Publications by authors named "Howard C Crawford"

92 Publications

Pancreatic cancer is marked by complement-high blood monocytes and tumor-associated macrophages.

Life Sci Alliance 2021 06 29;4(6). Epub 2021 Mar 29.

Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA

Pancreatic ductal adenocarcinoma (PDA) is accompanied by reprogramming of the local microenvironment, but changes at distal sites are poorly understood. We implanted biomaterial scaffolds, which act as an artificial premetastatic niche, into immunocompetent tumor-bearing and control mice, and identified a unique tumor-specific gene expression signature that includes high expression of , , , and Single-cell RNA sequencing mapped these genes to two distinct macrophage populations in the scaffolds, one marked by elevated , , and , the other with high , and In mice, expression of these genes in the corresponding populations was elevated in tumor-associated macrophages compared with macrophages in the normal pancreas. We then analyzed single-cell RNA sequencing from patient samples, and determined expression of , , and is elevated in human macrophages in primary tumors and liver metastases. Single-cell sequencing analysis of patient blood revealed a substantial enrichment of the same gene signature in monocytes. Taken together, our study identifies two distinct tumor-associated macrophage and monocyte populations that reflects systemic immune changes in pancreatic ductal adenocarcinoma patients.
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http://dx.doi.org/10.26508/lsa.202000935DOI Listing
June 2021

Inhibition of Hedgehog Signaling Alters Fibroblast Composition in Pancreatic Cancer.

Clin Cancer Res 2021 Apr 25;27(7):2023-2037. Epub 2021 Jan 25.

Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease characterized by an extensive fibroinflammatory stroma, which includes abundant cancer-associated fibroblast (CAF) populations. PDAC CAFs are heterogeneous, but the nature of this heterogeneity is incompletely understood. The Hedgehog pathway functions in PDAC in a paracrine manner, with ligands secreted by cancer cells signaling to stromal cells in the microenvironment. Previous reports investigating the role of Hedgehog signaling in PDAC have been contradictory, with Hedgehog signaling alternately proposed to promote or restrict tumor growth. In light of the newly discovered CAF heterogeneity, we investigated how Hedgehog pathway inhibition reprograms the PDAC microenvironment.

Experimental Design: We used a combination of pharmacologic inhibition, gain- and loss-of-function genetic experiments, cytometry by time-of-flight, and single-cell RNA sequencing to study the roles of Hedgehog signaling in PDAC.

Results: We found that Hedgehog signaling is uniquely activated in fibroblasts and differentially elevated in myofibroblastic CAFs (myCAF) compared with inflammatory CAFs (iCAF). Sonic Hedgehog overexpression promotes tumor growth, while Hedgehog pathway inhibition with the smoothened antagonist, LDE225, impairs tumor growth. Furthermore, Hedgehog pathway inhibition reduces myCAF numbers and increases iCAF numbers, which correlates with a decrease in cytotoxic T cells and an expansion in regulatory T cells, consistent with increased immunosuppression.

Conclusions: Hedgehog pathway inhibition alters fibroblast composition and immune infiltration in the pancreatic cancer microenvironment.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-3715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026631PMC
April 2021

ATDC binds to KEAP1 to drive NRF2-mediated tumorigenesis and chemoresistance in pancreatic cancer.

Genes Dev 2021 Feb 14;35(3-4):218-233. Epub 2021 Jan 14.

Perlmutter Cancer Center, New York University, New York, New York 10016, USA.

Pancreatic ductal adenocarcinoma is a lethal disease characterized by late diagnosis, propensity for early metastasis and resistance to chemotherapy. Little is known about the mechanisms that drive innate therapeutic resistance in pancreatic cancer. The ataxia-telangiectasia group D-associated gene (ATDC) is overexpressed in pancreatic cancer and promotes tumor growth and metastasis. Our study reveals that increased ATDC levels protect cancer cells from reactive oxygen species (ROS) via stabilization of nuclear factor erythroid 2-related factor 2 (NRF2). Mechanistically, ATDC binds to Kelch-like ECH-associated protein 1 (KEAP1), the principal regulator of NRF2 degradation, and thereby prevents degradation of NRF2 resulting in activation of a NRF2-dependent transcriptional program, reduced intracellular ROS and enhanced chemoresistance. Our findings define a novel role of ATDC in regulating redox balance and chemotherapeutic resistance by modulating NRF2 activity.
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http://dx.doi.org/10.1101/gad.344184.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849366PMC
February 2021

Disease-induced immunomodulation at biomaterial scaffolds detects early pancreatic cancer in a spontaneous model.

Biomaterials 2021 Feb 23;269:120632. Epub 2020 Dec 23.

Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA; Department of Chemical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA. Electronic address:

Pancreatic cancer has the worst prognosis of all cancers due to disease aggressiveness and paucity of early detection platforms. We developed biomaterial scaffolds that recruit metastatic tumor cells and reflect the immune dysregulation of native metastatic sites. While this platform has shown promise in orthotopic breast cancer models, its potential in other models is untested. Herein, we demonstrate that scaffolds recruit disseminated pancreatic cells in the KPCY model of spontaneous pancreatic cancer prior to adenocarcinoma formation (3-fold increase in scaffold YFP + cells). Furthermore, immune cells at the scaffolds differentiate early- and late-stage disease with greater accuracy (0.83) than the natural metastatic site (liver, 0.50). Early disease was identified by an approximately 2-fold increase in monocytes. Late-stage disease was marked by a 1.5-2-fold increase in T cells and natural killer cells. The differential immune response indicated that the scaffolds could distinguish spontaneous pancreatic cancer from spontaneous breast cancer. Collectively, our findings demonstrate the utility of scaffolds to reflect immunomodulation in two spontaneous models of tumorigenesis, and their particular utility for identifying early disease stages in the aggressive KPCY pancreatic cancer model. Such scaffolds may serve as a platform for early detection of pancreatic cancer to improve treatment and prognosis.
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http://dx.doi.org/10.1016/j.biomaterials.2020.120632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870556PMC
February 2021

Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer.

