Publications by authors named "Paul M Grandgenett"

33 Publications

Plexin-B3 Regulates Cellular Motility, Invasiveness, and Metastasis in Pancreatic Cancer.

Cancers (Basel) 2021 Feb 16;13(4). Epub 2021 Feb 16.

Department of Pathology and Microbiology, Nebraska Medical Center, Omaha, NE 68198, USA.

The Plexins family of proteins are well-characterized transmembrane receptors of semaphorins, axon guidance cue molecules, that mediate the cell attraction or repelling effects for such cues. Plexins and their ligands are involved in numerous cellular activities, such as motility, invasion, and adhesion to the basement membrane. The detachment of cells and the gain in motility and invasion are hallmarks of the cancer metastasis cascade, thus generating interest in exploring the role of plexins in cancer metastasis. Semaphorin-plexin complexes can act as tumor promoters or suppressors, depending upon the cancer type, and are under investigation for therapeutic purposes. Our group has identified Semaphorin-5A (SEMA5A)/Plexin-B3 as an attractive targetable complex for pancreatic cancer (PC) metastasis. However, our understanding of the Plexin-B3 function and pathological expression in PC is limited, and our present study delineates the role of Plexin-B3 in PC malignancy. We examined the pathological expression of Plexin-B3 in PC tumors and metastasis using a human tissue microarray, disease progression model of PDX-Cre-Kras (KC) mice, and different metastatic sites obtained from the Kras; Trp53; Pdx1-Cre (KPC) mice model. We observed a higher Plexin-B3 expression in PC tumor cores than the normal pancreas, and different metastatic sites were positive for Plexin-B3 expression. However, in the KC mice model, the Plexin-B3 expression increased initially and then decreased with the disease progression. Next, to evaluate the functional role of Plexin-B3, we utilized T3M-4- and CD18/HPAF-Control and -Plexin B3 knockdown cells for different in vivo and in vitro studies. The knockdown of Plexin-B3 enhanced the in vitro cellular migration, invasiveness, and impaired colony formation in three-dimensional culture, along with an increase in cellular spread and remodeling of the actin filaments. We also observed a higher metastasis in nude mice injected with T3M-4- and CD18/HPAF-shPlexin-B3 cells compared to their respective control cells. Furthermore, we observed a lower number of proliferating Ki-67-positive cells and higher ALDH1-A1-positive cells in the tumors formed by Plexin-B3 knockdown cells compared to tumors formed by the control cells. Together, our data suggest that the loss of Plexin-B3 is associated with the interference of cell division machinery and the induction of stem cell-like characteristics in PC cells.
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http://dx.doi.org/10.3390/cancers13040818DOI Listing
February 2021

Isoforms of MUC16 activate oncogenic signaling through EGF receptors to enhance the progression of pancreatic cancer.

Mol Ther 2020 Dec 25. Epub 2020 Dec 25.

Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198-6805, USA. Electronic address:

Aberrant expression of CA125/MUC16 is associated with pancreatic ductal adenocarcinoma (PDAC) progression and metastasis. However, knowledge of the contribution of MUC16 to pancreatic tumorigenesis is limited. Here, we show that MUC16 expression is associated with disease progression, basal-like and squamous tumor subtypes, increased tumor metastasis, and short-term survival of PDAC patients. MUC16 enhanced tumor malignancy through the activation of AKT and GSK3β oncogenic signaling pathways. Activation of these oncogenic signaling pathways resulted in part from increased interactions between MUC16 and epidermal growth factor (EGF)-type receptors, which were enhanced for aberrant glycoforms of MUC16. Treatment of PDAC cells with monoclonal antibody (mAb) AR9.6 significantly reduced MUC16-induced oncogenic signaling. mAb AR9.6 binds to a unique conformational epitope on MUC16, which is influenced by O-glycosylation. Additionally, treatment of PDAC tumor-bearing mice with either mAb AR9.6 alone or in combination with gemcitabine significantly reduced tumor growth and metastasis. We conclude that the aberrant expression of MUC16 enhances PDAC progression to an aggressive phenotype by modulating oncogenic signaling through ErbB receptors. Anti-MUC16 mAb AR9.6 blocks oncogenic activities and tumor growth and could be a novel immunotherapeutic agent against MUC16-mediated PDAC tumor malignancy.
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http://dx.doi.org/10.1016/j.ymthe.2020.12.029DOI Listing
December 2020

Metabolic programming of distinct cancer stem cells promotes metastasis of pancreatic ductal adenocarcinoma.

Oncogene 2021 Jan 27;40(1):215-231. Epub 2020 Oct 27.

Department of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical Center, Omaha, NE, 68198-5870, USA.

Pancreatic ductal adenocarcinoma (PDAC) metastasizes to distant organs, which is the primary cause of mortality; however, specific features mediating organ-specific metastasis remain unexplored. Emerging evidence demonstrates that cancer stem cells (CSCs) and cellular metabolism play a pivotal role in metastasis. Here we investigated the role of distinct subtypes of pancreatic CSCs and their metabolomic signatures in organ-specific metastatic colonization. We found that PDAC consists of ALDH+/CD133+ and drug-resistant (MDR1+) subtypes of CSCs with specific metabolic and stemness signatures. Human PDAC tissues with gemcitabine treatment, autochthonous mouse tumors from Kras; Pdx1-Cre (KC) and Kras; Trp53; Pdx-1 Cre (KPC) mice, and KPC- Liver/Lung metastatic cells were used to evaluate the CSC, EMT (epithelial-to-mesenchymal transition), and metabolic profiles. A strong association was observed between distinct CSC subtypes and organ-specific colonization. The liver metastasis showed drug-resistant CSC- and EMT-like phenotype with aerobic glycolysis and fatty acid β-oxidation-mediated oxidative (glyco-oxidative) metabolism. On the contrary, lung metastasis displayed ALDH+/CD133+ and MET-like phenotype with oxidative metabolism. These results were obtained by evaluating FACS-based side population (SP), autofluorescence (AF+) and Alde-red assays for CSCs, and Seahorse-based oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and fatty acid β-oxidation (FAO)-mediated OCR assays for metabolic features along with specific gene signatures. Further, we developed in vitro human liver and lung PDAC metastasis models by using a combination of liver or lung decellularized scaffolds, a co-culture, and a sphere culture methods. PDAC cells grown in the liver-mimicking model showed the enrichment of MDR1+ and CPT1A+ populations, whereas the PDAC cells grown in the lung-mimicking environment showed the enrichment of ALDH+/CD133+ populations. In addition, we observed significantly elevated expression of ALDH1 in lung metastasis and MDR1/LDH-A expression in liver metastasis compared to human primary PDAC tumors. Our studies elucidate that distinct CSCs adapt unique metabolic signatures for organotropic metastasis, which will pave the way for the development of targeted therapy for PDAC metastasis.
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http://dx.doi.org/10.1038/s41388-020-01518-2DOI Listing
January 2021

Suppression of pancreatic cancer liver metastasis by secretion-deficient ITIH5.

