Pancreatic Tissue Microarray Research Articles

Abstract B004: sCD-3BP: An MCT-1-“gated” therapeutic for pancreatic adenocarcinoma

Abstract Metabolic reprogramming is a hallmark of Pancreatic Ductal Adenocarcinomas (PDAC) and offers unique opportunities for therapeutic development. TCGA datasets and pancreatic tissue microarrays reveal that nearly 30% of PDACs exhibit a marked overexpression of Monocarboxylate Transporter 1 (MCT-1), a known lactate/pyruvate transporter. In addition, several clinical studies have correlated MCT-1 expression to poor patient prognosis in other cancers and potential resistance to therapies in PDAC, making MCT-1 an intriguing therapeutic target for pancreatic cancer. In this study, we leverage 3-Bromopyruvate (3BP), a cytotoxic pyruvate analog, to target MCT-1 overexpressing PDACs and optimize it for systemic delivery. First, we establish that the sensitivity of PDAC cells to 3BP correlates to MCT-1 expression. In vitro studies with a panel of pancreatic cancer cell lines highlight that the cytotoxicity of 3BP is limited to PDAC cells with elevated MCT-1 and the mode of action of 3BP is distinctly different from AZD-3965, an MCT-1 inhibitor under clinical investigation. We demonstrate that 3BP exerts its effect on PDAC cells using MCT-1 as gate to enter a cell and then induce cytotoxicity. While 3BP is considered promising, poor serum stability, pharmacokinetics, and excessive in vivo toxicity have hampered its clinical development. We show here, a novel formulation where 3BP is entrapped inside a cyclodextrin cage (sCD-3BP) stabilizes 3BP in vivo, retains its MCT-1 specific activity, and delivers significantly more drug to the tumor with a single dose. Additionally, we show that even short exposures of sCD-3BP are adequate for induction of cell death when compared to current standard-of-care therapeutics such as gemcitabine and components of FOLFIRINOX. Finally, we test our MCT-1 gated approach in multiple in vivo models of pancreatic cancer: an orthotopic pancreatic cancer cell line model and two patient-derived orthotopic xenografts (PDoX) models originating from a primary pancreatic and a metastatic site. Arguably more clinically relevant, the PDoX models used in our study closely mimic clinical features of donor disease, causing significant metastasis to lung and liver, marked invasion into the pancreas, and development of duct-like structures within the tumor. In these models, sCD-3BP significantly reduces pancreatic cancer growth and metastatic spread. In summary, we show that intravenous administration of sCD-3BP is safe, causes no damage to normal tissue and effectively suppresses PDAC tumor growth and reduces metastatic burden. In addition, elucidating the mechanism of action of 3BP allows us to identify potential candidates for this therapy. Our study demonstrates that sCD-3BP can be a powerful therapeutic tool against MCT-1 overexpressed PDACs and present a strong rationale for future human clinical trials. Citation Format: Surojit Sur, Jordina Rincon-Toroella, Marco Dal Molin, Kathleen Gabrielson, Chetan Bettegowda, Shibin Zhou, Ken Kinzler, Bert Vogelstein. sCD-3BP: An MCT-1-“gated” therapeutic for pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B004.

KDM6A Regulates Cell Plasticity and Pancreatic Cancer Progression by Noncanonical Activin Pathway.

