Human Pancreatic Ductal Epithelial Cells Research Articles

Plectin-1 Targeted AAV Vector for the Molecular Imaging of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is highly malignant disease that is the fourth leading cause of cancer-related death in the US. Gene therapy using AAV vectors to selectively deliver genes to PDAC cells is an attractive treatment option for pancreatic cancer. However, most AAV serotypes display a broad spectrum of tissue tropism and none of the existing serotypes specifically target PDAC cells. This study tests the hypothesis that AAV2 can be genetically re-engineered to specifically target PDAC cells by modifying the capsid surface to display a peptide that has previously been shown to bind plectin-1. Toward this end, a Plectin-1 Targeting Peptide (PTP) was inserted into the loop IV region of the AAV2 capsid, and the resulting capsid (AAV-PTP) was used in a series of in vitro and in vivo experiments. In vitro, AAV-PTP was found to target all five human PDAC cell lines tested (PANC-1, MIA PaCa-2, HPAC, MPanc-96, and BxPC-3) preferentially over two non-neoplastic human pancreatic cell lines (human pancreatic ductal epithelial and human pancreatic stellate cells). In vivo, mice bearing subcutaneous tumor xenografts were generated using the PANC-1 cell line. Once tumors reached a size of ∼1–2 mm in diameter, the mice were injected intravenously with luciferase reporter vectors packaged in the either AAV-PTP or wild type AAV2 capsids. Luciferase expression was then monitored by bioluminescence imaging on days 3, 7, and 14 after vector injection. The results indicate that the AAV-PTP capsid displays a 37-fold preference for PANC-1 tumor xenographs over liver and other tissues; whereas the wild type AAV2 capsid displays a complementary preference for liver over tumors and other tissues. Together, these results establish proof-of-principle for the ability of PTP-modified AAV capsids to selectively target gene delivery to PDAC cells in vivo, which opens promising new avenues for the early detection, diagnosis, and treatment of pancreatic cancer.

Class I and Class II Histone Deacetylases Are Potential Therapeutic Targets for Treating Pancreatic Cancer

BackgroundPancreatic cancer is a highly malignant disease with an extremely poor prognosis. Histone deacetylase inhibitors (HDACIs) have shown promising antitumor activities against preclinical models of pancreatic cancer, either alone or in combination with chemotherapeutic agents. In this study, we sought to identify clinically relevant histone deacetylases (HDACs) to guide the selection of HDAC inhibitors (HDACIs) tailored to the treatment of pancreatic cancer.MethodologyHDAC expression in seven pancreatic cancer cell lines and normal human pancreatic ductal epithelial cells was determined by Western blotting. Antitumor interactions between class I- and class II-selective HDACIs were determined by MTT assays and standard isobologram/CompuSyn software analyses. The effects of HDACIs on cell death, apoptosis and cell cycle progression, and histone H4, alpha-tubulin, p21, and γH2AX levels were determined by colony formation assays, flow cytometry analysis, and Western blotting, respectively.ResultsThe majority of classes I and II HDACs were detected in the pancreatic cancer cell lines, albeit at variable levels. Treatments with MGCD0103 (a class I-selective HDACI) resulted in dose-dependent growth arrest, cell death/apoptosis, and cell cycle arrest in G2/M phase, accompanied by induction of p21 and DNA double-strand breaks (DSBs). In contrast, MC1568 (a class IIa-selective HDACI) or Tubastatin A (a HDAC6-selective inhibitor) showed minimal effects. When combined simultaneously, MC1568 significantly enhanced MGCD0103-induced growth arrest, cell death/apoptosis, and G2/M cell cycle arrest, while Tubastatin A only synergistically enhanced MGCD0103-induced growth arrest. Although MC1568 or Tubastatin A alone had no obvious effects on DNA DSBs and p21 expression, their combination with MGCD0103 resulted in cooperative induction of p21 in the cells.ConclusionOur results suggest that classes I and II HDACs are potential therapeutic targets for treating pancreatic cancer. Accordingly, treating pancreatic cancer with pan-HDACIs may be more beneficial than class- or isoform-selective inhibitors.