Br J Cancer 2021 Jan 7;124(1):166-175. Epub 2020 Oct 7.

Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS, USA.

Background: Previously, we identified ITIH5 as a suppressor of pancreatic ductal adenocarcinoma (PDAC) metastasis in experimental models. Expression of ITIH5 correlated with decreased cell motility, invasion and metastasis without significant inhibition of primary tumour growth. Here, we tested whether secretion of ITIH5 is required to suppress liver metastasis and sought to understand the role of ITIH5 in human PDAC.

Methods: We expressed mutant ITIH5 with deletion of the N-terminal secretion sequence (ITIH5Δs) in highly metastatic human PDAC cell lines. We used a human tissue microarray (TMA) to compare ITIH5 levels in uninvolved pancreas, primary and metastatic PDAC.

Results: Secretion-deficient ITIH5Δs was sufficient to suppress liver metastasis. Similar to secreted ITIH5, expression of ITIH5Δs was associated with rounded cell morphology, reduced cell motility and reduction of liver metastasis. Expression of ITIH5 is low in both human primary PDAC and matched metastases.

Conclusions: Metastasis suppression by ITIH5 may be mediated by an intracellular mechanism. In human PDAC, loss of ITIH5 may be an early event and ITIH5-low PDAC cells in primary tumours may be selected for liver metastasis. Further defining the ITIH5-mediated pathway in PDAC could establish future therapeutic exploitation of this biology and reduce morbidity and mortality associated with PDAC metastasis.
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http://dx.doi.org/10.1038/s41416-020-01093-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782545PMC
January 2021

Genomic and Epigenomic Landscaping Defines New Therapeutic Targets for Adenosquamous Carcinoma of the Pancreas.

Cancer Res 2020 10 14;80(20):4324-4334. Epub 2020 Sep 14.

Division of Hematology/Oncology, Department of Internal Medicine, Mayo Clinic, Scottsdale, Arizona.

Adenosquamous cancer of the pancreas (ASCP) is a subtype of pancreatic cancer that has a worse prognosis and greater metastatic potential than the more common pancreatic ductal adenocarcinoma (PDAC) subtype. To distinguish the genomic landscape of ASCP and identify actionable targets for this lethal cancer, we applied DNA content flow cytometry to a series of 15 tumor samples including five patient-derived xenografts (PDX). We interrogated purified sorted tumor fractions from these samples with whole-genome copy-number variant (CNV), whole-exome sequencing, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analyses. These identified a variety of somatic genomic lesions targeting chromatin regulators in ASCP genomes that were superimposed on well-characterized genomic lesions including mutations in (87%) and (73%), amplification of (47%), and homozygous deletion of (40%) that are common in PDACs. Furthermore, a comparison of ATAC-seq profiles of three ASCP and three PDAC genomes using flow-sorted PDX models identified genes with accessible chromatin unique to the ASCP genomes, including the lysine methyltransferase and the pancreatic cancer stem cell regulator in all three ASCPs, and a fusion associated with focal CNVs in both genes in a single ASCP. Finally, we demonstrate significant activity of a pan FGFR inhibitor against organoids derived from the fusion-positive ASCP PDX model. Our results suggest that the genomic and epigenomic landscape of ASCP provide new strategies for targeting this aggressive subtype of pancreatic cancer. SIGNIFICANCE: These data provide a unique description of the ASCP genomic and epigenomic landscape and identify candidate therapeutic targets for this dismal cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906529PMC
October 2020

Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers.

Authors:
Ayuko Hoshino Han Sang Kim Linda Bojmar Kofi Ennu Gyan Michele Cioffi Jonathan Hernandez Constantinos P Zambirinis Gonçalo Rodrigues Henrik Molina Søren Heissel Milica Tesic Mark Loïc Steiner Alberto Benito-Martin Serena Lucotti Angela Di Giannatale Katharine Offer Miho Nakajima Caitlin Williams Laura Nogués Fanny A Pelissier Vatter Ayako Hashimoto Alexander E Davies Daniela Freitas Candia M Kenific Yonathan Ararso Weston Buehring Pernille Lauritzen Yusuke Ogitani Kei Sugiura Naoko Takahashi Maša Alečković Kayleen A Bailey Joshua S Jolissant Huajuan Wang Ashton Harris L Miles Schaeffer Guillermo García-Santos Zoe Posner Vinod P Balachandran Yasmin Khakoo G Praveen Raju Avigdor Scherz Irit Sagi Ruth Scherz-Shouval Yosef Yarden Moshe Oren Mahathi Malladi Mary Petriccione Kevin C De Braganca Maria Donzelli Cheryl Fischer Stephanie Vitolano Geraldine P Wright Lee Ganshaw Mariel Marrano Amina Ahmed Joe DeStefano Enrico Danzer Michael H A Roehrl Norman J Lacayo Theresa C Vincent Martin R Weiser Mary S Brady Paul A Meyers Leonard H Wexler Srikanth R Ambati Alexander J Chou Emily K Slotkin Shakeel Modak Stephen S Roberts Ellen M Basu Daniel Diolaiti Benjamin A Krantz Fatima Cardoso Amber L Simpson Michael Berger Charles M Rudin Diane M Simeone Maneesh Jain Cyrus M Ghajar Surinder K Batra Ben Z Stanger Jack Bui Kristy A Brown Vinagolu K Rajasekhar John H Healey Maria de Sousa Kim Kramer Sujit Sheth Jeanine Baisch Virginia Pascual Todd E Heaton Michael P La Quaglia David J Pisapia Robert Schwartz Haiying Zhang Yuan Liu Arti Shukla Laurence Blavier Yves A DeClerck Mark LaBarge Mina J Bissell Thomas C Caffrey Paul M Grandgenett Michael A Hollingsworth Jacqueline Bromberg Bruno Costa-Silva Hector Peinado Yibin Kang Benjamin A Garcia Eileen M O'Reilly David Kelsen Tanya M Trippett David R Jones Irina R Matei William R Jarnagin David Lyden

Cell 2020 Aug 13;182(4):1044-1061.e18. Epub 2020 Aug 13.

Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA. Electronic address:

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.
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http://dx.doi.org/10.1016/j.cell.2020.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522766PMC
August 2020

SIRT1-NOX4 signaling axis regulates cancer cachexia.

J Exp Med 2020 07;217(7)

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE.

Approximately one third of cancer patients die due to complexities related to cachexia. However, the mechanisms of cachexia and the potential therapeutic interventions remain poorly studied. We observed a significant positive correlation between SIRT1 expression and muscle fiber cross-sectional area in pancreatic cancer patients. Rescuing Sirt1 expression by exogenous expression or pharmacological agents reverted cancer cell-induced myotube wasting in culture conditions and mouse models. RNA-seq and follow-up analyses showed cancer cell-mediated SIRT1 loss induced NF-κB signaling in cachectic muscles that enhanced the expression of FOXO transcription factors and NADPH oxidase 4 (Nox4), a key regulator of reactive oxygen species production. Additionally, we observed a negative correlation between NOX4 expression and skeletal muscle fiber cross-sectional area in pancreatic cancer patients. Knocking out Nox4 in skeletal muscles or pharmacological blockade of Nox4 activity abrogated tumor-induced cachexia in mice. Thus, we conclude that targeting the Sirt1-Nox4 axis in muscles is an effective therapeutic intervention for mitigating pancreatic cancer-induced cachexia.
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http://dx.doi.org/10.1084/jem.20190745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336299PMC
July 2020

Macrophages potentiate STAT3 signaling in skeletal muscles and regulate pancreatic cancer cachexia.

Cancer Lett 2020 08 25;484:29-39. Epub 2020 Apr 25.

The Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA. Electronic address:

Incidence of cachexia is highly prevalent in pancreatic ductal adenocarcinoma (PDAC); advanced disease stage directly correlates with decreased muscle and fat mass in PDAC patients. The pancreatic tumor microenvironment is central to the release of systemic factors that govern lipolysis, proteolysis, and muscle and fat degeneration leading to the cachectic phenotype in cancer patients. The current study explores the role of macrophages, a key immunosuppressive player in the pancreatic tumor microenvironment, in regulating cancer cachexia. We observed a negative correlation between CD163-positive macrophage infiltration and muscle-fiber cross sectional area in human PDAC patients. To investigate the role of macrophages in myodegeneration, we utilized conditioned media transplant assays and orthotopic models of PDAC-induced cachexia in immune-competent mice with and without macrophage depletion. We observed that macrophage-derived conditioned medium, in combination with tumor cell-conditioned medium, promoted muscle atrophy through STAT3 signaling. Furthermore, macrophage depletion attenuated systemic inflammation and muscle wasting in pancreatic tumor-bearing mice. Targeting macrophage-mediated STAT3 activation or macrophage-derived interleukin-1 alpha or interleukin-6 diminished myofiber atrophy. Taken together, the current study identified the critical association between macrophages and cachexia phenotype in pancreatic cancer.
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http://dx.doi.org/10.1016/j.canlet.2020.04.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7286478PMC
August 2020

Upregulation of ZIP14 and Altered Zinc Homeostasis in Muscles in Pancreatic Cancer Cachexia.

Cancers (Basel) 2019 Dec 18;12(1). Epub 2019 Dec 18.

Institute for Cancer Genetics, Columbia University Irving Medical Center, New York, NY 10032, USA.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer type in which the mortality rate approaches the incidence rate. More than 85% of PDAC patients experience a profound loss of muscle mass and function, known as cachexia. PDAC patients with this condition suffer from decreased tolerance to anti-cancer therapies and often succumb to premature death due to respiratory and cardiac muscle wasting. Yet, there are no approved therapies available to alleviate cachexia. We previously found that upregulation of the metal ion transporter, , and altered zinc homeostasis are critical mediators of cachexia in metastatic colon, lung, and breast cancer models. Here, we show that a similar mechanism is likely driving the development of cachexia in PDAC. In two independent experimental metastasis models generated from the murine PDAC cell lines, Pan02 and FC1242, we observed aberrant expression and increased zinc ion levels in cachectic muscles. Moreover, in advanced PDAC patients, high levels of ZIP14 in muscles correlated with the presence of cachexia. These studies underscore the importance of altered ZIP14 function in PDAC-associated cachexia development and highlight a potential therapeutic opportunity for improving the quality of life and prolonging survival in PDAC patients.
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http://dx.doi.org/10.3390/cancers12010003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016633PMC
December 2019

The Proteomic Landscape of Pancreatic Ductal Adenocarcinoma Liver Metastases Identifies Molecular Subtypes and Associations with Clinical Response.

Clin Cancer Res 2020 03 17;26(5):1065-1076. Epub 2019 Dec 17.

Eppley Institute for Research in Cancer, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease that can be separated into distinct subtypes based on molecular signatures. Identifying PDAC subtype-specific therapeutic vulnerabilities is necessary to develop precision medicine approaches to treat PDAC.

Experimental Design: A total of 56 PDAC liver metastases were obtained from the UNMC Rapid Autopsy Program and analyzed with quantitative proteomics. PDAC subtypes were identified by principal component analysis based on protein expression profiling. Proteomic subtypes were further characterized by the associated clinical information, including but not limited to survival analysis, drug treatment response, and smoking and drinking status.

