Two Highly Specific Mouse Monoclonal Antibodies to the Putative C-Telopeptide of Human Collagen XIα1, a Cancer Biomarker

Objective To investigate the expression of Neuropilin-1 in human pancreatic ductal adenocarcinoma (PDAC) specimens and cell lines. Methods Expression and distribution of Neuropilin-1 were detected in operation tissue specimens of human pancreatic ductal adenocarcinoma with HE, immunohistochemistry and RT-PCR. Results Immunohistochemical analysis revealed that Neuropilin-1 were expressed in human pancreatic adenocarcinoma specimens, precancerous tissue and MIA PaCa-Ⅱ cell lines, but was absent in normal pancreatic tissue, mRNA of Neuropilin-1 could also be detected the in human pancreatic adenocarcinoma specimens, precancerous tissue and MIA PaCaⅡ cell Lines, while could not be detected in normal pancreatic tissue. Neuropilin-1 expression in pancreatic adenocarcinoma were higher than that in normal pancreas, and the differences were significant( P < 0. 01 ). Conclusions Neuropilin-1 might play an potential role in carcinogenesis for pancreatic cancer. Neuropilin-1 may be relevant to perineural invasion of pancreatic carcinoma by some signal transduction. Key words: Neuropilin-l/Bl; Pancreatic neoplasms/ME

Objective To investigate the expression of KL-6 mucin in pancreatic ducatal tissue,and to explore its clinical significance.Methods All specimens form 38 patients who were diagnosed as pancreatic ductal adenocarcinoma were subjected to immunohistochemical analysis using KL-6 monoclonal antibody.Results 100％ specimens of pancreatic adenocarcinoma were found positive staining,and marked positive staining was 78.9％,remarkable positive staining was not found in the surrounding noncancer regions of the pancreatic tissues (72.3％).KL-6 mucin expression was statistical significance with the tumor development and differentiation.Conclusion Most of all pancreatic adenocarcinoma cases showed remarkable positive staining for KL-6 mucin,while the majority of surrounding non-cancer regions did not.The aberrant expression of KL-6 mucin is significantly related to aggressive tumor behaviors of pancreatic adenocarcinoma.KL-6 mucin may be a specific target to detect the pancreatic adenocarcinoma in molecular imaging study. Key words: Pancreatic ductal adenocarcinoma; KL-6 mucin; Immunohistochemical

The effective treatment of pancreatic cancer relies on the diagnosis of the disease at an early stage, a difficult challenge. One major obstacle in the development of diagnostic biomarkers of early pancreatic cancer has been the dual expression of potential biomarkers in both chronic pancreatitis and cancer. To better understand the limitations of potential protein biomarkers, we used ICAT technology and tandem mass spectrometry-based proteomics to systematically study protein expression in chronic pancreatitis. Among the 116 differentially expressed proteins identified in chronic pancreatitis, most biological processes were responses to wounding and inflammation, a finding consistent with the underlining inflammation and tissue repair associated with chronic pancreatitis. Furthermore 40% of the differentially expressed proteins identified in chronic pancreatitis have been implicated previously in pancreatic cancer, suggesting some commonality in protein expression between these two diseases. Biological network analysis further identified c-MYC as a common prominent regulatory protein in pancreatic cancer and chronic pancreatitis. Lastly five proteins were selected for validation by Western blot and immunohistochemistry. Annexin A2 and insulin-like growth factor-binding protein 2 were overexpressed in cancer but not in chronic pancreatitis, making them promising biomarker candidates for pancreatic cancer. In addition, our study validated that cathepsin D, integrin beta1, and plasminogen were overexpressed in both pancreatic cancer and chronic pancreatitis. The positive involvement of these proteins in chronic pancreatitis and pancreatic cancer will potentially lower the specificity of these proteins as biomarker candidates for pancreatic cancer. Altogether our study provides some insights into the molecular events in chronic pancreatitis that may lead to diverse strategies for diagnosis and treatment of these diseases.