Background & AimsInactivating mutations of KDM6A, a histone demethylase, were frequently found in pancreatic ductal adenocarcinoma (PDAC). We investigated the role of KDM6A (lysine demethylase 6A) in PDAC development.MethodsWe performed a pancreatic tissue microarray analysis of KDM6A protein levels. We used human PDAC cell lines for KDM6A knockout and knockdown experiments. We performed bromouridine sequencing analysis to elucidate the effects of KDM6A loss on global transcription. We performed studies with Ptf1aCre; LSL-KrasG12D; Trp53R172H/+; Kdm6afl/fl or fl/Y, Ptf1aCre; Kdm6afl/fl or fl/Y, and orthotopic xenograft mice to investigate the impacts of Kdm6a deficiency on pancreatic tumorigenesis and pancreatitis.ResultsLoss of KDM6A was associated with metastasis in PDAC patients. Bromouridine sequencing analysis showed up-regulation of the epithelial–mesenchymal transition pathway in PDAC cells deficient in KDM6A. Loss of KDM6A promoted mesenchymal morphology, migration, and invasion in PDAC cells in vitro. Mechanistically, activin A and subsequent p38 activation likely mediated the role of KDM6A loss. Inhibiting either activin A or p38 reversed the effect. Pancreas-specific Kdm6a-knockout mice pancreata showed accelerated PDAC progression, developed a more aggressive undifferentiated type of PDAC, and increased metastases in the background of Kras and p53 mutations. Kdm6a-deficient pancreata in a pancreatitis model had a delayed recovery with increased PDAC precursor lesions compared with wild-type pancreata.ConclusionsLoss of KDM6A accelerates PDAC progression and metastasis, most likely by a noncanonical p38-dependent activin A pathway. KDM6A also promotes pancreatic tissue recovery from pancreatitis. Activin A might be used as a therapeutic target for KDM6A-deficient PDACs.

MicroRNA-7 as a Potential Biomarker for Prognosis in Pancreatic Cancer.

MicroRNAs play critical roles in tumor progression. Our recent study has indicated that microRNA-7 (miR-7) impairs autophagy-derived pools of glucose to suppress the glycolysis in pancreatic cancer progression. However, the roles of miR-7 in clinical significance and chemoresistance of pancreatic cancer remain unexplored. The aim of this study was to assess the expression of miR-7 in patients with pancreatic cancer and to evaluate the possibility of its usage as a prognostic molecular biomarker. MicroRNA array-based quantification analysis of 372 miRNAs was compared in serum between pancreatic cancer and healthy individuals, gemcitabine-sensitive and gemcitabine-resistance patients. We identified miR-7 showed the potential predictive power for gemcitabine-sensitive patients with pancreatic cancer. Then, the results were validated in pancreatic tissue microarray and The Cancer Genome Atlas (TCGA) dataset, demonstrating that lower miR-7 expression was correlated with more advanced tumor stages and worse prognosis in pancreatic cancer. The Cox proportional-hazards model analysis identified miR-7 to be an independent variable for prediction of the survival. Furthermore, the mechanistic exploration suggested the clinical significance of miR-7 involved its interference effect on autophagy and glycolysis in pancreatic cancer using pancreatic cancer tissue microarrays and TCGA data. Therefore, the results of the present study provide evidences that low microRNA-7 expression may contribute to tumor progression and poor prognosis in pancreatic cancer.

Protein Kinase D1, Reduced in Human Pancreatic Tumors, Increases Secretion of Small Extracellular Vesicles From Cancer Cells That Promote Metastasis to Lung in Mice

Pancreatic tumor cells release small extracellular vesicles (sEVs, exosomes) that contain lipids and proteins, RNA, and DNA molecules that might promote formation of metastases. It is not clear what cargo these vesicles contain and how they are released. Protein kinase D1 (PRKD1) inhibits cell motility and is believed to be dysregulated in pancreatic ductal adenocarcinomas. We investigated whether it regulates production of sEVs in pancreatic cancer cells and their ability to form premetastatic niches for pancreatic cancer cells in mice. We analyzed data from UALCAN and human pancreatic tissue microarrays to compare levels of PRKD1 between tumor and nontumor tissues. We studied mice with pancreas-specific disruption of Prkd1 (PRKD1KO mice), mice that express oncogenic KRAS (KC mice), and KC mice with disruption of Prkd1 (PRKD1KO-KC mice). Subcutaneous xenograft tumors were grown in NSG mice from Panc1 cells; some mice were then given injections of sEVs. Pancreata and lung tissues from mice were analyzed by histology, immunohistochemistry, and/or quantitative polymerase chain reaction; we performed nanoparticle tracking analysis of plasma sEVs. The Prkd1 gene was disrupted in Panc1 cells using CRISPR-Cas9 or knocked down with small hairpin RNAs, or PRKD1 activity was inhibited with the selective inhibitor CRT0066101. Pancreatic cancer cell lines were analyzed by gene-expression microarray, quantitative polymerase chain reaction, immunoblot, and immunofluorescence analyses. sEVs secreted by Panc1 cell lines were analyzed by flow cytometry, transmission electron microscopy, and mass spectrometry. Levels of PRKD1 were reduced in human pancreatic ductal adenocarcinoma tissues compared with nontumor tissues. PRKD1KO-KC mice developed more pancreatic intraepithelial neoplasia, at a faster rate, than KC mice, and had more lung metastases and significantly shorter average survival time. Serum from PRKD1KO-KC mice had increased levels of sEVs compared with KC mice. Pancreatic cancer cells with loss or inhibition of PRKD1 increased secretion of sEVs; loss of PRKD1 reduced phosphorylation of its substrate, cortactin, resulting in increased F-actin levels at the plasma membrane. sEVs from cells with loss or reduced expression of PRKD1 had altered content, and injection of these sEVs into mice increased metastasis of xenograft tumors to lung, compared with sEVs from pancreatic cells that expressed PRKD1. PRKD1-deficient pancreatic cancer cells showed increased loading of integrin α6β4 into sEVs-a process that required CD82. Human pancreatic ductal adenocarcinoma has reduced levels of PRKD1 compared with nontumor pancreatic tissues. Loss of PRKD1 results in reduced phosphorylation of cortactin in pancreatic cancer cell lines, resulting in increased in F-actin at the plasma membrane and increased release of sEVs, with altered content. These sEVs promote metastasis of xenograft and pancreatic tumors to lung in mice.