Results: Over 3,960 proteins were identified and used to delineate four distinct PDAC microenvironment subtypes: (i) metabolic; (ii) progenitor-like; (iii) proliferative; and (iv) inflammatory. PDAC risk factors of alcohol and tobacco consumption correlate with subtype classifications. Enhanced survival is observed in FOLFIRINOX treated metabolic and progenitor-like subtypes compared with the proliferative and inflammatory subtypes. In addition, TYMP, PDCD6IP, ERAP1, and STMN showed significant association with patient survival in a subtype-specific manner. Gemcitabine-induced alterations in the proteome identify proteins, such as serine hydroxymethyltransferase 1, associated with drug resistance.

Conclusions: These data demonstrate that proteomic analysis of clinical PDAC liver metastases can identify molecular signatures unique to disease subtypes and point to opportunities for therapeutic development to improve the treatment of PDAC.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-1496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056493PMC
March 2020

Truncated O-glycans promote epithelial-to-mesenchymal transition and stemness properties of pancreatic cancer cells.

J Cell Mol Med 2019 10 7;23(10):6885-6896. Epub 2019 Aug 7.

Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA.

Aberrant expression of Sialyl-Tn (STn) antigen correlates with poor prognosis and reduced patient survival. We demonstrated that expression of Tn and STn in pancreatic ductal adenocarcinoma (PDAC) is due to hypermethylation of Core 1 synthase specific molecular chaperone (COSMC) and enhanced the malignant properties of PDAC cells with an unknown mechanism. To explore the mechanism, we have genetically deleted COSMC in PDAC cells to express truncated O-glycans (SimpleCells, SC) which enhanced cell migration and invasion. Since epithelial-to-mesenchymal transition (EMT) play a vital role in metastasis, we have analysed the induction of EMT in SC cells. Expressions of the mesenchymal markers were significantly high in SC cells as compared to WT cells. Equally, we found reduced expressions of the epithelial markers in SC cells. Re-expression of COSMC in SC cells reversed the induction of EMT. In addition to this, we also observed an increased cancer stem cell population in SC cells. Furthermore, orthotopic implantation of T3M4 SC cells into athymic nude mice resulted in significantly larger tumours and reduced animal survival. Altogether, these results suggest that aberrant expression of truncated O-glycans in PDAC cells enhances the tumour aggressiveness through the induction of EMT and stemness properties.
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http://dx.doi.org/10.1111/jcmm.14572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6787448PMC
October 2019

Role of keratan sulfate expression in human pancreatic cancer malignancy.

Sci Rep 2019 07 4;9(1):9665. Epub 2019 Jul 4.

Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA.

Keratan sulfate (KS) is a sulfated linear polymer of N-acetyllactosamine. Proteoglycans carrying keratan sulfate epitopes were majorly observed in cornea, cartilage and brain; and mainly involved in embryonic development, cornea transparency, and wound healing process. Recently, expression of KS in cancer has been shown to be highly associated with advanced tumor grade and poor prognosis. Therefore, we aimed to identify the expression of KS epitope in human pancreatic cancer primary and metastatic tumor lesions. Immunohistochemical analysis of KS expression was performed on primary pancreatic tumors and metastatic tissues. We observed an increased expression of KS epitope on primary tumor tissues compared to uninvolved normal and tumor stroma; and is associated with worse overall survival. Moreover, lung metastatic tumors show a higher-level expression of KS compared to primary tumors. Interestingly, KS biosynthesis specific glycosyltransferases expression was differentially regulated in metastatic pancreatic tumors. Taken together, these results indicate that aberrant expression of KS is predictive of pancreatic cancer progression and metastasis and may serve as a novel prognostic biomarker for pancreatic cancer.
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http://dx.doi.org/10.1038/s41598-019-46046-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609602PMC
July 2019

Targeting LIF-mediated paracrine interaction for pancreatic cancer therapy and monitoring.

Nature 2019 05 17;569(7754):131-135. Epub 2019 Apr 17.

Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Activation of pancreatic stellate cells (PSCs) and consequent development of dense stroma are prominent features accounting for this aggressive biology. The reciprocal interplay between PSCs and pancreatic cancer cells (PCCs) not only enhances tumour progression and metastasis but also sustains their own activation, facilitating a vicious cycle to exacerbate tumorigenesis and drug resistance. Furthermore, PSC activation occurs very early during PDAC tumorigenesis, and activated PSCs comprise a substantial fraction of the tumour mass, providing a rich source of readily detectable factors. Therefore, we hypothesized that the communication between PSCs and PCCs could be an exploitable target to develop effective strategies for PDAC therapy and diagnosis. Here, starting with a systematic proteomic investigation of secreted disease mediators and underlying molecular mechanisms, we reveal that leukaemia inhibitory factor (LIF) is a key paracrine factor from activated PSCs acting on cancer cells. Both pharmacologic LIF blockade and genetic Lifr deletion markedly slow tumour progression and augment the efficacy of chemotherapy to prolong survival of PDAC mouse models, mainly by modulating cancer cell differentiation and epithelial-mesenchymal transition status. Moreover, in both mouse models and human PDAC, aberrant production of LIF in the pancreas is restricted to pathological conditions and correlates with PDAC pathogenesis, and changes in the levels of circulating LIF correlate well with tumour response to therapy. Collectively, these findings reveal a function of LIF in PDAC tumorigenesis, and suggest its translational potential as an attractive therapeutic target and circulating marker. Our studies underscore how a better understanding of cell-cell communication within the tumour microenvironment can suggest novel strategies for cancer therapy.
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http://dx.doi.org/10.1038/s41586-019-1130-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6565370PMC
May 2019

Trefoil factor(s) and CA19.9: A promising panel for early detection of pancreatic cancer.

EBioMedicine 2019 Apr 5;42:375-385. Epub 2019 Apr 5.

Department of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA; Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA; Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA. Electronic address:

Background: Trefoil factors (TFF1, TFF2, and TFF3) are small secretory molecules that recently have gained significant attention in multiple studies as an integral component of pancreatic cancer (PC) subtype-specific gene signature. Here, we comprehensively investigated the diagnostic potential of all the member of trefoil family, i.e., TFF1, TFF2, and TFF3 in combination with CA19.9 for detection of PC.