Role of the protein kinase C signaling pathway in the regulation of claudin-4 in normal human pancreatic duct epithelial cells and cancer cells

Role of the protein kinase C signaling pathway in the regulation of claudin-4 in normal human pancreatic duct epithelial cells and cancer cells

Selective Inhibitors of Nuclear Export Block Pancreatic Cancer Cell Proliferation and Reduce Tumor Growth in Mice

Tumor-suppressor proteins are inactivated by many different mechanisms, including nuclear exclusion by chromosome region maintenance (CRM)-1. Increased tumor levels of CRM-1 have been correlated with poor prognosis of patients with pancreatic cancer, making it a therapeutic target. Selective inhibitors of nuclear export (SINEs) bind to CRM-1 to irreversibly inhibit its ability to export proteins; we investigated a new class of SINEs in pancreatic cancer cells. We studied the effects of SINE analogs in a panel of pancreatic cancer cell lines and nontransformed human pancreatic ductal epithelial cells using proliferation, apoptosis, immunoblot, co-immunoprecipitation, small inhibitor RNA, and fluorescence microscopy analyses. The effects of the SINEs also were investigated in mice with subcutaneous and orthotopic tumors. SINEs (KPT-185, KPT-127, KPT-205, and KPT-227) inhibited proliferation and promoted apoptosis of pancreatic cancer cells, but did not affect human pancreatic ductal epithelial cells. The nuclei of cells incubated with KPT-185 accumulated tumor-suppressor proteins (p27, FOXO, p73, and prostate apoptosis response-4 [PAR-4]) and inhibited interactions between CRM-1 and these proteins. Mutations in the region of CRM-1 that bind to SINEs (Cys-528), or small inhibitor RNA knockdown of PAR-4, prevented the ability of KPT-185 to block proliferation and induce apoptosis of pancreatic cancer cells. Oral administration of KPT-330 to mice reduced growth of subcutaneous and orthotopic xenograft tumors without major toxicity. Analysis of tumor remnants showed that KPT-330 disrupted the interaction between CRM-1 and PAR-4, activated PAR-4 signaling, and reduced proliferation of tumor cells. We identified SINEs that inhibit CRM-1 and promote nuclear accumulation of tumor-suppressor proteins in pancreatic cancer cells. Oral administration of the drug to mice reduces growth of xenograft tumors.

A Potential Window onto Early Pancreatic Cancer Development: Evidence of Cancer Stem Cell Growth after Exposure to Cadmium Chloride in Vitro

Cancer stem cells are a small subset of tumor cells that are postulated to underlie tumor initiation, growth, and metastasis. Research suggests that these cells may have begun as normal stem cells that underwent mutation or other changes that derail cellular programming and alter growth control pathways. Investigating a previously established link between cadmium exposure and pancreatic cancer, a new study finds that normal human pancreatic cells chronically exposed in vitro to a low level of cadmium chloride acquired cancer cell characteristics and generated what appeared to be cancer stem cells [EHP 120(9):1265–1271; Qu et al.]. Normal human pancreatic ductal epithelial cells were cultured with or without 1.0 µM of cadmium chloride for 29 weeks. The treated cells were sampled periodically and assessed for cancer cell characteristics, including increased secretion of matrix metalloproteinase-9 (MMP-9), increased invasiveness, increased anchorage-independent growth (i.e., ability to grow while floating freely in medium), and altered colony formation. Various subpopulations of pancreatic cancer stem cells have been shown to overexpress the genes CD44, CXCR4, OCT4, and S100P, which are involved in various aspects of cancer growth and spread. Samples therefore were also analyzed for expression of these genes and their associated proteins. The results showed that, compared with control cells, those cells chronically exposed to cadmium (which the authors dubbed CCE cells) demonstrated characteristics typical of cancer cells. Spheres derived from CCE cells (bottom) rapidly outpaced those derived from control cells (top). Stem cells and cancer stem cells, when studied in vitro, often form free-floating spheres of cells for reasons not completely understood. The investigators isolated and further cultured spheres generated by control and CCE cells, then submitted them to the same analyses as earlier samples. Spheres derived from CCE cell cultures exhibited cancer cell characteristics and were more numerous and larger than control spheres. They also exhibited increased transcription and expression of OCT4, CD44, and CXCR4. In gel cultures CCE spheres grew aggressively and formed more intricate structures than control spheres; microscopically, these structures appeared malignant, with poorly differentiated cells of many shapes and sizes. The study shows that in vitro exposure of normal human pancreatic cells to a nontoxic level of cadmium chloride can promote the development of cancer characteristics, thus strengthening the evidence for cadmium as a potential cause of pancreatic cancer in humans. Additionally, the study may help investigators define the very early stages of pancreatic cancer, a major advantage for a disease that typically is diagnosed only after it has metastasized.