Bioinformatic Analysis Suggests That Three Hub Genes May Be a Vital Prognostic Biomarker in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies worldwide due to its ineffective diagnosis and poor prognosis. It is essential to identify differentially expressed genes (DEGs) in PDAC to gain new insights into its underlying molecular mechanisms, as well as identify potential diagnostic and therapeutic targets. We screened 135 DEGs from the GSE15417, GSE16515, and GSE28735 PDAC and normal pancreatic tissue microarray data sets, and identified 16 DEGs that were correlated with PDAC prognosis through the Kaplan-Meier survival analysis and log-rank tests. The Cancer Genome Atlas and Oncomine databases validated the expression levels of 16 candidate genes (SLC6A14, GPRC5A, IFI27, ERP27, SDR16C5, SIDT2, TCN1, COL12A1, MMP1, CEACAM6, DKK1, ITGA2, KRT19, PLAU, ANO1, and GABRP). Weighted gene coexpression network analysis (WGCNA) and protein and protein interaction (PPI) analysis identified three hub genes-ERP27, ITGA2, and MMP1-that are likely important in PDAC prognosis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that they were enriched in functions of extracellular matrix organization, extracellular structure organization, and positive regulation of cell migration. Taken together, we identified three pivotal genes for PDAC, which can improve our understanding of its pathogenesis, progression, and prognosis.

Abstract C28: KMT2D mediates TGF-β-induced epithelial-to-mesenchymal transition to promote more aggressive pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is predicted to be the second leading cause of cancer-related death in the United States by 2030. Surgery is the only potential curative treatment for patients with PDAC. However, less than 20% of patients are operable due to early metastasis. The mechanism of metastasis in PDAC is not fully understood. Recent studies failed to find metastasis-specific genetic mutations in PDAC, suggesting that aberrant epigenetic regulation may be one of the crucial mechanisms. Lysine-specific histone methyltransferase 2D (KMT2D) is one of the most frequently mutated epigenetic genes in human cancers, including PDAC. TCGA database analysis showed that KMT2D mutation is associated with poor survival in PDAC patients. However, the role of KMT2D in PDAC development remains elusive. Using human pancreatic tissue microarray containing both primary and metastatic PDAC tissues, we have found that the expression of KMT2D is specifically and significantly suppressed in metastatic PDAC, suggesting an important role of KMT2D in metastasis. Furthermore, treating PDAC cells with TGF-β resulted in decrease in KMT2D expression. In order to investigate the role of KMT2D in PDAC, we knocked out KMT2D (KMT2D KO) expression using CRISPR/Cas9 system in PDAC cell lines. We performed a novel Bru-seq analysis to investigate the effect of KMT2D on global nascent RNA transcript profile. Interestingly, epithelial-to-mesenchymal transition (EMT) pathway was one of the pathways that were highly upregulated upon KMT2D loss. RNA and protein assays confirmed that KMT2D KO cells had decreased epithelial marker, E-cadherin, and increased mesenchymal markers, vimentin and Snail. KMT2D KO cells also displayed enhanced ability to migrate and invade. Orthotopic xenograft mouse model using KMT2D KO cells showed less differentiated and more mesenchymal tumor cell morphology and larger primary and metastatic tumors compared to control cells. Taken together, loss of KMT2D in PDAC cells mediates TGF-β-induced EMT and enhances tumor growth and metastatic ability. These findings link epigenetic regulation to TGF-β signaling in PDAC and lay the foundation for designing novel therapeutic strategies. Citation Format: Hong S. Kim, Shuang Lu, Yubo Cao, Zhujun Yi, Sivakumar Jeyarajan, Lili Zhao, Karan Bedi, Ishwarya V. Narayanan, Michelle T. Paulsen, Mats Ljungman, Jiaqi Shi. KMT2D mediates TGF-β-induced epithelial-to-mesenchymal transition to promote more aggressive pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C28.