Methods: Trefoil factors (TFFs) gene expression was analyzed in publicly available cancer genome datasets, followed by assessment of their expression in genetically engineered spontaneous mouse model (GEM) of PC (KrasG12D; Pdx1-Cre (KC)) and in human tissue microarray consisting of normal pancreas adjacent to tumor (NAT), precursor lesions (PanIN), and various pathological grades of PC by immunohistochemistry (IHC). Serum TFFs and CA19.9 levels were evaluated via ELISA in comprehensive sample set (n = 362) comprised of independent training and validation sets each containing benign controls (BC), chronic pancreatitis (CP), and various stages of PC. Univariate and multivariate logistic regression and receiver operating characteristic curves (ROC) were used to examine their diagnostic potential both alone and in combination with CA19.9.

Findings: The publicly available datasets and expression analysis revealed significant increased expression of TFF1, TFF2, and TFF3 in human PanINs and PC tissues. Assessment of KC mouse model also suggested upregulated expression of TFFs in PanIN lesions and early stage of PC. In serum analyses studies, TFF1 and TFF2 were significantly elevated in early stages of PC in comparison to benign and CP control group while significant elevation in TFF3 levels were observed in CP group with no further elevation in its level in early stage PC group. In receiver operating curve (ROC) analyses, combination of TFFs with CA19.9 emerged as promising panel for discriminating early stage of PC (EPC) from BC (AUC = 0.93) as well as CP (AUC = 0.93). Notably, at 90% specificity (desired for blood-based biomarker panel), TFFs combination improved CA19.9 sensitivity by 10% and 25% to differentiate EPC from BC and CP respectively. In an independent blinded validation set, the combination of TFFs and CA19.9 (AUC = 0.82) also improved the overall efficacy of CA19.9 (AUC = 0.66) to differentiate EPC from CP proving unique biomarker capabilities of TFFs to distinguish early stage of this deadly lethal disease.

Interpretation: In silico, tissue and serum analyses validated significantly increased level of all TFFs in precursor lesions and early stages of PC. The combination of TFFs enhanced sensitivity and specificity of CA19.9 to discriminate early stage of PC from benign control and chronic pancreatitis groups.
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http://dx.doi.org/10.1016/j.ebiom.2019.03.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6491718PMC
April 2019

Metastatic cancers promote cachexia through ZIP14 upregulation in skeletal muscle.

Nat Med 2018 06 6;24(6):770-781. Epub 2018 Jun 6.

Institute for Cancer Genetics, Columbia University, New York, NY, USA.

Patients with metastatic cancer experience a severe loss of skeletal muscle mass and function known as cachexia. Cachexia is associated with poor prognosis and accelerated death in patients with cancer, yet its underlying mechanisms remain poorly understood. Here, we identify the metal-ion transporter ZRT- and IRT-like protein 14 (ZIP14) as a critical mediator of cancer-induced cachexia. ZIP14 is upregulated in cachectic muscles of mice and in patients with metastatic cancer and can be induced by TNF-α and TGF-β cytokines. Strikingly, germline ablation or muscle-specific depletion of Zip14 markedly reduces muscle atrophy in metastatic cancer models. We find that ZIP14-mediated zinc uptake in muscle progenitor cells represses the expression of MyoD and Mef2c and blocks muscle-cell differentiation. Importantly, ZIP14-mediated zinc accumulation in differentiated muscle cells induces myosin heavy chain loss. These results highlight a previously unrecognized role for altered zinc homeostasis in metastatic cancer-induced muscle wasting and implicate ZIP14 as a therapeutic target for its treatment.
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http://dx.doi.org/10.1038/s41591-018-0054-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6015555PMC
June 2018

Organoid Profiling Identifies Common Responders to Chemotherapy in Pancreatic Cancer.

Cancer Discov 2018 09 31;8(9):1112-1129. Epub 2018 May 31.

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

Pancreatic cancer is the most lethal common solid malignancy. Systemic therapies are often ineffective, and predictive biomarkers to guide treatment are urgently needed. We generated a pancreatic cancer patient-derived organoid (PDO) library that recapitulates the mutational spectrum and transcriptional subtypes of primary pancreatic cancer. New driver oncogenes were nominated and transcriptomic analyses revealed unique clusters. PDOs exhibited heterogeneous responses to standard-of-care chemotherapeutics and investigational agents. In a case study manner, we found that PDO therapeutic profiles paralleled patient outcomes and that PDOs enabled longitudinal assessment of chemosensitivity and evaluation of synchronous metastases. We derived organoid-based gene expression signatures of chemosensitivity that predicted improved responses for many patients to chemotherapy in both the adjuvant and advanced disease settings. Finally, we nominated alternative treatment strategies for chemorefractory PDOs using targeted agent therapeutic profiling. We propose that combined molecular and therapeutic profiling of PDOs may predict clinical response and enable prospective therapeutic selection. New approaches to prioritize treatment strategies are urgently needed to improve survival and quality of life for patients with pancreatic cancer. Combined genomic, transcriptomic, and therapeutic profiling of PDOs can identify molecular and functional subtypes of pancreatic cancer, predict therapeutic responses, and facilitate precision medicine for patients with pancreatic cancer. .
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http://dx.doi.org/10.1158/2159-8290.CD-18-0349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125219PMC
September 2018

Enhancer Reprogramming Promotes Pancreatic Cancer Metastasis.

Cell 2017 Aug 27;170(5):875-888.e20. Epub 2017 Jul 27.

Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA. Electronic address:

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal human malignancies, owing in part to its propensity for metastasis. Here, we used an organoid culture system to investigate how transcription and the enhancer landscape become altered during discrete stages of disease progression in a PDA mouse model. This approach revealed that the metastatic transition is accompanied by massive and recurrent alterations in enhancer activity. We implicate the pioneer factor FOXA1 as a driver of enhancer activation in this system, a mechanism that renders PDA cells more invasive and less anchorage-dependent for growth in vitro, as well as more metastatic in vivo. In this context, FOXA1-dependent enhancer reprogramming activates a transcriptional program of embryonic foregut endoderm. Collectively, our study implicates enhancer reprogramming, FOXA1 upregulation, and a retrograde developmental transition in PDA metastasis.
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http://dx.doi.org/10.1016/j.cell.2017.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5726277PMC
August 2017

MUC1 and HIF-1alpha Signaling Crosstalk Induces Anabolic Glucose Metabolism to Impart Gemcitabine Resistance to Pancreatic Cancer.