P2Y Receptors Regulate Proliferation of Human Pancreatic Duct Epithelial Cells

The aim of this study was to investigate the effect of P2Y receptor activation on proliferation of human pancreatic duct epithelial cells. Proliferation was measured by immunoassay for bromodeoxyuridine incorporation into a pancreatic duct epithelial cell line, PANC-1. Expression of P2Y receptors was examined using quantitative reverse transcription-polymerase chain reaction and Western blot. Extracellular nucleotides, adenosine diphosphate (ADP) and uridine diphosphate (UDP), stimulated proliferation of pancreatic duct cells in a concentration-dependent manner. The nucleotide efficacy order was ADP > UDP > uridine triphosphate (UTP) > adenosine triphosphate. P2Y(1) and P2Y(6) receptor blockers, MRS2500 and MRS2578, blocked the effect of ADP and UDP. The signal that transmitted the proliferative activity of ADP and UDP was transducted to phospholipase C, inositol 1,4,5-triphosphate receptor, and protein kinase C. These results indicate involvement of P2Y(1) and P2Y(6) receptors in ADP- and UDP-stimulated proliferation. Pancreatic duct cells expressed the messenger RNA transcripts of P2Y receptors, P2Y(1) , P2Y(2), and P2Y(6), and P2Y(1) and P2Y(6) receptor protein. Extracellular nucleotides increase proliferation of human pancreatic duct epithelial cells by activation of P2Y(1) and P2Y(6) receptors. This provides the basic model for the effect of P2Y receptors on the proliferation of pancreatic duct epithelial cells.

Regulation of tight junctions in human normal pancreatic duct epithelial cells and cancer cells

To investigate the regulation of tight junction molecules in normal human pancreatic duct epithelial (HPDE) cells and pancreatic cancer cells, we introduced the human telomerase reverse transcriptase (hTERT) gene into HPDE cells in primary culture and compared them to pancreatic cancer cell lines. The hTERT-transfected HPDE cells were positive for PDE markers and expressed claudin-1, claudin-4, claudin-7, and claudin-18, occludin, tricellulin, marvelD3, JAM-A, zonula occludens (ZO)-1, and ZO-2. The tight junction molecules, including claudin-4 and claudin-18 of normal HPDE cells, were in part regulated via a protein kinase C signal pathway by transcriptional control. In addition, claudin-18 in normal HPDE cells and pancreatic cancer cells was markedly induced by a PKC activator, and claudin-18 in pancreatic cancer cells was also modified by DNA methylation. In the marvel family of normal HPDE cells and pancreatic cancer cells, tricellulin was upregulated via a c-Jun N-terminal kinase pathway, and marvelD3 was downregulated during Snail-induced epithelial-mesenchymal transition.

Chronic Cadmium Exposure in Vitro Causes Acquisition of Multiple Tumor Cell Characteristics in Human Pancreatic Epithelial Cells

Background: Cancer may be a stem cell (SC)–based disease involving formation of cancer SCs (CSCs) potentially arising from transformation of normal SCs. Cadmium has been linked to human pancreatic cancer.Objective: We studied cadmium exposure of human pancreatic ductal epithelial (HPDE) cells and whether SCs may be targeted in this process.Methods: We chronically exposed HPDE cells to low level cadmium (1 μM) for ≤ 29 weeks. Nonadherent spheroid formation was used to indicate CSC-like cell production, and we assessed tumor cell characteristics in such spheres. Assessed tumor cell characteristics including secretion of matrix metalloproteinase-9 (MMP-9), invasion, and colony formation were fortified by evaluating expression of relevant genes by real-time reverse transcription polymerase chain reaction and by Western blot.Results: Increased MMP-9 secretion and overexpression of the pancreatic cancer marker S100P occurred in chronic (29 weeks of exposure) cadmium-exposed (CCE) cells. CCE cells also showed markedly higher colony formation and invasion, typical of cancer cells. Floating “spheres” of viable cells, known to contain an abundance of normal SCs or CSCs, form in vitro with many cell types. CCE cells produced 3-fold more spheres than control cells and were more invasive, secreted more MMP-9, and overexpressed markers for pancreatic SCs/CSCs (i.e., CXCR4, OCT4, CD44) and S100P, a marker for pancreatic cancer. CCE-derived spheres rapidly produced aggressive, highly branched, and poorly differentiated glandular-like structures in Matrigel.Conclusions: Chronic cadmium exposure produced multiple tumor cell characteristics in HPDE cells and CCE cell–derived spheres. These data support the plausibility of cadmium as a human pancreatic carcinogen.