Desmoplasia of Pancreatic Ductal Adenocarcinoma

Sno(RNA)wing and Pancreatic Cancer Metastasis

Background: Pancreatic cancer is associated with 6.9% and 4% of all cancer-related deaths in the United States and Brazil, respectively. Pancreatic ductal carcinoma comprises 90% of cases, the majority being of adenocarcinoma subtype. Approximately 12% of periampullary tumors are adenocarcinomas of Vater papilla (ampullary adenocarcinomas); ampullary tumors are often associated with a better prognosis than ductal adenocarcinomas. Although genetic alterations were previously identified in pancreatic carcinomas, there is still a lack of effective treatment strategies. Therefore, the identification of new biomarkers, such as alterations in non-coding RNAs, is urgently needed for the development of novel molecularly targeted therapies for these cancers. microRNAs (miRNAs) are frequently deregulated and contribute to cancer development and progression and have potential prognostic and predictive value. Global miRNA expression profiling analysis in pancreatic cancer, followed by the identification of miRNA target genes may lead to the identification of clinically applicable biomarkers. The novel aspect of our work is the investigation of pancreatic tumors from Brazilian patients, with the inclusion of ampullary adenocarcinomas, a rare subtype. Objectives: To identify global miRNA expression profiles and miRNA target genes in pancreatic ductal and ampullary adenocarcinomas compared to paired histologically normal pancreatic tissue. Patients and Methods: 30 formalin fixed, paraffin embedded (FFPE) pancreatic carcinoma samples were used, including 24 pancreatic ductal adenocarcinomas (PDAC) and 6 ampullary adenocarcinomas (AMP). Paired histologically normal pancreatic tissues were used as controls. All tumor and normal tissues were needle microdissected (Leica EZ4 stereomicroscope). Global miRNA expression profiles were determined using the TaqMan Array Human MicroRNA Cards (TLDA) (card A, v3.0) (Life Technologies) platform. Data analysis was performed using the ExpressionSuite Software v1.0.3. Statistical analysis was performed to correlate miRNA expression with relevant clinical data, using SAS 9.3 software. Computational bioinformatics analysis was performed to identify miRNA target genes, as well as to construct protein-protein interaction and miRNA-gene targets networks. Results and Discussion: We identified 63 significantly deregulated (FC≥2 and p&amp;lt;0.05) miRNAs in PDAC (33 over- and 30 under-expressed) compared to paired histologically normal pancreatic tissue. In AMP, a group of 7 miRNAs was significantly deregulated (4 over- and 3 under-expressed) compared to normal pancreas. Our results showed differentially expressed miRNAs and a complexity of miRNA changes potentially associated to PDAC and AMP tumorigenesis. 3/7 miRNAs (miR-222, 148a and 375) were commonly deregulated in PDAC and AMP tumors. Furthermore, miRNA-gene targets networks were distinct in these different histological subtypes of pancreatic carcinomas. Global miRNA expression profiles showed that PDAC have a significantly higher number of altered miRNAs and a higher number of predicted miRNA target genes than AMP tumors, which could be potentially associated to disease progression and tumor aggressiveness in PDAC compared to AMP. Although these tumors have biological differences, commonly deregulated miRNAs in PDAC and AMP suggest that PDAC and AMP tumorigenesis may share commonly deregulated pathways. Conclusion: miRNAs identified herein may be associated to the biology of PDAC and AMP. Among the miRNAs exclusively deregulated in PDAC, we identified known and not previously reported (novel) miRNAs. In addition, we identified several miRNA target genes associated with tumor invasion, metastasis and poor patient prognosis. Functional in vitro and in vivo validation studies may elucidate the role of identified miRNAs as modulators of oncogenesis mechanisms in PDAC and AMP. T. Felix was funded through São Paulo Research Foundation (FAPESP), MSc. fellowship (2014/00367-4) Citation Format: Tainara F. Felix, Tomas Tokar, Maria A. M. Rodrigues, Rogerio A. Oliveira, Claudia N. Hasimoto, Juan C. Llanos, Robson F. Carvalho, Silvia R. Rogatto, Wan Lam, Igor Jurisica, Sandra A. Drigo, Patricia P. Reis.{Authors}. Differentially expressed microRNA profiles in pancreatic ductal and ampullary adenocarcinomas. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A28.