Cancer Cell 2017 07;32(1):71-87.e7

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198-5950, USA; Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA. Electronic address:

Poor response to cancer therapy due to resistance remains a clinical challenge. The present study establishes a widely prevalent mechanism of resistance to gemcitabine in pancreatic cancer, whereby increased glycolytic flux leads to glucose addiction in cancer cells and a corresponding increase in pyrimidine biosynthesis to enhance the intrinsic levels of deoxycytidine triphosphate (dCTP). Increased levels of dCTP diminish the effective levels of gemcitabine through molecular competition. We also demonstrate that MUC1-regulated stabilization of hypoxia inducible factor-1α (HIF-1α) mediates such metabolic reprogramming. Targeting HIF-1α or de novo pyrimidine biosynthesis, in combination with gemcitabine, strongly diminishes tumor burden. Finally, reduced expression of TKT and CTPS, which regulate flux into pyrimidine biosynthesis, correlates with better prognosis in pancreatic cancer patients on fluoropyrimidine analogs.
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http://dx.doi.org/10.1016/j.ccell.2017.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5533091PMC
July 2017

Identification of FRA-1 as a novel player in pancreatic cancer in cooperation with a MUC1: ERK signaling axis.

Oncotarget 2016 Jun;7(26):39996-40011

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA.

The MUC1 glycoprotein is overexpressed and aberrantly glycosylated in >90% of pancreatic ductal adenocarcinoma cases and impacts tumor progression by initiating downstream signaling through phosphorylation of its cytoplasmic tail. Previous studies have demonstrated that MUC1 alters expression of known targets of activator protein 1 (AP-1); however, no studies have evaluated the precise impact of MUC1 signaling on the activity and formation of AP-1. Given the known role of these proteins in modulating migration, invasion, and tumor progression, we explored the effects of MUC1 on AP-1 dimer formation and function. We determined that MUC1 increased the protein levels of c-Jun, the major component of AP-1, and promoted dimerization of c-Jun with the Fos-protein FRA-1. We demonstrate that FRA-1 acts as a potent mediator of migration and invasion in a manner that is modulated by signals through MUC1, which acts as a dominant regulator of specific AP-1 and FRA-1 target genes. Our results provide the first in vivo evidence of a FRA-1 mediated expression profile that impacts pancreatic tumor growth properties. In summary, we show that MUC1 enhancement of ERK activation influences FRA-1 activity to modulate tumor migration, invasion and metastasis in a subset of pancreatic cancer cases.
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http://dx.doi.org/10.18632/oncotarget.9557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5129987PMC
June 2016

Oncofetal Chondroitin Sulfate Glycosaminoglycans Are Key Players in Integrin Signaling and Tumor Cell Motility.

Mol Cancer Res 2016 12 21;14(12):1288-1299. Epub 2016 Sep 21.

Centre for Medical Parasitology at Department of International Health, Immunology and Microbiology, University of Copenhagen, Denmark.

Many tumors express proteoglycans modified with oncofetal chondroitin sulfate glycosaminoglycan chains (ofCS), which are normally restricted to the placenta. However, the role of ofCS in cancer is largely unknown. The function of ofCS in cancer was analyzed using the recombinant ofCS-binding VAR2CSA protein (rVAR2) derived from the malaria parasite, Plasmodium falciparum We demonstrate that ofCS plays a key role in tumor cell motility by affecting canonical integrin signaling pathways. Binding of rVAR2 to tumor cells inhibited the interaction of cells with extracellular matrix (ECM) components, which correlated with decreased phosphorylation of Src kinase. Moreover, rVAR2 binding decreased migration, invasion, and anchorage-independent growth of tumor cells in vitro Mass spectrometry of ofCS-modified proteoglycan complexes affinity purified from tumor cell lines on rVAR2 columns revealed an overrepresentation of proteins involved in cell motility and integrin signaling, such as integrin-β1 (ITGB1) and integrin-α4 (ITGA4). Saturating concentrations of rVAR2 inhibited downstream integrin signaling, which was mimicked by knockdown of the core chondroitin sulfate synthesis enzymes β-1,3-glucuronyltransferase 1 (B3GAT1) and chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGALNACT1). The ofCS modification was highly expressed in both human and murine metastatic lesions in situ and preincubation or early intravenous treatment of tumor cells with rVAR2 inhibited seeding and spreading of tumor cells in mice. This was associated with a significant increase in survival of the animals. These data functionally link ofCS modifications with cancer cell motility and further highlights ofCS as a novel therapeutic cancer target.

Implications: The cancer-specific expression of ofCS aids in metastatic phenotypes and is a candidate target for therapy. Mol Cancer Res; 14(12); 1288-99. ©2016 AACR.
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http://dx.doi.org/10.1158/1541-7786.MCR-16-0103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5136311PMC
December 2016

Reflections on MUC1 glycoprotein: the hidden potential of isoforms in carcinogenesis.

APMIS 2016 Nov 19;124(11):913-924. Epub 2016 Aug 19.

i3S-Institute of Research and Innovation in Health, University of Porto, Porto, Portugal.

Mucin 1 (MUC1) has been described as the renaissance molecule due to the large set of functions it displays in both normal and neoplastic cells. This membrane-tethered glycoprotein is overexpressed and aberrantly glycosylated in most epithelial cancers, being involved in several processes related with malignant phenotype acquisition. With a highly polymorphic structure, both in the polypeptide and glycan counterparts, MUC1 variability has been associated with susceptibility to several diseases, including cancer. Biochemical features and biological functions have been characterized upon the full-length MUC1 protein, remaining to clarify the real impact on cell dynamics of the plethora of MUC1 isoforms. This review aims to encompass a detailed characterization of MUC1 role in carcinogenesis, highlighting recent findings in cell differentiation and uncovering new evidences of MUC1 isoforms involvement in malignant phenotype.
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http://dx.doi.org/10.1111/apm.12587DOI Listing
November 2016

Image-based detection and targeting of therapy resistance in pancreatic adenocarcinoma.