Abstract 89: Reduced kinase D-interacting substrate of 220kDa (Kidins220) in pancreatic cancer promotes EGFR/ERK signaling and disease progression

Abstract Kidins220 (Kinase D-Interacting Substrate, 220kDa), also known as ARMS (ankyrin repeat-rich membrane spanning) is a transmembrane scaffolding protein. Kidins220 acts as a platform for protein-protein interactions to coordinate cell plasticity, migration and neural growth receptor signalling. Present study aims to investigate the involvement of Kidins220 in the disease progression of pancreatic cancer. Pancreatic tumors (n=149) together with paired adjacent background tissues were collected at Peking University Cancer Hospital with written consent from the patients and also an ethical approval from the Peking University Cancer Hospital Research Ethics Committee. Kidins220 transcript levels were determined using real-time PCR. Relevance of Kidins220 transcript levels and pathologic/clinical features was analyzed. Knockdown of Kidins220 was carried out with a lentiviral Kidins220 shRNA. Immunohistochemical (IHC) staining of Kidins220 was performed on a pancreatic tissue microarray (TMA) (PA2081a, US Biomax, Inc., Rockville, MD, USA). A decreased expression of Kidins220 transcripts was observed in pancreatic cancer tissues in comparison with the paired background control (P=0.029). According to the TNM staging, early stage tumors (TNM 1&2) expressed higher levels of Kidins220 transcripts compared with its expression in more advanced tumors (TNM 3&4 stage, p=0.0034). In line with the finding from the Beijing pancreatic cancer cohort, a reduced expression of Kidins220 protein was seen in the pancreatic carcinomas of the TMA using IHC, p<0.001 compared with normal control. Moreover, based on the IHC results, primary tumors with distance metastases exhibited a decreased level of Kidins220 expression compared to those without metastasis. Knockdown of Kidins220 in pancreatic cancer cell lines PANC1 and MIA-PaCa-2 promoted migration and invasion of both cell lines. A protein array analysis (KinexTM) revealed an increased EGFR phosphorylation in the Kidins220 knockdown pancreatic cancer cell lines which was confirmed with Western blot. The Kidins220 knockdown-resulted increased invasion was eliminated by targeting EGFR using both gefitinib or with the irreversible pan-HER tyrosine kinase inhibitor, neratinib. Proliferation assays showed that neratinib was more effective to inhibit proliferation of PANC-1 and Mia-PaCa-2 cells at a range of concentration from 20nM to 2μM. In conclusion, the expression of Kidins220 was reduced in pancreatic cancer and the reduced expression in primary tumors was associated with distant metastases. Reduced Kidins220 expression promoted migration and invasion of pancreatic cancer cells through an upregulation of EGFR. The therapeutic potential of neratinib or gefitinib for reduced kidins220 relevant disease progression and metastasis in pancreatic cancers is yet to fully investigated. Citation Format: Shuo Cai, Ping-Hui Sun, Xiangyu Gao, Ke Ji, Xiuyun Tian, Chunyi Hao, Bilal Al-Sarireh, Paul Griffiths, Steven Hiscox, Jared Tonkington, Jun Cai, Wen G. Jiang, Lin Ye. Reduced kinase D-interacting substrate of 220kDa (Kidins220) in pancreatic cancer promotes EGFR/ERK signaling and disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 89.