Gastroenterology 2021 Jan 9;160(1):362-377.e13. Epub 2020 Oct 9.

Barts Cancer Institute, Queen Mary University of London, London, United Kingdom.

Background & Aims: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarker-driven therapeutic strategy targeting DDR and replication stress in PC.

Methods: We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids.

Results: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P < .001) and PARP inhibitor therapy (P < .001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P < .018) and WEE1 inhibitor (P < .029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < .001) but was not associated with DDR deficiency.

Conclusions: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy.
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http://dx.doi.org/10.1053/j.gastro.2020.09.043DOI Listing
January 2021

The Gustatory Sensory G-Protein GNAT3 Suppresses Pancreatic Cancer Progression in Mice.

Cell Mol Gastroenterol Hepatol 2021 31;11(2):349-369. Epub 2020 Aug 31.

Rogel Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan; Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan. Electronic address:

Background & Aims: Pancreatic ductal adenocarcinoma (PDA) initiation and progression are accompanied by an immunosuppressive inflammatory response. Here, we evaluated the immunomodulatory role of chemosensory signaling in metaplastic tuft cells (MTCs) by analyzing the role of GNAT3, a gustatory pathway G-protein expressed by MTCs, during PDA progression.

Methods: Gnat3-null (Gnat3) mice were crossbred with animals harboring a Cre-inducible Kras allele with either Ptf1a (KC) or tamoxifen-inducible Ptf1a (KC) mice to drive oncogenic KRAS expression in the pancreas. Ex vivo organoid conditioned medium generated from KC and Gnat3;KC acinar cells was analyzed for cytokine secretion. Experimental pancreatitis was induced in KC and Gnat3;KC mice to accelerate tumorigenesis, followed by analysis using mass cytometry and single-cell RNA sequencing. To study PDA progression, KC and Gnat3;KC mice were aged to morbidity or 52 weeks.

Results: Ablation of Gnat3 in KC organoids increased release of tumor-promoting cytokines in conditioned media, including CXCL1 and CXCL2. Analysis of Gnat3;KC pancreata found altered expression of immunomodulatory genes in Cxcr2 expressing myeloid-derived suppressor cells (MDSCs) and an increased number of granulocytic MDSCs, a subset of tumor promoting MDSCs. Importantly, expression levels of CXCL1 and CXCL2, known ligands for CXCR2, were also elevated in Gnat3;KC pancreata. Consistent with the tumor-promoting role of MDSCs, aged Gnat3;KC mice progressed more rapidly to metastatic carcinoma compared with KC controls.

Conclusions: Compromised gustatory sensing, achieved by Gnat3 ablation, enhanced the CXCL1/2-CXCR2 axis to alter the MDSC population and promoted the progression of metastatic PDA.
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http://dx.doi.org/10.1016/j.jcmgh.2020.08.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7779788PMC
August 2020

Beta 1 integrin signaling mediates pancreatic ductal adenocarcinoma resistance to MEK inhibition.

Sci Rep 2020 07 7;10(1):11133. Epub 2020 Jul 7.

Department of Surgery, University of Michigan Cancer Center, University of Michigan, 1500 East Medical Center Drive, Cancer Center Room 6306, Ann Arbor, MI, 48109-0944, USA.

Pancreatic cancer, one of the deadliest human malignancies, has a dismal 5-year survival rate of 9%. KRAS is the most commonly mutated gene in pancreatic cancer, but clinical agents that directly target mutant KRAS are not available. Several effector pathways are activated downstream of oncogenic Kras, including MAPK signaling. MAPK signaling can be inhibited by targeting MEK1/2; unfortunately, this approach has been largely ineffective in pancreatic cancer. Here, we set out to identify mechanisms of MEK inhibitor resistance in pancreatic cancer. We optimized the culture of pancreatic tumor 3D clusters that utilized Matrigel as a basement membrane mimetic. Pancreatic tumor 3D clusters recapitulated mutant KRAS dependency and recalcitrance to MEK inhibition. Treatment of the clusters with trametinib, a MEK inhibitor, had only a modest effect on these cultures. We observed that cells adjacent to the basement membrane mimetic Matrigel survived MEK inhibition, while the cells in the interior layers underwent apoptosis. Our findings suggested that basement membrane attachment provided survival signals. We thus targeted integrin β1, a mediator of extracellular matrix contact, and found that combined MEK and integrin β1 inhibition bypassed trametinib resistance. Our data support exploring integrin signaling inhibition as a component of combination therapy in pancreatic cancer.
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http://dx.doi.org/10.1038/s41598-020-67814-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340786PMC
July 2020

An essential role for Argonaute 2 in EGFR-KRAS signaling in pancreatic cancer development.

Nat Commun 2020 06 4;11(1):2817. Epub 2020 Jun 4.

Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, MI, 48109, USA.

Both KRAS and EGFR are essential mediators of pancreatic cancer development and interact with Argonaute 2 (AGO2) to perturb its function. Here, in a mouse model of mutant KRAS-driven pancreatic cancer, loss of AGO2 allows precursor lesion (PanIN) formation yet prevents progression to pancreatic ductal adenocarcinoma (PDAC). Precursor lesions with AGO2 ablation undergo oncogene-induced senescence with altered microRNA expression and EGFR/RAS signaling, bypassed by loss of p53. In mouse and human pancreatic tissues, PDAC progression is associated with increased plasma membrane localization of RAS/AGO2. Furthermore, phosphorylation of AGO2 disrupts both the wild-type and oncogenic KRAS-AGO2 interaction, albeit under different conditions. ARS-1620 (G12C-specific inhibitor) disrupts the KRAS-AGO2 interaction, suggesting that the interaction is targetable. Altogether, our study supports a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR-RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical for PDAC progression.
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http://dx.doi.org/10.1038/s41467-020-16309-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272436PMC
June 2020

Differential Contribution of Pancreatic Fibroblast Subsets to the Pancreatic Cancer Stroma.

Cell Mol Gastroenterol Hepatol 2020 23;10(3):581-599. Epub 2020 May 23.

Department of Surgery, University of Michigan, Ann Arbor, Michigan; Rogel Cancer Center, University of Michigan, Ann Arbor, Michigan; Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan. Electronic address:

Background & Aims: Although the healthy pancreas consists mostly of epithelial cells, pancreatic cancer and the precursor lesions known as pancreatic intraepithelial neoplasia, are characterized by an extensive accumulation of fibroinflammatory stroma that includes a substantial and heterogeneous fibroblast population. The cellular origin of fibroblasts within the stroma has not been determined. Here, we show that the Gli1 and Hoxb6 markers label distinct fibroblast populations in the healthy mouse pancreas. We then set out to determine whether these distinct fibroblast populations expanded during carcinogenesis.

Methods: We developed genetically engineered models using a dual-recombinase approach that allowed us to induce pancreatic cancer formation through codon-optimized Flp recombinase-driven epithelial recombination of Kirsten rat sarcoma viral oncogene homolog while labeling Gli1 or Hoxb6 fibroblasts in an inducible manner. By using these models, we lineage-traced these 2 fibroblast populations during the process of carcinogenesis.

Results: Although in the healthy pancreas Gli1 fibroblasts and Hoxb6 fibroblasts are present in similar numbers, they contribute differently to the stroma in carcinogenesis. Namely, Gli1 fibroblasts expand dramatically, whereas Hoxb6 cells do not.

Conclusions: Fibroblasts present in the healthy pancreas expand during carcinogenesis, but with a different prevalence for different subtypes. Here, we compared Gli1 and Hoxb6 fibroblasts and found only Gli1 expanded to contribute to the stroma during pancreatic carcinogenesis.
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http://dx.doi.org/10.1016/j.jcmgh.2020.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7399194PMC
May 2020

HNF4A and GATA6 Loss Reveals Therapeutically Actionable Subtypes in Pancreatic Cancer.

Cell Rep 2020 05;31(6):107625

The Kinghorn Cancer Centre, 370 Victoria Street, Darlinghurst and Garvan Institute of Medical Research, Sydney, NSW 2010, Australia; St Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia.

Pancreatic ductal adenocarcinoma (PDAC) can be divided into transcriptomic subtypes with two broad lineages referred to as classical (pancreatic) and squamous. We find that these two subtypes are driven by distinct metabolic phenotypes. Loss of genes that drive endodermal lineage specification, HNF4A and GATA6, switch metabolic profiles from classical (pancreatic) to predominantly squamous, with glycogen synthase kinase 3 beta (GSK3β) a key regulator of glycolysis. Pharmacological inhibition of GSK3β results in selective sensitivity in the squamous subtype; however, a subset of these squamous patient-derived cell lines (PDCLs) acquires rapid drug tolerance. Using chromatin accessibility maps, we demonstrate that the squamous subtype can be further classified using chromatin accessibility to predict responsiveness and tolerance to GSK3β inhibitors. Our findings demonstrate that distinct patterns of chromatin accessibility can be used to identify patient subgroups that are indistinguishable by gene expression profiles, highlighting the utility of chromatin-based biomarkers for patient selection in the treatment of PDAC.
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http://dx.doi.org/10.1016/j.celrep.2020.107625DOI Listing
May 2020

Discoidin Domain Receptor 1 (DDR1) Is Necessary for Tissue Homeostasis in Pancreatic Injury and Pathogenesis of Pancreatic Ductal Adenocarcinoma.

Am J Pathol 2020 08 24;190(8):1735-1751. Epub 2020 Apr 24.

Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan; Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan; Department of Surgery, University of Michigan, Ann Arbor, Michigan. Electronic address:

Pancreatic ductal adenocarcinoma (PDA) and chronic pancreatitis are characterized by a dense collagen-rich desmoplastic reaction. Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase activated by collagens that can regulate cell proliferation, migration, adhesion, and remodeling of the extracellular matrix. To address the role of DDR1 in PDA, Ddr1-null (Ddr) mice were crossed with the Kras; Trp53; Ptf1a (KPC) model of metastatic PDA. Ddr1; KPC mice progress to differentiated PDA but resist progression to poorly differentiated cancer compared with KPC control mice. Strikingly, severe pancreatic atrophy accompanied tumor progression in Ddr1; KPC mice. To further explore the effects of Ddr1 ablation, Ddr1 mice were crossed with the Kras; Ptf1a neoplasia model and subjected to cerulein-induced experimental pancreatitis. Similar to KPC mice, tissue atrophy was a hallmark of both neoplasia and pancreatitis models in the absence of Ddr1. Compared with controls, Ddr1 models had increased acinar cell dropout and reduced proliferation with no difference in apoptotic cell death between control and Ddr1 animals. In most models, organ atrophy was accompanied by increased fibrillar collagen deposition, suggesting a compensatory response in the absence of this collagen receptor. Overall, these data suggest that DDR1 regulates tissue homeostasis in the neoplastic and injured pancreas.
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http://dx.doi.org/10.1016/j.ajpath.2020.03.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416078PMC
August 2020

Carcinogenesis of Pancreatic Ductal Adenocarcinoma.

Gastroenterology 2020 06 19;158(8):2072-2081. Epub 2020 Mar 19.

Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan. Electronic address:

Although the estimated time for development of pancreatic ductal adenocarcinoma (PDA) is more than 20 years, PDAs are usually detected at late, metastatic stages. PDAs develop from duct-like cells through a multistep carcinogenesis process, from low-grade dysplastic lesions to carcinoma in situ and eventually to metastatic disease. This process involves gradual acquisition of mutations in oncogenes and tumor suppressor genes, as well as changes in the pancreatic environment from a pro-inflammatory microenvironment that favors the development of early lesions, to a desmoplastic tumor microenvironment that is highly fibrotic and immune suppressive. This review discusses our current understanding of how PDA originates.
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http://dx.doi.org/10.1053/j.gastro.2020.02.059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7282937PMC
June 2020

Early detection and imaging strategies to reveal and target developing pancreatic cancer.

Expert Rev Anticancer Ther 2020 02 27;20(2):81-83. Epub 2020 Jan 27.

Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Mayo Clinic, Jacksonville, FL, USA.

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http://dx.doi.org/10.1080/14737140.2020.1720654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7380330PMC
February 2020

Regulatory T-cell Depletion Alters the Tumor Microenvironment and Accelerates Pancreatic Carcinogenesis.

Cancer Discov 2020 03 7;10(3):422-439. Epub 2020 Jan 7.

Department of Surgery, University of Michigan, Ann Arbor, Michigan.

Regulatory T cells (Treg) are abundant in human and mouse pancreatic cancer. To understand the contribution to the immunosuppressive microenvironment, we depleted Tregs in a mouse model of pancreatic cancer. Contrary to our expectations, Treg depletion failed to relieve immunosuppression and led to accelerated tumor progression. We show that Tregs are a key source of TGFβ ligands and, accordingly, their depletion reprogramed the fibroblast population, with loss of tumor-restraining, smooth muscle actin-expressing fibroblasts. Conversely, we observed an increase in chemokines , and leading to increased myeloid cell recruitment, restoration of immune suppression, and promotion of carcinogenesis, an effect that was inhibited by blockade of the common CCL3/6/8 receptor CCR1. Further, Treg depletion unleashed pathologic CD4 T-cell responses. Our data point to new mechanisms regulating fibroblast differentiation in pancreatic cancer and support the notion that fibroblasts are a heterogeneous population with different and opposing functions in pancreatic carcinogenesis. SIGNIFICANCE: Here, we describe an unexpected cross-talk between Tregs and fibroblasts in pancreatic cancer. Treg depletion resulted in differentiation of inflammatory fibroblast subsets, in turn driving infiltration of myeloid cells through CCR1, thus uncovering a potentially new therapeutic approach to relieve immunosuppression in pancreatic cancer...
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http://dx.doi.org/10.1158/2159-8290.CD-19-0958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7224338PMC
March 2020

Tissue of origin dictates GOT1 dependence and confers synthetic lethality to radiotherapy.

Cancer Metab 2020 2;8. Epub 2020 Jan 2.

1Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI 48109 USA.

Background: Metabolic programs in cancer cells are influenced by genotype and the tissue of origin. We have previously shown that central carbon metabolism is rewired in pancreatic ductal adenocarcinoma (PDA) to support proliferation through a glutamate oxaloacetate transaminase 1 (GOT1)-dependent pathway.

Methods: We utilized a doxycycline-inducible shRNA-mediated strategy to knockdown GOT1 in PDA and colorectal cancer (CRC) cell lines and tumor models of similar genotype. These cells were analyzed for the ability to form colonies and tumors to test if tissue type impacted GOT1 dependence. Additionally, the ability of GOT1 to impact the response to chemo- and radiotherapy was assessed. Mechanistically, the associated specimens were examined using a combination of steady-state and stable isotope tracing metabolomics strategies and computational modeling. Statistics were calculated using GraphPad Prism 7. One-way ANOVA was performed for experiments comparing multiple groups with one changing variable. Student's test (unpaired, two-tailed) was performed when comparing two groups to each other. Metabolomics data comparing three PDA and three CRC cell lines were analyzed by performing Student's test (unpaired, two-tailed) between all PDA metabolites and CRC metabolites.

Results: While PDA exhibits profound growth inhibition upon GOT1 knockdown, we found CRC to be insensitive. In PDA, but not CRC, GOT1 inhibition disrupted glycolysis, nucleotide metabolism, and redox homeostasis. These insights were leveraged in PDA, where we demonstrate that radiotherapy potently enhanced the effect of GOT1 inhibition on tumor growth.