Nature 2016 06 6;534(7607):407-411. Epub 2016 Jun 6.

Departments of Pharmacology and Medicine, University of California San Diego School of Medicine La Jolla, CA.

Pancreatic intraepithelial neoplasia is a pre-malignant lesion that can progress to pancreatic ductal adenocarcinoma, a highly lethal malignancy marked by its late stage at clinical presentation and profound drug resistance. The genomic alterations that commonly occur in pancreatic cancer include activation of KRAS2 and inactivation of p53 and SMAD4 (refs 2-4). So far, however, it has been challenging to target these pathways therapeutically; thus the search for other key mediators of pancreatic cancer growth remains an important endeavour. Here we show that the stem cell determinant Musashi (Msi) is a critical element of pancreatic cancer progression both in genetic models and in patient-derived xenografts. Specifically, we developed Msi reporter mice that allowed image-based tracking of stem cell signals within cancers, revealing that Msi expression rises as pancreatic intraepithelial neoplasia progresses to adenocarcinoma, and that Msi-expressing cells are key drivers of pancreatic cancer: they preferentially harbour the capacity to propagate adenocarcinoma, are enriched in circulating tumour cells, and are markedly drug resistant. This population could be effectively targeted by deletion of either Msi1 or Msi2, which led to a striking defect in the progression of pancreatic intraepithelial neoplasia to adenocarcinoma and an improvement in overall survival. Msi inhibition also blocked the growth of primary patient-derived tumours, suggesting that this signal is required for human disease. To define the translational potential of this work we developed antisense oligonucleotides against Msi; these showed reliable tumour penetration, uptake and target inhibition, and effectively blocked pancreatic cancer growth. Collectively, these studies highlight Msi reporters as a unique tool to identify therapy resistance, and define Msi signalling as a central regulator of pancreatic cancer.
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http://dx.doi.org/10.1038/nature17988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4998062PMC
June 2016

Tumour exosome integrins determine organotropic metastasis.

Nature 2015 Nov 28;527(7578):329-35. Epub 2015 Oct 28.

Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, New York 10021, USA.

Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.
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http://dx.doi.org/10.1038/nature15756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4788391PMC
November 2015

Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver.

Nat Cell Biol 2015 Jun 18;17(6):816-26. Epub 2015 May 18.

1] Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medical College, New York, New York 10021, USA [2] Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA.

Pancreatic ductal adenocarcinomas (PDACs) are highly metastatic with poor prognosis, mainly due to delayed detection. We hypothesized that intercellular communication is critical for metastatic progression. Here, we show that PDAC-derived exosomes induce liver pre-metastatic niche formation in naive mice and consequently increase liver metastatic burden. Uptake of PDAC-derived exosomes by Kupffer cells caused transforming growth factor β secretion and upregulation of fibronectin production by hepatic stellate cells. This fibrotic microenvironment enhanced recruitment of bone marrow-derived macrophages. We found that macrophage migration inhibitory factor (MIF) was highly expressed in PDAC-derived exosomes, and its blockade prevented liver pre-metastatic niche formation and metastasis. Compared with patients whose pancreatic tumours did not progress, MIF was markedly higher in exosomes from stage I PDAC patients who later developed liver metastasis. These findings suggest that exosomal MIF primes the liver for metastasis and may be a prognostic marker for the development of PDAC liver metastasis.
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http://dx.doi.org/10.1038/ncb3169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5769922PMC
June 2015

MicroRNA-200c modulates the expression of MUC4 and MUC16 by directly targeting their coding sequences in human pancreatic cancer.

PLoS One 2013 25;8(10):e73356. Epub 2013 Oct 25.

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.

Transmembrane mucins, MUC4 and MUC16 are associated with tumor progression and metastatic potential in human pancreatic adenocarcinoma. We discovered that miR-200c interacts with specific sequences within the coding sequence of MUC4 and MUC16 mRNAs, and evaluated the regulatory nature of this association. Pancreatic cancer cell lines S2.028 and T3M-4 transfected with miR-200c showed a 4.18 and 8.50 fold down regulation of MUC4 mRNA, and 4.68 and 4.82 fold down regulation of MUC16 mRNA compared to mock-transfected cells, respectively. A significant reduction of glycoprotein expression was also observed. These results indicate that miR-200c overexpression regulates MUC4 and MUC16 mucins in pancreatic cancer cells by directly targeting the mRNA coding sequence of each, resulting in reduced levels of MUC4 and MUC16 mRNA and protein. These data suggest that, in addition to regulating proteins that modulate EMT, miR-200c influences expression of cell surface mucins in pancreatic cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0073356PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3808362PMC
August 2014

Expression of core 3 synthase in human pancreatic cancer cells suppresses tumor growth and metastasis.

Int J Cancer 2013 Dec 5;133(12):2824-33. Epub 2013 Aug 5.

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE.

Core 3-derived glycans, a major type of O-glycan expressed by normal epithelial cells of the gastrointestinal tract, are downregulated during malignancy because of loss of expression of functional β3-N-acetylglucosaminyltransferase-6 (core 3 synthase). We investigated the expression of core 3 synthase in normal pancreas and pancreatic cancer and evaluated the biological effects of re-expressing core 3 synthase in pancreatic cancer cells that had lost expression. We determined that pancreatic tumors and tumor cell lines have lost expression of core 3 synthase. Therefore, we re-expressed core 3 synthase in human pancreatic cancer cells (Capan-2 and FG) to investigate the contribution of core 3 glycans to malignant progression. Pancreatic cancer cells expressing core 3 synthase showed reduced in vitro cell proliferation, migration and invasion compared to vector control cells. Expression of core 3 O-glycans induced altered expression of β1 integrin, decreased activation of focal adhesion kinase, led to the downregulation of expression of several genes including REG1α and FGFR3 and altered lamellipodia formation. The addition of a GlcNAc residue by core 3 synthase leads to the extension of the tumor-associated Tn structure on MUC1. Orthotopic injection of FG cells expressing core 3 synthase into the pancreas of nude mice produced significantly smaller tumors and decreased metastasis to the surrounding tissues compared to vector control FG cells. These findings indicate that expression of core 3-derived O-glycans in pancreatic cancer cells suppresses tumor growth and metastasis through modulation of glycosylation of mucins and other cell surface and extracellular matrix proteins.
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http://dx.doi.org/10.1002/ijc.28322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3873636PMC
December 2013

Targeting the NF-κB and mTOR pathways with a quinoxaline urea analog that inhibits IKKβ for pancreas cancer therapy.