Micro/nanoscale technologies for the development of hormone-expressing islet-like cell clusters

Insulin-expressing islet-like cell clusters derived from precursor cells have significant potential in the treatment of type-I diabetes. Given that cluster size and uniformity are known to influence islet cell behavior, the ability to effectively control these parameters could find applications in the development of anti-diabetic therapies. In this work, we combined micro and nanofabrication techniques to build a biodegradable platform capable of supporting the formation of islet-like structures from pancreatic precursors. Soft lithography and electrospinning were used to create arrays of microwells (150-500 μm diameter) structurally interfaced with a porous sheet of micro/nanoscale polyblend fibers (~0.5-10 μm in cross-sectional size), upon which human pancreatic ductal epithelial cells anchored and assembled into insulin-expressing 3D clusters. The microwells effectively regulated the spatial distribution of the cells on the platform, as well as cluster size, shape and homogeneity. Average cluster cross-sectional area (~14000-17500 μm(2)) varied in proportion to the microwell dimensions, and mean circularity values remained above 0.7 for all microwell sizes. In comparison, clustering on control surfaces (fibers without microwells or tissue culture plastic) resulted in irregularly shaped/sized cell aggregates. Immunoreactivity for insulin, C-peptide and glucagon was detected on both the platform and control surfaces; however, intracellular levels of C-peptide/cell were ~60 % higher on the platform.

Su1249 Non-Oxidative Ethanol Metabolites Induce Intracellular ATP Depletion and Inhibit Pancreatic Ductal Bicarbonate Secretion in Human Pancreatic Ductal Epithelial Cell Line

Su1249 Non-Oxidative Ethanol Metabolites Induce Intracellular ATP Depletion and Inhibit Pancreatic Ductal Bicarbonate Secretion in Human Pancreatic Ductal Epithelial Cell Line

Abstract 4866: The atypical kinase SgK223 is a novel mediator of cell invasion in pancreatic cancer

Abstract Pancreatic cancer still remains one of the most aggressive and lethal of human cancers, with a lack of effective treatments contributing to its poor prognosis and high mortality. Perturbations in tyrosine kinase signaling are characteristic of many human cancers. In order to detect aberrant protein tyrosine phosphorylation events in pancreatic cancer, and to subclassify pancreatic cancer cell lines based on their tyrosine phosphorylation patterns, tryptic digests from a wide panel of pancreatic cancer cell lines were subjected to anti-phosphotyrosine immunoprecipitation, then analysed by liquid chromatography coupled with tandem mass spectrometry (MS/MS). Proteins were ranked according to differences in median phosphorylation levels across the cell line panel, and those that exhibited significantly variable phosphorylation levels were selected as candidates for further evaluation as biomarkers and therapeutic targets. One candidate identified by this approach was SgK223, a 175 kDa atypical kinase with substitutions in the DFG motif of its kinase domain. Since this is one of the conserved motifs critical for full catalytic activity, SgK223 may be catalytically inactive. Western blotting revealed that SgK223 protein expression varied across the panel, with a subset of pancreatic cancer lines exhibiting overexpression relative to normal human pancreatic duct epithelial (HPDE) cells. To further interrogate the role of SgK223 in pancreatic cancer, SgK223 was overexpressed in HPDE cells. This resulted in an elongated cell morphology and increased cellular migration and invasion in transwell assays, with no effect on cellular proliferation or Erk and Akt activity. MS/MS analysis determined that SgK223 was phosphorylated on tyrosine residues 159 and 413. Since the former is a candidate Stat3 binding site, we characterised Stat3 signalling in this model. SgK223 overexpression led to enhanced Stat3 tyrosine phosphorylation and transcriptional activity. These data identify a role for SgK223 as a mediator of cell invasion in pancreatic cancer, and support the use of MS-based phosphoproteomic profiling as an effective discovery tool for cancer-associated signaling proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4866. doi:1538-7445.AM2012-4866

Abstract LB-150: Binding selectivity of RNAs by eukaryotic initiation factor 3f in pancreatic cells