Aberrant over-expression of TRPM7 ion channels in pancreatic cancer: required for cancer cell invasion and implicated in tumor growth and metastasis.

Our previous studies in zebrafish development have led to identification of the novel roles of the transient receptor potential melastatin-subfamily member 7 (TRPM7) ion channels in human pancreatic cancer. However, the biological significance of TRPM7 channels in pancreatic neoplasms was mostly unexplored. In this study, we determined the expression levels of TRPM7 in pancreatic tissue microarrays and correlated these measurements in pancreatic adenocarcinoma with the clinicopathological features. We also investigated the role of TRPM7 channels in pancreatic cancer cell invasion using the MatrigelTM-coated transwell assay. In normal pancreas, TRPM7 is expressed at a discernable level in the ductal cells and centroacinar cells and at a relatively high level in the islet endocrine cells. In chronic pancreatitis, pre-malignant tissues, and malignant neoplasms, there is variable expression of TRPM7. In the majority of pancreatic adenocarcinoma specimens examined, TRPM7 is expressed at either moderate-level or high-level. Anti-TRPM7 immunoreactivity in pancreatic adenocarcinoma significantly correlates with the size and stages of tumors. In human pancreatic adenocarcinoma cells in which TRPM7 is highly expressed, short hairpin RNA-mediated suppression of TRPM7 impairs cell invasion. The results demonstrate that TRPM7 channels are over-expressed in a proportion of the pre-malignant lesions and malignant tumors of the pancreas, and they are necessary for invasion by pancreatic cancer cells. We propose that TRPM7 channels play important roles in development and progression of pancreatic neoplasm, and they may be explored as clinical biomarkers and targets for its prevention and treatment.

CRM1 expression in pancreatic carcinoma.

e15115 Background: Increased expression of chromosomal region maintenance (CRM1) protein, also known as exportin 1, has been described in several human cancers. It is an important regulator of subcellular localization of tumor suppressor proteins and growth regulatory proteins. Direct inhibition of CRM1 blocks cell proliferation and induces apoptosis leading to the current evaluation of CRM1 inhibitors as anticancer agents in early phase clinical trials. There is a paucity of data regarding the prevalence of CRM1 expression in pancreatic cancer. Methods: We analyzed the expression of CRM1 by immunohistochemistry (IHC) in pancreatic tissue microarray (TMA) samples (malignant and non-malignant) obtained from patients who underwent potentially curative resection for pancreatic ductal adenocarcinoma. CRM1 antibody (Santa Cruz Biotechnology) was used for immunostaining the formalin fixed paraffin embedded core sections in the TMA samples. The intensity of staining and percentage of cells stained were graded on a scale of 0 to 3, with 3 being highest. The final IHC score was obtained using the product of immunostain intensity and percentage of cells stained (Range: 0-9). Low and high CRM1 expression was considered if the IHC score was 0 to 4 and 6 to 9 respectively. Results: Seventy nonmalignant and 91 pancreatic carcinoma samples were evaluated in this study. The median IHC score was 6 (range: 0-9) and 3 (range: 1-9) in malignant and nonmalignant pancreatic tissue samples respectively (P<0.0001). High CRM1 expression was found in 11% (8/70) of normal tissue samples and 69% (63/91) of tumor samples (P<0.0001). There were 48 paired samples of pancreatic cancer tissue and normal tissue obtained from same patient. Among these patients, 33 (69%) patients had higher CRM1 expression, 7 (15%) patients had similar expression and 8 (17%) patients had lower expression in malignant tissue sample as compared to their adjacent normal tissues. Conclusions: Higher CRM1 expression occurs frequently in pancreatic cancer as compared to nonmalignant pancreatic tissue, reinforcing its putative tumor oncogenic activity, and raising the value of targeting it for pancreatic cancer therapy.