Clin Cancer Res 2013 Apr 26;19(8):2025-35. Epub 2013 Feb 26.

Eppley Institute for Cancer Research and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.

Purpose: The presence of TNF-α in approximately 50% of surgically resected tumors suggests that the canonical NF-κB and the mTOR pathways are activated. Inhibitor of IκB kinase β (IKKβ) acts as the signaling node that regulates transcription via the p-IκBα/NF-κB axis and regulates translation via the mTOR/p-S6K/p-eIF4EBP axis. A kinome screen identified a quinoxaline urea analog 13-197 as an IKKβ inhibitor. We hypothesized that targeting the NF-κB and mTOR pathways with 13-197 will be effective in malignancies driven by these pathways.

Experimental Design: Retrospective clinical and preclinical studies in pancreas cancers have implicated NF-κB. We examined the effects of 13-197 on the downstream targets of the NF-κB and mTOR pathways in pancreatic cancer cells, pharmacokinetics, toxicity and tumor growth, and metastases in vivo.

Results: 13-197 inhibited the kinase activity of IKKβ in vitro and TNF-α-mediated NF-κB transcription in cells with low-μmol/L potency. 13-197 inhibited the phosphorylation of IκBα, S6K, and eIF4EBP, induced G1 arrest, and downregulated the expression of antiapoptotic proteins in pancreatic cancer cells. Prolonged administration of 13-197 did not induce granulocytosis and protected mice from lipopolysaccharide (LPS)-induced death. Results also show that 13-197 is orally available with extensive distribution to peripheral tissues and inhibited tumor growth and metastasis in an orthotopic pancreatic cancer model without any detectable toxicity.

Conclusion: These results suggest that 13-197 targets IKKβ and thereby inhibits mTOR and NF-κB pathways. Oral availability along with in vivo efficacy without obvious toxicities makes this quinoxaline urea chemotype a viable cancer therapeutic.
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http://dx.doi.org/10.1158/1078-0432.CCR-12-2909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3630250PMC
April 2013

Rosiglitazone and Gemcitabine in combination reduces immune suppression and modulates T cell populations in pancreatic cancer.

Cancer Immunol Immunother 2013 Feb 5;62(2):225-36. Epub 2012 Aug 5.

Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, NE 68198, USA.

Pancreatic ductal adenocarcinoma is a leading cause of cancer mortality with a dismal 2-5 % 5-year survival rate. Monotherapy with Gemcitabine has limited success, highlighting the need for additional therapies that enhance the efficacy of current treatments. We evaluated the combination of Gemcitabine and Rosiglitazone, an FDA-approved drug for the treatment of type II diabetes, in an immunocompetent transplantable mouse model of pancreatic cancer. Tumor progression, survival, and metastases were evaluated in immunocompetent mice with subcutaneous or orthotopic pancreatic tumors treated with Pioglitazone, Rosiglitazone, Gemcitabine, or combinations of these. We characterized the impact of high-dose Rosiglitazone and Gemcitabine therapy on immune suppressive mediators, including MDSC and T regulatory cells, and on modulation of peripheral and intra-tumoral T cell populations. Combinations of Rosiglitazone and Gemcitabine significantly reduced tumor progression and metastases, enhanced apoptosis, and significantly extended overall survival compared to Gemcitabine alone. Rosiglitazone altered tumor-associated immune suppressive mediators by limiting early MDSC accumulation and intra-tumoral T regulatory cells. Combination therapy with Rosiglitazone and Gemcitabine modulated T cell populations by enhancing circulating CD8(+) T cells and intra-tumoral CD4(+) and CD8(+) T cells while limiting T regulatory cells. The results suggest that Rosiglitazone, in combination with Gemcitabine, decreases immune suppressive mechanisms in immunocompetent animals and provides pre-clinical data in support of combining Rosiglitazone and Gemcitabine as a clinical therapy for pancreatic cancer.
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http://dx.doi.org/10.1007/s00262-012-1324-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3873637PMC
February 2013

Cyclin-dependent kinase 5 is amplified and overexpressed in pancreatic cancer and activated by mutant K-Ras.

Clin Cancer Res 2011 Oct 8;17(19):6140-50. Epub 2011 Aug 8.

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.

Purpose: To evaluate the nature of cyclin-dependent kinase 5 (CDK5) hyperactivity in pancreatic cancer progression.

Experimental Design: We used genetic, biochemical, and molecular biology methods to investigate the nature and function of overexpression of CDK5 and its activators p35 and p39 during the progression of pancreatic cancer.

Results: Amplification of the CDK5 gene or either of its main activators, p35 and p39, was observed in 67% of human pancreatic ductal adenocarcinoma (PDAC). CDK5, p35, and p39 were rarely expressed in pancreatic ducts whereas more than 90% of PDACs had increased levels of CDK5 and p35. Increased levels of CDK5, p35, and p39 protein were observed in several pancreatic cancer cell lines. Inhibition of CDK5 kinase activity using a CDK5 dominant-negative mutant or the drug roscovitine significantly decreased the migration and invasion of pancreatic cancer cells in vitro. Increased CDK5 kinase activity was also observed in immortalized human pancreatic nestin-expressing (HPNE) cells expressing a mutant form of K-Ras (G12D) compared with HPNE cells expressing native K-Ras. G12D K-Ras increased cleavage of p35 to p25, a stable and greater activator of CDK5, thus implicating a role for CDK5 in early progression of PDAC. Inhibition of the signaling cascade downstream of mutant K-Ras (G12D) that involves mitogen-activated protein/extracellular signal-regulated kinase, phosphoinositide 3-kinase, or CDK5 decreased p25 protein levels.

Conclusion: These results suggest that mutant K-Ras acts in concert with CDK5 and its activators to increase malignant progression, migration, and invasion of pancreatic cancer cells.
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http://dx.doi.org/10.1158/1078-0432.CCR-10-2288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3425449PMC
October 2011