Previously, we have identified an overexpression of IL-13Rα2 in 71% of human pancreatic ductal adenocarcinoma (PDA) specimens. We have also demonstrated that these receptors can be a target for a receptor directed chimeric cytotoxin, IL-13PE, which consists of IL-13 and a truncated Pseudomonas exotoxin (IL13-PE). In the present study, we examined the anti-tumor activities of IL13-PE in combination with gemcitabine, an established first line therapy for advanced pancreatic cancer. Both these agents were cytotoxic to two pancreatic cancer cell lines (HS766T and MIA-PaCa2) with IC50 of 1.1 - 51 ng/ml and 19 - 98 nmol/L, respectively. Upon combination of these two agents, we observed a synergistic cytotoxicity in these two cell lines (IC50: 0.4 ng/ml with 0.01 nmol/L gemcitabine in HS766T and 21 ng/ml with 1 nmol/L gemcitabine in MIA-PaCa2). In addition, we have demonstrated a synergistic anti-tumor activity in an orthotopic mouse model of human PDA developed in athymic nude mice. This study showed complete eradiation of tumors as assessed by whole body imaging of GFP-transfected tumors in 57% of mice in an early cancer model and also resulted in a prolongation in survival. In contrast, single therapy with either IL13-PE or gemcitabine did not produce complete eradiation, but tumor volumes were significantly decreased. In advanced cancer models, IL13-PE and gemcitabine also produced a dramatic reduction and retardation of tumor growth and enhanced survival compared to animals treated with either agent alone. Furthermore, silencing of IL-13Rα2 by RNAi in tumor cells prior to tumor implantation revealed that the combination of IL13-PE and gemcitabine was less effective in this model indicating that IL-13Rα2 is an essential target in PDA for complete elimination of tumors. Mechanistically, gemcitabine increased IL-13Rα2 mRNA expression in vitro and in vivo, which in turn, resulted in a synergism of combination therapy and improved induction of apoptosis in PDA cell lines. Based on these data, we conclude that combination therapy of IL13-PE and gemcitabine may be an additional therapeutic option for management of pancreatic ductal cancer patients for better clinical outcome. 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 5539.

Increased expression of alpha-1-antitrypsin, glutathione S-transferase π and vascular endothelial growth factor in human pancreatic adenocarcinoma

The aim of this study was to determine whether the number of argyrophilic nucleolar organizer regions (AgNORs) could be of diagnostic significance in differentiating between chronic pancreatitis and pancreatic ductal adenocarcinoma. The number of AgNORs was enumerated in biopsy specimens of normal pancreas, chronic pancreatitis, and pancreatic ductal adenocarcinoma. The number of AgNORs was lower in patients with normal pancreas than in patients with chronic pancreatitis or pancreatic adenocarcinoma. In addition, the number of AgNORs was significantly lower in chronic pancreatitis than in pancreatic ductal adenocarcinoma (p < 0.001). The diagnosis of pancreatic adenocarcinoma is usually clear. Difficulties can be encountered, however, in cases of chronic pancreatitis, specially when biopsy material is small. Our results suggest that the number of AgNORs may help in distinguishing between chronic pancreatitis and pancreatic ductal adenocarcinoma, especially in diagnostically difficult specimens.