Abstract Deregulation of translation is necessary for an increase in protein production that benefits cancer cells to increase proliferation and tumor growth. Eukaryotic initiation factor 3f (eIF3f), a subunit in the eIF3 protein complex, has been shown to be a regulator for translation initiation. Our group was the first to describe eIF3f as a tumor suppressor, where we show its expression is lost in melanoma and pancreatic tumor tissues. In this study, we show that eIF3f binds to various RNAs for genes involved in GTP-binding, RNA binding, RNA capping and other cellular processes, through the use of RNA-Immunoprecipitation assay followed by a microarray analysis, coined RIP-Chip in normal human pancreatic ductal epithelial (HPDE) cells. We confirmed the binding of eIF3f to these RNAs through RNA-Immunoprecipitation (RIP) assay using an antibody against eIF3f to pulldown of our sample RNA and visualized by RT-PCR analysis. To determine the role of this selective binding of eIF3f to these particular RNAs, we observed their expression in the HPDE cells, were eIF3f was stably knocked-down with lentivirus containing eIF3f-siRNA. Preliminary results demonstrate some correlation between the absence of eIF3f and the protein levels of the genes encoding for the eIF3f pulled-down RNA. Our results demonstrate that eIF3f may be involved in directly targeting specific oncogenic RNA sequences for degradation. Further investigations will be needed to elucidate the role of these genes and eIF3f expression in the transformation of pancreatic tissues to the aggressive phenotype of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-150. doi:1538-7445.AM2012-LB-150

Abstract 2717: Reolysin: A novel reovirus-based agent that induces endoplasmic reticular stress in RAS-activated pancreatic cancer

Abstract Mutations in the RAS oncogene are associated with transformation and proliferation and are prevalent in many tumor types. Since RAS is frequently hyperactivated in pancreatic cancer and plays an essential role in tumor progression, it is an ideal therapeutic target. Reoviruses are naturally occurring viruses that are non-pathogenic and have been reported to specifically replicate in cancer cells with an activated RAS pathway, but not in normal tissue. To take advantage of this observation therapeutically, the reovirus-based anticancer agent Reolysin was recently developed. Reolysin has already progressed into clinical trials, but its mechanisms of action have not been fully elucidated. We hypothesized that Reolysin exposure would result in the production of viral products, stimulate endoplasmic reticular (ER) stress, and induce apoptosis. Reolysin treatment triggered the selective replication of reoviruses as visualized by immunocytochemistry and electron microscopy in KRAS-transformed immortalized human pancreatic duct epithelial cells and in a panel of pancreatic cancer cell lines. Reovirus accumulation was associated with an increase in the expression of ER stress-related genes as assessed by microarray and quantitative RT-PCR analyses, cleavage of caspase-4, and induction of apoptosis. In support of our hypothesis that Reolysin induces ER stress-mediated apoptosis, co-treatment with tunicamycin or brefeldin A further augmented Reolysin-induced cell death. Previous studies have demonstrated that the proteasome inhibitor bortezomib causes ER stress-mediated apoptosis. Here we show that co-treatment with bortezomib and Reolysin stimulates the simultaneous accumulation of ubiquitinated and viral proteins resulting in enhanced levels of ER stress-related gene expression, caspase-4 cleavage, and apoptosis. The role of caspase-4 as a regulator of therapeutic sensitivity to these agents was investigated by targeting caspase-4 expression with siRNA. Knockdown of caspase-4 blunted apoptosis stimulated by Reolysin, bortezomib, and the combination. Importantly, bortezomib also dramatically augmented the anticancer activity of Reolysin in xenograft models of pancreatic cancer in vivo. Analysis of tumor specimens from mice treated with Reolysin and bortezomib displayed reovirus accumulation, enhanced expression of GRP78/Bip, a marker of ER stress and increased levels of apoptosis. Collectively, our results demonstrate that the abnormal protein accumulation induced by Reolysin and bortezomib promotes heightened ER stress and apoptosis in pancreatic cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2717. doi:1538-7445.AM2012-2717

Constitutive K‐RasG12D Activation of ERK2 Specifically Regulates 3D Invasion of Human Pancreatic Cancer Cells via MMP‐1