Conclusions: Taken together, these results illustrate the role of tissue type in dictating metabolic dependencies and provide new insights for targeting metabolism to treat PDA.
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http://dx.doi.org/10.1186/s40170-019-0202-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941320PMC
January 2020

Interleukin 22 Signaling Regulates Acinar Cell Plasticity to Promote Pancreatic Tumor Development in Mice.

Gastroenterology 2020 04 14;158(5):1417-1432.e11. Epub 2019 Dec 14.

Department of Surgery, University of Michigan, Ann Arbor, Michigan; Rogel Cancer Center, University of Michigan, Ann Arbor, Michigan. Electronic address:

Background & Aims: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy that invades surrounding structures and metastasizes rapidly. Although inflammation is associated with tumor formation and progression, little is known about the mechanisms of this connection. We investigate the effects of interleukin (IL) 22 in the development of pancreatic tumors in mice.

Methods: We performed studies with Pdx1-Cre;LSL-Kras;Trp53;Rosa26 (PKCY) mice, which develop pancreatic tumors, and PKCY mice with disruption of IL22 (PKCY Il22mice). Pancreata were collected at different stages of tumor development and analyzed by immunohistochemistry, immunoblotting, real-time polymerase chain reaction, and flow cytometry. Some mice were given cerulean to induce pancreatitis. Pancreatic cancer cell lines (PD2560) were orthotopically injected into C57BL/6 mice or Il22mice, and tumor development was monitored. Pancreatic cells were injected into the tail veins of mice, and lung metastases were quantified. Acini were collected from C57BL/6 mice and resected human pancreata and were cultured. Cell lines and acini cultures were incubated with IL22 and pharmacologic inhibitors, and protein levels were knocked down with small hairpin RNAs. We performed immunohistochemical analyses of 26 PDACs and 5 nonneoplastic pancreas specimens.

Results: We observed increased expression of IL22 and the IL22 receptor (IL22R) in the pancreas compared with other tissues in mice; IL22 increased with pancreatitis and tumorigenesis. Flow cytometry indicated that the IL22 was produced primarily by T-helper 22 cells. PKCY Il22mice did not develop precancerous lesions or pancreatic tumors. The addition of IL22 to cultured acinar cells increased their expression of markers of ductal metaplasia; these effects of IL22 were prevented with inhibitors of Janus kinase signaling to signal transducer and activator of transcription (STAT) (ruxolitinib) or mitogen-activated protein kinase kinase (MEK) (trametinib) and with STAT3 knockdown. Pancreatic cells injected into Il22 mice formed smaller tumors than those injected into C57BL/6. Incubation of IL22R-expressing PDAC cells with IL22 promoted spheroid formation and invasive activity, resulting in increased expression of stem-associated transcription factors (GATA4, SOX2, SOX17, and NANOG), and increased markers of the epithelial-mesenchymal transition (CDH1, SNAI2, TWIST1, and beta catenin); ruxolitinib blocked these effects. Human PDAC tissues had higher levels of IL22, phosphorylated STAT3, and markers of the epithelial-mesenchymal transition than nonneoplastic tissues. An increased level of STAT3 in IL22R-positive cells was associated with shorter survival times of patients.

Conclusions: We found levels of IL22 to be increased during pancreatitis and pancreatic tumor development and to be required for tumor development and progression in mice. IL22 promotes acinar to ductal metaplasia, stem cell features, and increased expression of markers of the epithelial-mesenchymal transition; inhibitors of STAT3 block these effects. Increased expression of IL22 by PDACs is associated with reduced survival times.
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http://dx.doi.org/10.1053/j.gastro.2019.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7197347PMC
April 2020

Glutamine Anabolism Plays a Critical Role in Pancreatic Cancer by Coupling Carbon and Nitrogen Metabolism.

Cell Rep 2019 10;29(5):1287-1298.e6

Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854, USA; Rutgers Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA. Electronic address:

Glutamine is thought to play an important role in cancer cells by being deaminated via glutaminolysis to α-ketoglutarate (aKG) to fuel the tricarboxylic acid (TCA) cycle. Supporting this notion, aKG supplementation can restore growth/survival of glutamine-deprived cells. However, pancreatic cancers are often poorly vascularized and limited in glutamine supply, in alignment with recent concerns on the significance of glutaminolysis in pancreatic cancer. Here, we show that aKG-mediated rescue of glutamine-deprived pancreatic ductal carcinoma (PDAC) cells requires glutamate ammonia ligase (GLUL), the enzyme responsible for de novo glutamine synthesis. GLUL-deficient PDAC cells are capable of the TCA cycle but defective in aKG-coupled glutamine biosynthesis and subsequent nitrogen anabolic processes. Importantly, GLUL expression is elevated in pancreatic cancer patient samples and in mouse PDAC models. GLUL ablation suppresses the development of Kras-driven murine PDAC. Therefore, GLUL-mediated glutamine biosynthesis couples the TCA cycle with nitrogen anabolism and plays a critical role in PDAC.
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http://dx.doi.org/10.1016/j.celrep.2019.09.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886125PMC
October 2019

Activation of the intrinsic fibroinflammatory program in adult pancreatic acinar cells triggered by Hippo signaling disruption.