Background: Arginine (Arg) deprivation is a promising therapeutic approach for tumors with low argininosuccinate synthetase 1 (ASS1) expression. However, its efficacy as a single agent therapy needs to be improved as resistance is frequently observed. Methods: A tissue microarray was performed to assess ASS1 expression in surgical specimens of pancreatic ductal adenocarcinoma (PDAC) and its correlation with disease prognosis. An RNA-Seq analysis examined the role of ASS1 in regulating global gene transcriptome. A high throughput screen of FDA-approved oncology drugs identified synthetic lethality between histone deacetylase (HDAC) inhibitors and Arg deprivation in PDAC cells with low ASS1 expression. We examined HDAC inhibitor panobinostat (PAN) and Arg deprivation in a panel of human PDAC cell lines, in ASS1-high and -knockdown/knockout isogenic models, in both anchorage-dependent and -independent cultures, and in multicellular complex cultures that model the PDAC tumor microenvironment. We examined the effects of combined Arg deprivation and PAN on DNA damage and the protein levels of key DNA repair enzymes. We also evaluate the efficacy of PAN and ADI-PEG20 (an Arg-degrading agent currently in Phase II clinical trials) in xenograft models with ASS1-low and -high PDAC tumors. Results: Low ASS1 protein level is a negative prognostic indicator in PDAC. Arg deprivation in ASS1-deficient PDAC cells upregulated asparagine synthetase (ASNS) which redirected aspartate (Asp) from being used for de novo nucleotide biosynthesis, thus causing nucleotide insufficiency and impairing cell cycle S-phase progression. Comprehensively validated, HDAC inhibitors and Arg deprivation showed synthetic lethality in ASS1-low PDAC cells. Mechanistically, combined Arg deprivation and HDAC inhibition triggered degradation of a key DNA repair enzyme C-terminal-binding protein interacting protein (CtIP), resulting in DNA damage and apoptosis. In addition, S-phase-retained ASS1-low PDAC cells (due to Arg deprivation) were also sensitized to DNA damage, thus yielding effective cell death. Compared to single agents, the combination of PAN and ADI-PEG20 showed better efficacy in suppressing ASS1-low PDAC tumor growth in mouse xenograft models. Conclusion: The combination of PAN and ADI-PEG20 is a rational translational therapeutic strategy for treating ASS1-low PDAC tumors through synergistic induction of DNA damage. Citation Format: Stephanie S Kim, JING CUI, Shili Xu, Soumya Poddar, Thuc M. Le, Luyi Li, Nanping Wu, Alexandra Moore, Lei Zhou, Alice Yu, Amanda M. Dann, Irmina A. Elliott, Evan R. Abt, Woosuk Kim, David W. Dawson, Caius G. Radu, Timothy R. Donahue. Histone deacetylase inhibition is synthetically lethal with arginine deprivation in pancreatic cancers with low argininosuccinate synthetase 1 expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 554.

Recent advances in cancer biology have emerged important roles for microRNAs (miRNAs) in regulating tumor responses. However, their function in mediating intercellular communication within the tumor microenvironment is thus far poorly explored. Here, we found miR-206 to be abrogated in human pancreatic ductal adenocarcinoma (PDAC) specimens and cell lines. We show that miR-206 directly targets the oncogenes KRAS and annexin a2 (ANXA2), thereby acting as tumor suppressor in PDAC cells by blocking cell cycle progression, cell proliferation, migration and invasion. Importantly, we identified miR-206 as a negative regulator of oncogenic KRAS-induced nuclear factor-κB transcriptional activity, resulting in a concomitant reduction of the expression and secretion of pro-angiogenic and pro-inflammatory factors including the cytokine interleukin-8, the chemokines (C-X-C motif) ligand 1 and (C–C motif) ligand 2, and the granulocyte macrophage colony-stimulating factor. We further show that miR-206 abrogates the expression and secretion of the potent pro-lymphangiogenic factor vascular endothelial growth factor C in pancreatic cancer cells through an NF-κB-independent mechanism. By using in vitro and in vivo approaches, we reveal that re-expression of miR-206 in PDAC cells is sufficient to inhibit tumor blood and lymphatic vessel formation, thus leading to a significant delay of tumor growth and progression. Taken together, our study sheds light onto the role of miR-206 as a pleiotropic modulator of different hallmarks of cancer, and as such raising the intriguing possibility that miR-206 may be an attractive candidate for miRNA-based anticancer therapies.

To investigate the expression and clinical significance of Sonic hedgehog (Shh) in pancreatic adenocarcinoma. The expression of Shh protein was examined in 34 surgical specimens of primary pancreatic adenocarcinoma, 21 nonmalignant specimens of the pancreas by immunohistochemistry streptavidin-perosidase (SP) method. In addition, semiquantitative reverse transcriptase polymerase chain reaction was carried out to analyze Shh mRNA expression in 22 pairs of freshly resected pancreatic adenocarcinoma tissues and their adjacent nontumorous tissues. The positive expression rate of Shh protein was 64.7% (22/34) in 34 surgical specimens of primary pancreatic adenocarcinoma and 0% (0/21) in 21 nonmalignant specimens of the pancreas. The expression rate of Shh was higher in pancreatic adenocarcinoma tissues than that of nonmalignant pancreatic tissues (χ2 = 22.647, P = 0.000). Sonic hedgehog protein expression correlated with TNM stages and distant metastasis. Moreover, the expression levels of Shh mRNA were higher in pancreatic adenocarcinoma tissues than that of the matched adjacent nontumorous tissues. Sonic hedgehog might play a pivotal role during tumorigenesis of pancreatic adenocarcinoma, and high Shh expression might be associated with the malignant potential of pancreatic cancer.

