by microvilli and separated from the basolateral pole by tight junctions. In this stationary phase, they form domes, which are thought to result from the exchange of water and electrolytes. In this study, we demonstrated, using patch-clamp techniques, that HCO3- ions exit via the g350 high conductance anionic channel we observed recently at the Capan 1 cell surface. This g350 channel was thought to be either a Cl-/HCO3- antiport or a simple HCO3- channel. The stilbene derivatives 4-acetamido-4 isothiocyano-2-2'-disulfonic acid (SITS) and 4,4' diisothiocyano stilbene-2,2' disulfonic acid (DIDS) reduced both the number of domes and the Cl- and HCO3- flux through the g350 channel. Moreover, using histochemical, immunocytochemical and biochemical methods we showed that Capan 1 cells express a specific pattern of carbonic anhydrases (CA). Two types of CA were detected: the CA II isozyme mainly localized in the cytoplasm, but also found beneath the inner leaflet of the apical plasma membrane, and the CA IV isozyme localized on the outer leaflet of the apical plasma membrane and microvilli. Their molecular masses were 30 (CA II) and 55 kDa (CA IV), respectively. They were expressed continuously during the exponential growth phase, although their activity increased greatly during the stationary phase. Inhibition of dome formation by acetazolamide indicated the existence of a direct relationship between dome formation and CA. Characteristic structures with a central electron-dense core surrounded by a light halo were observed on the surface of cell membrane using histochemical and immunocytochemical methods. These structures were thought to represent a channel, corresponding possibly to CA IV. Our observations suggest that Capan 1 cells, despite their neoplasic transformation, produce HCO3- ions in the same way as normal human pancreatic ductal cells. Capan 1 cells in culture may therefore represent a suitable model for studying pancreatic duct HCO3- secretion at the cellular and molecular levels.