Pancreatic ductal adenocarcinomas (PDAC) are highly invasive & metastatic neoplasms unresponsive to current therapies. Overwhelmingly, PDAC harbors constitutive, oncogenic mutations in K‐RasG12D that exist prior to invasion. Histologic & genetic analyses of human PDAC exhibit increased expression of ERK1/2 and pro‐invasive MMPs; indicators of poor prognosis. However, the distinct molecular mechanisms necessary for K‐Ras – ERK1/2 signaling & its influence on MMP‐directed stromal invasion in primary human pancreatic ductal epithelial cells (PDECs) has yet to be elucidated in 3D. Expression of oncogenic K‐RasG12D alone in genetically‐defined PDECs reveals increased invadopodia & epithelial‐to‐mesenchymal transition markers, but only when cultured in a 3D basement membrane (BM) model. Activation of ERK2, but not ERK1, occurs only in K‐RasG12D mutated PDECs cultured in 3D & is necessary for invasion based upon pharmacologic & shRNA inhibition. Increased invasion of K‐RasG12D PDECs through the BM is associated with a specific microarray gene signature & induction of MMPs. Specifically, MMP‐1 RNA, protein, & proteolysis are amplified in K‐RasG12D PDECs when assayed by RT‐PCR, ELISA, & FRET. Importantly, MMP‐1 silencing mimics ERK2 inhibition & disrupts vertical invasion. ERK2‐isoform & MMP‐1 targeting in 3D are shown to be viable strategies to attenuate invasion of K‐RasG12D mutated human pancreatic cancer cells. GPB is funded by an F30 DK088402‐01.

The Tumor Suppressive Role of eIF3f and Its Function in Translation Inhibition and rRNA Degradation

Deregulated translation plays an important role in human cancer. We previously reported decreased eukaryotic initiation factor 3 subunit f (eIF3f) expression in pancreatic cancer. Whether decreased eIF3f expression can transform normal epithelial cells is not known. In our current study, we found evidence that stable knockdown of eIF3f in normal human pancreatic ductal epithelial cells increased cell size, nuclear pleomorphism, cytokinesis defects, cell proliferation, clonogenicity, apoptotic resistance, migration, and formation of 3-dimensional irregular masses. Our findings support the tumor suppressive role of eIF3f in pancreatic cancer. Mechanistically, we found that eIF3f inhibited both cap-dependent and cap-independent translation. An increase in the ribosomal RNA (rRNA) level was suggested to promote the generation of cancer. The regulatory mechanism of rRNA degradation in mammals is not well understood. We demonstrated here that eIF3f promotes rRNA degradation through direct interaction with heterogeneous nuclear ribonucleoprotein (hnRNP) K. We showed that hnRNP K is required for maintaining rRNA stability: under stress conditions, eIF3f dissociates hnRNP K from rRNA, thereby preventing it from protecting rRNA from degradation. We also demonstrated that rRNA degradation occurred in non-P body, non-stress granule cytoplasmic foci that contain eIF3f. Our findings established a new mechanism of rRNA decay regulation mediated by hnRNP K/eIF3f and suggest that the tumor suppressive function of eIF3f may link to impaired rRNA degradation and translation.

Regulation of pancreatic cancer growth by superoxide.

K-ras mutations have been identified in up to 95% of pancreatic cancers, implying their critical role in the molecular pathogenesis. Expression of K-ras oncogene in an immortalized human pancreatic ductal epithelial cell line, originally derived from normal pancreas (H6c7), induced the formation of carcinoma in mice. We hypothesized that K-ras oncogene correlates with increased non-mitochondrial-generated superoxide (O 2.-), which could be involved in regulating cell growth contributing to tumor progression. In the H6c7 cell line and its derivatives, H6c7er-Kras+ (H6c7 cells expressing K-ras oncogene), and H6c7eR-KrasT (tumorigenic H6c7 cells expressing K-ras oncogene), there was an increase in hydroethidine fluorescence in cell lines that express K-ras. Western blots and activity assays for the antioxidant enzymes that detoxify O 2.- were similar in these cell lines suggesting that the increase in hydroethidine fluorescence was not due to decreased antioxidant capacity. To determine a possible non-mitochondrial source of the increased levels of O 2.-, Western analysis demonstrated the absence of NADPH oxidase-2 (NOX2) in H6c7 cells but present in the H6c7 cell lines expressing K-ras and other pancreatic cancer cell lines. Inhibition of NOX2 decreased hydroethidine fluorescence and clonogenic survival. Furthermore, in the cell lines with the K-ras oncogene, overexpression of superoxide dismutases that detoxify non-mitochondrial sources of O 2.-, and treatment with the small molecule O 2.- scavenger Tempol, also decreased hydroethidine fluorescence, inhibited clonogenic survival and inhibited growth of tumor xenografts. Thus, O 2.- produced by NOX2 in pancreatic cancer cells with K-ras, may regulate pancreatic cancer cell growth.