SUCLA2-coupled regulation of GLS succinylation and activity counteracts oxidative stress in tumor cells

Krüppel-Like Factor 10 Expression as a Prognostic Indicator for Pancreatic Adenocarcinoma

Background: Pancreatic adenocarcinoma (PA) is the fourth leading cause of cancer related death in the USA. Patients diagnosed with this disease can expect a 1-year survival rate of approximately 10%. One of the main reasons of the high mortality observed within PA is failure on first-line therapies. Current PA treatment involves Gemcitabine, a nucleoside analog that showed improvement in overall survival. Recently this drug has been used in combination with nab-paclitaxel, 5-FU, Erlotinib and MEK inhibitors. However, a growing number of patients have shown resistance to these regimes. A more comprehensive understanding of resistance mechanisms will enhance treatment choice and clinical responses, and this remains an area of intense investigation. However much of the data available on PA drug targets and efficacy come from commercially available pancreatic cell lines, a main limitation in PA research, as these lines do not accurately represent a given patients' tumor, underscoring the need for the development and use of patient-specific primary epithelial PA cells. In our current study we present the use of patient-derived primary PA cells as a model system for basic and translational research as well as personalized therapeutic approaches. Methods: Patients' biopsies were collected after surgery and long-term cultures of PA cells were established using the conditional reprogramming of cells (CRC) approach. To date, six primary lines have been continuous culture for over 6 month. KRAS and p53 sequencing verified the PA origin of both the patient sample and the matched CRC lines. CRC karyotyping was also performed to confirm the absence of normal cells as well as to validate the long-term genomic integrity of the lines in culture. In order to evaluate patient-specific differences in treatment response, the IC50's for gemcitabine, nab-paclitaxel (Abraxane) and the MEK inhibitor, Trametinib, were determined. To better understand treatment resistance mechanisms, new drug resistance approaches were developed and multiple drug-resistant clones per primary cell line were established, and their resistance verified. Gemcitabine activity was also evaluated in combination with Abraxane and Trematenib in both parental and resistant-clone cell lines. In addition, novel three-dimensional (3D) organoid cultures have been established from the two dimensional (2D) CRC cultures in order to verify the constancy of the model. Results: We established KRAS-mutant primary cell lines derived from patients' PA specimens. The cell lines' karyotype showed stability over multiple passages covering more than 6 months in continuous 2D culture. Five Abraxane resistant clones have been derived to date from two different parent cell lines and two gemcitabine resistant clones derived from one of these cell lines. The clones tested were 3 to 1000 times less sensitive to the drugs when compared to the parents. Notably, the Abraxane resistant clones also showed a greater resistance to Gemcitabine as compared to the parent line. In addition, the PA lines also showed an overall increasing sensitivity to Gemcitabine when pre- or co-treated with Trematenib. Molecular and genetic analyses are being performed to identify potential biomarkers of high therapeutic value. Conclusion: Taken together, the ease of culture, the genetic stability, the medium to high throughput ability to identify differences between patients sensitivity to FDA approved drugs, all confirm the power of this technology for on-demand in vitro use in PA research. Our approach now enables the high-resolution experiments necessary to better understand the underlying drug sensitivity and resistance mechanisms that directly affect clinical outcomes. Citation Format: Erika M. Parasido, Praathibha Sripadhan, Richard Schlegel, Michael J. Pishvaian, Jonathan Brody, Jordan Winter, Christopher Albanese. Patient-derived Pancreatic Adenocarcinoma cells: A new model system to define chemotherapy resistance mechanisms and to improve targeted personalized treatment. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B15.