PLoS Biol 2019 09 12;17(9):e3000418. Epub 2019 Sep 12.

Department of Cell Systems & Anatomy, UT Health San Antonio, San Antonio, Texas, United States of America.

Damaged acinar cells play a passive role in activating pancreatic stellate cells (PSCs) via recruitment of immune cells that subsequently activate PSCs. However, whether acinar cells directly contribute to PSC activation is unknown. Here, we report that the Hippo pathway, a well-known regulator of proliferation, is essential for suppression of expression of inflammation and fibrosis-associated genes in adult pancreatic acinar cells. Hippo inactivation in acinar cells induced yes-associated protein 1 (YAP1)/transcriptional coactivator with PDZ binding motif (TAZ)-dependent, irreversible fibrosis and inflammation, which was initiated by Hippo-mediated acinar-stromal communications and ameliorated by blocking YAP1/TAZ target connective tissue growth factor (CTGF). Hippo disruption promotes acinar cells to secrete fibroinflammatory factors and induce stromal activation, which precedes acinar proliferation and metaplasia. We found that Hippo disruption did not induce cell-autonomous proliferation but primed acinar cells to exogenous pro-proliferative stimuli, implying a well-orchestrated scenario in which Hippo signaling acts as an intrinsic link to coordinate fibroinflammatory response and proliferation for maintenance of the tissue integrity. Our findings suggest that the fibroinflammatory program in pancreatic acinar cells is suppressed under normal physiological conditions. While transient activation of inflammatory gene expression during tissue injury may contribute to the control of damage and tissue repair, its persistent activation may result in tissue fibrosis and failure of regeneration.
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http://dx.doi.org/10.1371/journal.pbio.3000418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6742234PMC
September 2019

Putting the Cell of Origin for Pancreatic Cancer Into its Proper Context.

Cell Mol Gastroenterol Hepatol 2019 29;8(4):645-646. Epub 2019 Aug 29.

Departments of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan. Electronic address:

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http://dx.doi.org/10.1016/j.jcmgh.2019.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889687PMC
March 2020

Hiding in plain sight.

Science 2019 06;364(6446):1132-1133

Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA.

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http://dx.doi.org/10.1126/science.aax9341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6709523PMC
June 2019

Myeloid Cell-Derived HB-EGF Drives Tissue Recovery After Pancreatitis.

Cell Mol Gastroenterol Hepatol 2019 21;8(2):173-192. Epub 2019 May 21.

Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan; Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan; Rogel Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan. Electronic address:

Background & Aims: Pancreatitis is a major cause of morbidity and mortality and is a risk factor for pancreatic tumorigenesis. Upon tissue damage, an inflammatory response, made up largely of macrophages, provides multiple growth factors that promote repair. Here, we examine the molecular pathways initiated by macrophages to promote pancreas recovery from pancreatitis.

Methods: To induce organ damage, mice were subjected to cerulein-induced experimental pancreatitis and analyzed at various times of recovery. CD11b-DTR mice were used to deplete myeloid cells. Hbegf;LysM-Cre mice were used to ablate myeloid cell-derived heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF). To ablate EGFR specifically during recovery, pancreatitis was induced in Egfr;Ptf1a;FSF-Rosa26 mice followed by tamoxifen treatment.

Results: Macrophages infiltrating the pancreas in experimental pancreatitis make high levels of HB-EGF. Both depletion of myeloid cells and ablation of myeloid cell HB-EGF delayed recovery from experimental pancreatitis, resulting from a decrease in cell proliferation and an increase in apoptosis. Mechanistically, ablation of myeloid cell HB-EGF impaired epithelial cell DNA repair, ultimately leading to cell death. Soluble HB-EGF induced EGFR nuclear translocation and methylation of histone H4, facilitating resolution of DNA damage in pancreatic acinar cells in vitro. Consistent with its role as the primary receptor of HB-EGF, in vivo ablation of EGFR from pancreatic epithelium during recovery from pancreatitis resulted in accumulation of DNA damage.

Conclusions: By using novel conditional knockout mouse models, we determined that HB-EGF derived exclusively from myeloid cells induces epithelial cell proliferation and EGFR-dependent DNA repair, facilitating pancreas healing after injury.
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http://dx.doi.org/10.1016/j.jcmgh.2019.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6661420PMC
July 2020

ATDC is required for the initiation of KRAS-induced pancreatic tumorigenesis.

Genes Dev 2019 06 2;33(11-12):641-655. Epub 2019 May 2.

Department of Surgery, New York University School of Medicine, New York, New York 10016, USA.

Pancreatic adenocarcinoma (PDA) is an aggressive disease driven by oncogenic KRAS and characterized by late diagnosis and therapeutic resistance. Here we show that deletion of the ataxia-telangiectasia group D-complementing () gene, whose human homolog is up-regulated in the majority of pancreatic adenocarcinoma, completely prevents PDA development in the context of oncogenic KRAS. ATDC is required for KRAS-driven acinar-ductal metaplasia (ADM) and its progression to pancreatic intraepithelial neoplasia (PanIN). As a result, mice lacking ATDC are protected from developing PDA. Mechanistically, we show ATDC promotes ADM progression to PanIN through activation of β-catenin signaling and subsequent SOX9 up-regulation. These results provide new insight into PDA initiation and reveal ATDC as a potential target for preventing early tumor-initiating events.
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http://dx.doi.org/10.1101/gad.323303.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546061PMC
June 2019

Macrophage-Released Pyrimidines Inhibit Gemcitabine Therapy in Pancreatic Cancer.