Effect of three-dimensional collagen I and global histone acetylation on gemcitabine resistance in pancreatic cancer cells.

e14515 Background: Pancreatic ductal adenocarcinoma (PDAC) is associated with a pronounced collagen-rich stromal reaction that has been shown to contribute to chemo-resistance. PDAC is also associated with epigenetic changes. We have previously shown that PDAC cells are resistant to gemcitabine in the collagen microenvironment because of increased expression of the chromatin remodeling protein high mobility group A2 (HMGA2) and increased ERK1/2 signaling. Methods: Pancreatic tissue microarrays were stained with trichrome and for histone H3K9, H3K27 acetylation (Ac), and histone acetyltransferase (HATs) expression. PDAC cells were plated onto tissue culture plastic or in 3D collagen gels and protein expression was assessed by Western blotting. DNA damage response was assessed by comet and clonogenic assays. Results: Human PDAC tumors display in areas of fibrosis higher histone H3K9Ac and H3K27Ac. Moreover, PDAC cells upregulate H3K9Ac and H3K27Ac along with GCN5, PCAF and p300 HATs when grown in 3D collagen. Inhibiting ERK1/2 activity and/or decreasing HMGA2 expression attenuates the effect of collagen on H3Ac and HAT expression. Human PDAC tumors with HMGA2 also demonstrate H3Ac and HAT expression. Additionally, cells in 3D collagen demonstrate reduced tailing with the comet assay, increased clonogenic potential and increased γH2AX following gemcitabine treatment, suggesting an increased repair response to damaged DNA in the collagen microenvironment. Significantly, downregulation of HATs along with inhibition of ERK1/2 activity attenuates gemcitabine-induced γH2AX detected in 3D collagen. Conclusions: Collagen microenvironment limits the effectiveness of gemcitabine through ERK1/2 and HMGA2-dependent HAT expression. HMGA2 expression is associated with histone acetylation and HAT expression in human PDAC tumors, particularly in area of fibrosis, suggesting that fibrosis may contribute to chemo-resistance through increased HMGA2-HAT signaling. Given that very little progress has been made in the treatment of pancreatic cancer, targeting HATs could be a novel approach to sensitize pancreatic tumors to chemotherapy.

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.

MUC13 Mucin Augments Pancreatic Tumorigenesis

The high death rate of pancreatic cancer is attributed to the lack of reliable methods for early detection and underlying molecular mechanisms of its aggressive pathogenesis. Although MUC13, a newly identified transmembrane mucin, is known to be aberrantly expressed in ovarian and gastro-intestinal cancers, its role in pancreatic cancer is unknown. Herein, we investigated the expression profile and functions of MUC13 in pancreatic cancer progression. The expression profile of MUC13 in pancreatic cancer was investigated using a recently generated monoclonal antibody (clone PPZ0020) and pancreatic tissue microarrays. The expression of MUC13 was significantly (P < 0.005) higher in cancer samples compared with normal/nonneoplastic pancreatic tissues. For functional analyses, full-length MUC13 was expressed in MUC13 null pancreatic cancer cell lines, MiaPaca and Panc1. MUC13 overexpression caused a significant (P < 0.05) increase in cell motility, invasion, proliferation, and anchorage-dependent or -independent clonogenicity while decreasing cell-cell and cell-substratum adhesion. Exogenous MUC13 expression significantly (P < 0.05) enhanced pancreatic tumor growth and reduced animal survival in a xenograft mouse model. These tumorigenic characteristics correlated with the upregulation/phosphorylation of HER2, p21-activated kinase 1 (PAK1), extracellular signal-regulated kinase (ERK), Akt, and metastasin (S100A4), and the suppression of p53. Conversely, suppression of MUC13 in HPAFII pancreatic cancer cells by short hairpin RNA resulted in suppression of tumorigenic characteristics, repression of HER2, PAK1, ERK, and S100A4, and upregulation of p53. MUC13 suppression also significantly (P < 0.05) reduced tumor growth and increased animal survival. These results imply a role of MUC13 in pancreatic cancer and suggest its potential use as a diagnostic and therapeutic target.