S100P-Binding Protein, S100PBP, Mediates Adhesion through Regulation of Cathepsin Z in Pancreatic Cancer Cells

Several S100 proteins are up-regulated in pancreatic ductal adenocarcinoma (PDAC), the most significant being S100P. We previously reported on S100PBP, a binding partner of S100P, that shows no homology to any described protein and whose functions are completely unknown. To determine S100PBP expression across human tissues and organs, immunohistochemistry was performed using both multiorgan- and in-house-constructed pancreatic tissue microarrays. To establish S100PBP functions, cell lines with either stably overexpressed or silenced S100PBP were generated and investigated using Affymetrix gene expression arrays and complementary functional assays. We show that S100PBP is differentially expressed in various healthy and tumor specimens, which is both cancer- and tissue-type dependent. In healthy pancreas, S100PBP is expressed in the nuclear/perinuclear region of both exocrine and endocrine compartments. In early precancerous lesions, S100PBP is translocated to the cytoplasm, whereas in PDAC and metastatic lesions, its expression is significantly diminished. The most pronounced phenotypic change after manipulation of S100PBP expression was seen in adhesion; this was significantly reduced after S100PBP up-regulation and increased after S100PBP silencing. Up-regulation or silencing of S100PBP also led to a concomitant change in the levels of the protease cathepsin Z, the silencing of which significantly reduced PDAC cell adhesion. We further demonstrate that the interaction of cathepsin Z with arginine-glycine-aspartic acid-binding integrins, specifically αvβ5, mediates the changes seen in adhesion of PDAC cells.

Constitutive K-RasG12D Activation of ERK2 Specifically Regulates 3D Invasion of Human Pancreatic Cancer Cells via MMP-1

Pancreatic ductal adenocarcinomas (PDAC) are highly invasive and metastatic neoplasms commonly unresponsive to current drug therapy. Overwhelmingly, PDAC harbors early constitutive, oncogenic mutations in K-Ras(G12D) that exist prior to invasion. Histologic and genetic analyses of human PDAC biopsies also exhibit increased expression of extracellular signal-regulated kinase (ERK) 1/2 and proinvasive matrix metalloproteinases (MMP), indicators of poor prognosis. However, the distinct molecular mechanisms necessary for K-Ras/ERK1/2 signaling and its influence on MMP-directed stromal invasion in primary human pancreatic ductal epithelial cells (PDEC) have yet to be elucidated in three-dimensions. Expression of oncogenic K-Ras(G12D) alone in genetically defined PDECs reveals increased invadopodia and epithelial-to-mesenchymal transition markers, but only when cultured in a three-dimensional model incorporating a basement membrane analog. Activation of ERK2, but not ERK1, also occurs only in K-Ras(G12D)-mutated PDECs cultured in three-dimensions and is a necessary intracellular signaling event for invasion based upon pharmacologic and short hairpin RNA (shRNA) inhibition. Increased active invasion of K-Ras(G12D) PDECs through the basement membrane model is associated with a specific microarray gene expression signature and induction of MMP endopeptidases. Specifically, MMP-1 RNA, its secreted protein, and its proteolytic cleavage activity are amplified in K-Ras(G12D) PDECs when assayed by real-time quantitative PCR, ELISA, and fluorescence resonance energy transfer (FRET). Importantly, shRNA silencing of MMP-1 mimics ERK2 inhibition and disrupts active, vertical PDEC invasion. ERK2 isoform and MMP-1 targeting are shown to be viable strategies to attenuate invasion of K-Ras(G12D)-mutated human pancreatic cancer cells in a three-dimensional tumor microenvironment.

Metabolic Profiling Comparison of Human Pancreatic Ductal Epithelial Cells and Three Pancreatic Cancer Cell Lines using NMR Based Metabonomics.