Cell Metab 2019 06 28;29(6):1390-1399.e6. Epub 2019 Feb 28.

Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA; University of Michigan Rogel Cancer Center, University of Michigan, Ann Arbor, MI 48109, USA; Department of Internal Medicine, Division of Gastroenterology and Hepatology, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address:

Pancreatic ductal adenocarcinoma (PDA) is characterized by abundant infiltration of tumor-associated macrophages (TAMs). TAMs have been reported to drive resistance to gemcitabine, a frontline chemotherapy in PDA, though the mechanism of this resistance remains unclear. Profiling metabolite exchange, we demonstrate that macrophages programmed by PDA cells release a spectrum of pyrimidine species. These include deoxycytidine, which inhibits gemcitabine through molecular competition at the level of drug uptake and metabolism. Accordingly, genetic or pharmacological depletion of TAMs in murine models of PDA sensitizes these tumors to gemcitabine. Consistent with this, patients with low macrophage burden demonstrate superior response to gemcitabine treatment. Together, these findings provide insights into the role of macrophages in pancreatic cancer therapy and have potential to inform the design of future treatments. Additionally, we report that pyrimidine release is a general function of alternatively activated macrophage cells, suggesting an unknown physiological role of pyrimidine exchange by immune cells.
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http://dx.doi.org/10.1016/j.cmet.2019.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6602533PMC
June 2019

Signaling Networks That Control Cellular Plasticity in Pancreatic Tumorigenesis, Progression, and Metastasis.

Gastroenterology 2019 05 1;156(7):2073-2084. Epub 2019 Feb 1.

Department of Surgery, University of Miami School of Medicine, Miami, Florida; Sylvester Cancer Center, University of Miami, Miami, Florida. Electronic address:

Pancreatic ductal adenocarcinoma is one of the deadliest cancers, and its incidence on the rise. The major challenges in overcoming the poor prognosis with this disease include late detection and the aggressive biology of the disease. Intratumoral heterogeneity; presence of a robust, reactive, and desmoplastic stroma; and the crosstalk between the different tumor components require complete understanding of the pancreatic tumor biology to better understand the therapeutic challenges posed by this disease. In this review, we discuss the processes involved during tumorigenesis encompassing the inherent plasticity of the transformed cells, development of tumor stroma crosstalk, and enrichment of cancer stem cell population during tumorigenesis.
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http://dx.doi.org/10.1053/j.gastro.2018.12.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545585PMC
May 2019

Epithelial-Stromal Interactions in Pancreatic Cancer.

Annu Rev Physiol 2019 02 12;81:211-233. Epub 2018 Nov 12.

Department of Surgery, University of Michigan, Ann Arbor, Michigan 48109, USA; email:

Pancreatic cancer is characterized by an extensive fibroinflammatory reaction that includes immune cells, fibroblasts, extracellular matrix, vascular and lymphatic vessels, and nerves. Overwhelming evidence indicates that the pancreatic cancer microenvironment regulates cancer initiation, progression, and maintenance. Pancreatic cancer treatment has progressed little over the past several decades, and the prognosis remains one of the worst for any cancer. The contribution of the microenvironment to carcinogenesis is a key area of research, offering new potential targets for treating the disease. Here, we explore the composition of the pancreatic cancer stroma, discuss the network of interactions between different components, and describe recent attempts to target the stroma therapeutically. We also discuss current areas of active research related to the tumor microenvironment.
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http://dx.doi.org/10.1146/annurev-physiol-020518-114515DOI Listing
February 2019

STAT3 is a master regulator of epithelial identity and KRAS-driven tumorigenesis.

Genes Dev 2018 09 22;32(17-18):1175-1187. Epub 2018 Aug 22.

Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York 11794, USA.

A dichotomy exists regarding the role of signal transducer and activator of transcription 3 (STAT3) in cancer. Functional and genetic studies demonstrate either an intrinsic requirement for STAT3 or a suppressive effect on common types of cancer. These contrasting actions of STAT3 imply context dependency. To examine mechanisms that underlie STAT3 function in cancer, we evaluated the impact of STAT3 activity in KRAS-driven lung and pancreatic cancer. Our study defines a fundamental and previously unrecognized function of STAT3 in the maintenance of epithelial cell identity and differentiation. Loss of STAT3 preferentially associates with the acquisition of mesenchymal-like phenotypes and more aggressive tumor behavior. In contrast, persistent STAT3 activation through Tyr705 phosphorylation confers a differentiated epithelial morphology that impacts tumorigenic potential. Our results imply a mechanism in which quantitative differences of STAT3 Tyr705 phosphorylation, as compared with other activation modes, direct discrete outcomes in tumor progression.
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http://dx.doi.org/10.1101/gad.311852.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120712PMC
September 2018

HNF1A is a novel oncogene that regulates human pancreatic cancer stem cell properties.

Elife 2018 08 3;7. Epub 2018 Aug 3.

Department of Surgery, New York University Langone Health, New York, United States.