Computational prediction and experimental validation associating FABP-1 and pancreatic adenocarcinoma with diabetes

BackgroundPancreatic cancer, composed principally of pancreatic adenocarcinoma (PaC), is the fourth leading cause of cancer death in the United States. PaC-associated diabetes may be a marker of early disease. We sought to identify molecules associated with PaC and PaC with diabetes (PaC-DM) using a novel translational bioinformatics approach. We identified fatty acid binding protein-1 (FABP-1) as one of several candidates. The primary aim of this pilot study was to experimentally validate the predicted association between FABP-1 with PaC and PaC with diabetes.MethodsWe searched public microarray measurements for genes that were specifically highly expressed in PaC. We then filtered for proteins with known involvement in diabetes. Validation of FABP-1 was performed via antibody immunohistochemistry on formalin-fixed paraffin embedded pancreatic tissue microarrays (FFPE TMA). FFPE TMA were constructed using148 cores of pancreatic tissue from 134 patients collected between 1995 and 2002 from patients who underwent pancreatic surgery. Primary analysis was performed on 21 normal and 60 pancreatic adenocarcinoma samples, stratified for diabetes. Clinical data on samples was obtained via retrospective chart review. Serial sections were cut per standard protocol. Antibody staining was graded by an experienced pathologist on a scale of 0-3. Bivariate and multivariate analyses were conducted to assess FABP-1 staining and clinical characteristics.ResultsNormal samples were significantly more likely to come from younger patients. PaC samples were significantly more likely to stain for FABP-1, when FABP-1 staining was considered a binary variable. Compared to normals, there was significantly increased staining in diabetic PaC samples (p = 0.004) and there was a trend towards increased staining in the non-diabetic PaC group (p = 0.07). In logistic regression modeling, FABP-1 staining was significantly associated with diagnosis of PaC (OR 8.6 95% CI 1.1-68, p = 0.04), though age was a confounder.ConclusionsCompared to normal controls, there was a significant positive association between FABP-1 staining and PaC on FFPE-TMA, strengthened by the presence of diabetes. Further studies with closely phenotyped patient samples are required to understand the true relationship between FABP-1, PaC and PaC-associated diabetes. A translational bioinformatics approach has potential to identify novel disease associations and potential biomarkers in gastroenterology.

Development and evaluation of monoclonal antibodies against phosphatidylethanolamine binding protein 1 in pancreatic cancer patients

Phosphatidylethanolamine binding protein 1 (PEBP1), also known as Raf kinase inhibitor protein (RKIP), has been considered as a suppressor of metastasis and a prognostic marker in prostate cancer, breast cancer, gastrointestinal stromal tumors, melanoma, and epithelial ovarian cancer. In this report, recombinant PEBP1 was successfully expressed in an Escherichia coli system. A panel of monoclonal antibodies (mAbs) against PEBP1 with high specificity and affinity was generated and characterized using ELISA, western blot analysis, immunofluorescent staining and immunohistochemical staining. PEBP1 expression in normal 293 cells and a few pancreatic cancer cell lines was detected with mAb 7F12 in western blot analysis. To screen for a pair of mAbs with optimal binding affinity to soluble PEBP1, ForteBio's Octet system was used. Sandwich ELISA with mAb pair 4F10 and 8E2 showed a linear correlation between absorbance and PEBP1 protein concentration over a range of 7 to 100ng/ml. MAb 4A11 detected a high level expression of PEBP1 in normal pancreatic tissue, and cancer adjacent normal pancreatic tissue in a pancreatic tissue microarray (TMA) comprising 80 human tissue cores. Pancreatic cancer tissues show a no or very weak staining intensity of PEBP1. In 69 valid cases, PEBP1 expression was significantly lower in tumor than in normal pancreas (p=8.40E-14) and adjacent normal tissue (p=8.46E-17). PEBP1 expression in pancreatic cancer was not associated with pTMN stage, differentiation grade and pathologic diagnosis. In conclusion, our results suggest that PEBP1 overexpresses in normal pancreas but significantly decreases its expression in pancreatic cancer tissues. Anti-PEBP1 mAbs 4A11, 4F10, 7F12, and 8E2 are potential clinical diagnostic agents for pancreatic cancer.