Metabolic profiles of hydrophilic and lipophilic cell extracts from three cancer cell lines, Miapaca-2, Panc-1 and AsPC-1, and a non-cancerous pancreatic ductal epithelial cell line, H6C7, were examined by proton nuclear magnetic resonance spectroscopy. Over twenty five hydrophilic metabolites were identified by principal component and statistical significance analyses as distinguishing the four cell types. Fifteen metabolites were identified with significantly altered concentrations in all cancer cells, e.g. absence of phosphatidylgrycerol and phosphatidylcholine, and increased phosphatidylethanolamine and cholesterols. Altered concentrations of metabolites involved in glycerophospholipid metabolism, lipopolysaccharide and fatty acid biosynthesis indicated differences in cellular membrane composition between non-cancerous and cancer cells. In addition to cancer specific metabolites, several metabolite changes were unique to each cancer cell line. Increased N-acetyl groups in AsPC-1, octanoic acids in Panc-1, and UDP species in Miapaca-2 indicated differences in cellular membrane composition between the cancer cell lines. Induced glutamine metabolism and protein synthesis in cancer cells were indicated by absence of glutamine other metabolites such as acetate, lactate, serine, branched amino acids, and succinate. Knowledge of the specifically altered metabolic pathways identified in these pancreatic cancer cell lines may be useful for identifying new therapeutic targets and studying the effects of potential new therapeutic drugs.

Abstract C235: Interleukin-6 secreted by cancer-associated fibroblasts mediates gemcitabine resistance in pancreatic cancer.

Abstract Introduction: Pancreatic ductal adenocarcinoma is characterized by a dense stroma containing an abundance of cancer-associated fibroblasts (CAFs). It is also notably resistant to gemcitabine. We hypothesize that the large population of CAFs in pancreatic tumors secrete elevated levels of multiple soluble factors, which promote survival of pancreatic cancer cells and reduce their sensitivity to gemcitabine. Previously, we found that growth of MiaPaCa-2 and BxPC-3 tumors was suppressed with gemcitabine treatment, but that of tumors from MiaPaCa-2 and BxPC-3 co-injected with primary pancreatic CAFs was not. This suggests that the presence of CAFs causes pancreatic cancer cells to become more resistant to gemcitabine. Subsequent Affymetrix analyses showed that interleukin-6 (IL-6) is one of 382 genes that are differentially upregulated by >2-fold (P<0.05) in primary pancreatic CAFs as compared to normal fibroblasts. The objectives of the current study are to validate the expression/secretion of IL-6 as well as other components of the IL-6 signaling pathway, and to determine how IL-6 affects the sensitivity of pancreatic cancer cells to gemcitabine. Methods: Sixteen primary CAF lines were established in culture from resected pancreatic ductal adenocarcinoma tumor tissues. As well, eleven primary orthotopic tumor xenograft lines were established in SCID mice from the resected tissues. Subcutaneous tumors were also grown by injecting 5×106 MiaPaCa-2, BxPC-3, or Panc-1 pancreatic cancer cells into SCID mice. Using ELISA, the levels of IL-6, soluble IL-6 receptor (sIL-6R) and the soluble form of gp130 were evaluated in the primary CAFs and orthotopic tumor xenografts, as well as in several pancreatic cancer cell lines. Levels of mouse IL-6 in the serum of mice bearing orthotopic tumors and subcutaneous cell line xenografts were also assayed. Results: The levels of human IL-6 protein were 1.1- to 79-fold higher in the CAF lines than in normal human fibroblasts. However, human IL-6 protein was not detected in pancreatic cancer cell lines PK8, MiaPaCa-2, Panc-1, Capan-2, BxPC-3, or in normal human pancreatic ductal epithelial cells (HPDEs). The levels of mouse IL-6 protein were 1.2- to 94-fold higher in the serum of mice bearing orthotopic tumors as compared to the serum of normal healthy mice. Interestingly, no mouse IL-6 protein was detected in the serum of mice bearing subcutaneous cancer cell line tumors. Furthermore, high levels of mouse IL-6 (12–100 pg/mg) but not human IL-6 were evident in the lysates of the orthotopic tumors. Human sIL-6Rprotein levels were found to be high (24–65 pg/mg) in pancreatic cancer cell lines, but present only at low levels (∼12 pg/mg) in four of the sixteen CAF lines. It was also detected at 13–66 pg/mg in all of the orthotopic tumors. Soluble gp130 protein was observed at higher levels in the CAFs than in the pancreatic cancer cell lines. It was also detected in all of the orthotopic tumors. Conclusion: Our results demonstrate that CAFs are the major producers of IL-6 in pancreatic tumors. It is likely that IL-6 secreted by CAFs binds to IL-6 receptors on pancreatic cancer cells, resulting in the activation of downstream cell survival proteins. Co-culture experiments are underway to identify these proteins and to determine if inhibition of IL-6 signaling may increase the sensitivity of pancreatic cancer cells to gemcitabine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C235.