The biological properties of pancreatic cancer stem cells (PCSCs) remain incompletely defined and the central regulators are unknown. By bioinformatic analysis of a human PCSC-enriched gene signature, we identified the transcription factor HNF1A as a putative central regulator of PCSC function. Levels of HNF1A and its target genes were found to be elevated in PCSCs and tumorspheres, and depletion of HNF1A resulted in growth inhibition, apoptosis, impaired tumorsphere formation, decreased PCSC marker expression, and downregulation of expression. Conversely, HNF1A overexpression increased PCSC marker expression and tumorsphere formation in pancreatic cancer cells and drove pancreatic ductal adenocarcinoma (PDA) cell growth. Importantly, depletion of HNF1A in xenografts impaired tumor growth and depleted PCSC marker-positive cells in vivo. Finally, we established an HNF1A-dependent gene signature in PDA cells that significantly correlated with reduced survivability in patients. These findings identify HNF1A as a central transcriptional regulator of PCSC properties and novel oncogene in PDA.
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http://dx.doi.org/10.7554/eLife.33947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6122955PMC
August 2018

MYC regulates ductal-neuroendocrine lineage plasticity in pancreatic ductal adenocarcinoma associated with poor outcome and chemoresistance.

Nat Commun 2017 11 23;8(1):1728. Epub 2017 Nov 23.

Department of Molecular and Medical Genetics, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR, 97239, USA.

Intratumoral phenotypic heterogeneity has been described in many tumor types, where it can contribute to drug resistance and disease recurrence. We analyzed ductal and neuroendocrine markers in pancreatic ductal adenocarcinoma, revealing heterogeneous expression of the neuroendocrine marker Synaptophysin within ductal lesions. Higher percentages of Cytokeratin-Synaptophysin dual positive tumor cells correlate with shortened disease-free survival. We observe similar lineage marker heterogeneity in mouse models of pancreatic ductal adenocarcinoma, where lineage tracing indicates that Cytokeratin-Synaptophysin dual positive cells arise from the exocrine compartment. Mechanistically, MYC binding is enriched at neuroendocrine genes in mouse tumor cells and loss of MYC reduces ductal-neuroendocrine lineage heterogeneity, while deregulated MYC expression in KRAS mutant mice increases this phenotype. Neuroendocrine marker expression is associated with chemoresistance and reducing MYC levels decreases gemcitabine-induced neuroendocrine marker expression and increases chemosensitivity. Altogether, we demonstrate that MYC facilitates ductal-neuroendocrine lineage plasticity in pancreatic ductal adenocarcinoma, contributing to poor survival and chemoresistance.
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http://dx.doi.org/10.1038/s41467-017-01967-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701042PMC
November 2017

Epithelial-Myeloid cell crosstalk regulates acinar cell plasticity and pancreatic remodeling in mice.

Elife 2017 10 5;6. Epub 2017 Oct 5.

Department of Surgery, University of Michigan, Ann Arbor, United States.

Dedifferentiation of acini to duct-like cells occurs during the physiologic damage response in the pancreas, but this process can be co-opted by oncogenic Kras to drive carcinogenesis. Myeloid cells infiltrate the pancreas during the onset of pancreatic cancer, and promote carcinogenesis. Here, we show that the function of infiltrating myeloid cells is regulated by oncogenic Kras expressed in epithelial cells. In the presence of oncogenic Kras, myeloid cells promote acinar dedifferentiation and carcinogenesis. Upon inactivation of oncogenic Kras, myeloid cells promote re-differentiation of acinar cells, remodeling of the fibrotic stroma and tissue repair. Intriguingly, both aspects of myeloid cell activity depend, at least in part, on activation of EGFR/MAPK signaling, with different subsets of ligands and receptors in different target cells promoting carcinogenesis or repair, respectively. Thus, the cross-talk between epithelial cells and infiltrating myeloid cells determines the balance between tissue repair and carcinogenesis in the pancreas.
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http://dx.doi.org/10.7554/eLife.27388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5690281PMC
October 2017

Mitogen-activated Protein Kinase Kinase Activity Maintains Acinar-to-Ductal Metaplasia and Is Required for Organ Regeneration in Pancreatitis.

Cell Mol Gastroenterol Hepatol 2017 Jan;3(1):99-118

Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan; Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan.

Background & Aims: Mitogen-activated protein kinase (MAPK) signaling in the exocrine pancreas has been extensively studied in the context of pancreatic cancer, where its potential as a therapeutic target is limited by acquired drug resistance. However, its role in pancreatitis is less understood. We investigated the role of mitogen-activated protein kinase kinase (MEK)-initiated MAPK signaling in pancreatitis to determine the potential for MEK inhibition in treating pancreatitis patients.

Methods: To examine the role of MEK signaling in pancreatitis, we used both genetic and pharmacologic approaches to inhibit the MAPK signaling pathway in a murine model of cerulein-induced pancreatitis. We generated mice harboring inducible short hairpins targeting the MEK isoforms and/or specifically in the pancreatic epithelium. We also used the MEK inhibitor trametinib to determine the efficacy of systemic inhibition in mice with pancreatitis.

Results: We demonstrated an essential role for MEK signaling in the initiation of pancreatitis. We showed that both systemic and parenchyma-specific MEK inhibition in established pancreatitis induces epithelial differentiation and stromal remodeling. However, systemic MEK inhibition also leads to a loss of the proliferative capacity of the pancreas, preventing the restoration of organ mass.

Conclusions: MEK activity is required for the initiation and maintenance of pancreatitis. MEK inhibition may be useful in the treatment of chronic pancreatitis to interrupt the vicious cycle of destruction and repair but at the expense of organ regeneration.
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http://dx.doi.org/10.1016/j.jcmgh.2016.09.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5235341PMC
January 2017