Mucins and CD56 as markers of tumour invasion and prognosis in periampullary cancer

This study investigated the association of mucins and cluster of differentiation (CD) 56 with vascular and perineural invasion and survival in patients with periampullary cancer. Immunohistochemical staining was performed on formalin-fixed pancreatic tissue microarrays (cancer, chronic pancreatitis and normal pancreatic tissue) constructed from 126 pancreatic resections (cancer, 104; chronic pancreatitis, 22). Mucin (MUC) 1, MUC4 and MUC5AC expression was quantified using the immunohistochemical score (range 0-300), MUC3 expression was described as membranous or cytoplasmic, and expression of CD56, MUC2 and MUC6 as present or absent. In cancers, vascular invasion correlated with overexpression (immunohistochemical score of 100 or more) of MUC1 (P = 0.003) and presence of MUC6 (P = 0.024), and perineural invasion correlated with overexpression of MUC5AC (P = 0.015). Reduced survival was observed with overexpression of MUC4 (P = 0.032) and MUC5AC (P = 0.048), membranous expression of MUC3 (P = 0.048), and presence of CD56 (P = 0.041). Perineural invasion also correlated with CD56 expression (P = 0.001). Overexpression of MUC4 and MUC5AC correlated with tumour recurrence (P = 0.001 and P = 0.034 respectively). Multivariable analysis identified membranous expression of MUC3 (P = 0.023), lymphatic invasion (P = 0.015) and perineural invasion (P = 0.004) as independent predictors of poor survival. Mucins and CD56 may be markers of prognosis in patients with periampullary cancer.

Abstract 2358: MUC13 mucin augments pancreatic tumorigenesis

Abstract Pancreatic cancer is one of the most lethal malignancies with extremely poor prognosis. The high death rate of pancreatic cancer is attributed to a lack of reliable methods of early diagnosis and underlying molecular mechanisms associated with aggressive pathogenesis. MUC13, a newly identified transmembrane mucin, is known to be aberrantly expressed in ovarian, gastric and colon cancer. However, the expression and functions of MUC13 in pancreatic cancer are unknown. Herein, we have investigated the expression and functions of MUC13 mucin, in pancreatic cancer to determine its potential for early cancer diagnosis and its role in pancreatic cancer pathogenesis. The expression profile of MUC13 in pancreatic cancer was investigated using a recently generated monoclonal antibody (MAb, clone PPZ0020) and pancreatic tissue microarrays. The expression of MUC13 was significantly (p&amp;lt;0.005) higher in cancer samples compared to the normal/non-neoplastic pancreatic tissues. For the functional analyses, a full length MUC13 gene cloned in pcDNA3.1 was expressed in MUC13 null pancreatic cancer cell lines, MiaPaca and Panc1. The exogenous MUC13 expression induced morphological changes, including scattering of cells. These changes were abrogated through c-jun NH2-terminal kinase (JNK) chemical inhibitor (SP600125) or JNK2 siRNA. Additionally, a marked reduction in cell-cell adhesion and significant (p&amp;lt;0.05) increases in cell motility, invasion, proliferation, clonogenicity and tumorigenesis in a xenograft mouse model system were observed upon exogenous MUC13 expression. These cellular characteristics were correlated with the up-regulation of HER2, p21-activated kinase1 (PAK1), extracellular signal-regulated kinase (ERK) and S100A4 (metastasin) and suppression of p53. Inhibition of MUC13 expression by MUC13 siRNA or shRNA resulted in suppression of tumorigenic characteristics in HPAFII pancreatic cancer cells. These results, for the first time, suggest that MUC13 expression augments pancreatic cancer progression and has potential as a diagnostic and/or therapeutic target. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2358.

Identification of human polo-like kinase 1 as a potential therapeutic target in pancreatic cancer

Pancreas cancer is the fourth leading cause of cancer-related death in adults in the United States. New molecular targets for diagnosis and therapy of this disease are desperately needed. In this study, we report on the mitotic serine-threonine kinase polo-like kinase 1 (Plk1) in pancreatic cancer. Plk1 mRNA was found to be overexpressed in 9 of 10 tested pancreatic cancer cell lines and in 4 of 4 tested human tumors. Immunohistochemical staining of a pancreatic tissue microarray showed that 26 of the 35 tumors taken directly from patients overexpressed Plk1. We also examined the effects of depleting Plk1 in pancreatic cancer cells by the use of antisense oligonucleotides. Antisense-treated pancreatic cancer cells showed cell cycle arrest in G(2)-M as well as a drastic reduction in proliferation rates. These data suggest that Plk1 is a potential therapeutic target in devising a treatment for patients with pancreatic cancer.