Publications by authors named "Stephen F Konieczny"

60 Publications

CRISPR/Cas12a-mediated CHO genome engineering can be effectively integrated at multiple stages of the cell line generation process for bioproduction.

Biotechnol J 2021 Apr 25;16(4):e2000308. Epub 2021 Jan 25.

Bioprocess Research and Development, Eli Lilly and Company, Indianapolis, Indiana, USA.

Most biopharmaceuticals produced today are generated using Chinese hamster ovary (CHO) cells, therefore significant attention is focused on methods to improve CHO cell productivity and product quality. The discovery of gene-editing tools, such as CRISPR/Cas9, offers new opportunities to improve CHO cell bioproduction through cell line engineering. Recently an additional CRISPR-associated protein, Cas12a (Cpf1), was shown to be effective for gene editing in eukaryotic cells, including CHO. In this study, we demonstrate the successful application of CRISPR/Cas12a for the generation of clonally derived CHO knockout (KO) cell lines with improved product quality attributes. While we found Cas12a efficiency to be highly dependent on the targeting RNA used, we were able to generate CHO KO cell lines using small screens of only 96-320 clonally derived cell lines. Additionally, we present a novel bulk culture analysis approach that can be used to quickly assess CRISPR RNA efficiency and determine ideal screen sizes for generating genetic KO cell lines. Most critically, we find that Cas12a can be directly integrated into the cell line generation process through cotransfection with no negative impact on titer or screen size. Overall, our results show CRISPR/Cas12a to be an efficient and effective CHO genome editing tool.
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http://dx.doi.org/10.1002/biot.202000308DOI Listing
April 2021

Thrombin-PAR1 signaling in pancreatic cancer promotes an immunosuppressive microenvironment.

J Thromb Haemost 2021 01 25;19(1):161-172. Epub 2020 Oct 25.

Department of Biological Sciences and The Purdue Center for Cancer Research, Purdue University, West Lafayette, IN, USA.

Essentials Elimination of PDAC tumor cell PAR1 increased cytotoxic T cells and reduced tumor macrophages. PAR1 PDAC cells are preferentially eliminated from growing tumors. Thrombin-PAR1 signaling in PDAC tumor cells drives an immunosuppressive gene signature. Csf2 and Ptgs2 are thrombin-PAR1 downstream immune suppressor genes in PDAC tumor cells. ABSTRACT: Background Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prothrombotic state and a lack of host antitumor immune responsiveness. Linking these two key features, we previously demonstrated that tumor-derived coagulation activity promotes immune evasion. Specifically, thrombin-protease-activated receptor-1 (PAR1) signaling in mouse PDAC cells drives tumor growth by evading cytotoxic CD8a cells. Methods Syngeneic mixed cell tumor growth, transcriptional analyses, and functional tests of immunosuppressive response genes were used to identify cellular and molecular immune evasion mechanisms mediated by thrombin-PAR-1 signaling in mouse PDAC tumor cells. Results Elimination of tumor cell PAR1 in syngeneic graft studies increased cytotoxic T lymphocyte (CTL) infiltration and decreased tumor-associated macrophages in the tumor microenvironment. Co-injection of PAR1-expressing and PAR1-knockout (PAR-1 ) tumor cells into immunocompetent mice resulted in preferential elimination of PAR-1 cells from developing tumors, suggesting that PAR1-dependent immune evasion is not reliant on CTL exclusion. Transcriptomics analyses revealed no PAR1-dependent changes in the expression of immune checkpoint proteins and no difference in major histocompatibility complex-I cell surface expression. Importantly, thrombin-PAR1 signaling in PDAC cells upregulated genes linked to immunosuppression, including Csf2 and Ptgs2. Functional analyses confirmed that both Csf2 and Ptgs2 are critical for PDAC syngeneic graft tumor growth and overexpression of each factor partially restored tumor growth of PAR1 cells in immunocompetent mice. Conclusions Our results provide novel insight into the mechanisms of a previously unrecognized pathway coupling coagulation to PDAC immune evasion by identifying PAR1-dependent changes in the tumor microenvironment, a PAR1-driven immunosuppressive gene signature, and Csf2 and Ptgs2 as critical PAR1 downstream targets.
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http://dx.doi.org/10.1111/jth.15115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790967PMC
January 2021

An engineered pancreatic cancer model with intra-tumoral heterogeneity of driver mutations.

Lab Chip 2020 10 10;20(20):3720-3732. Epub 2020 Sep 10.

School of Mechanical Engineering, Purdue University, West Lafayette, IN, USA.

Pancreatic ductal adenocarcinoma (PDAC) is a complex disease with significant intra-tumoral heterogeneity (ITH). Currently, no reliable PDAC tumor model is available that can present ITH profiles in a controlled manner. We develop an in vitro microfluidic tumor model mimicking the heterogeneous accumulation of key driver mutations of human PDAC using cancer cells derived from genetically engineered mouse models. These murine pancreatic cancer cell lines have KPC (Kras and Trp53 mutations) and KIC genotypes (Kras mutation and Cdkn2a deletion). Also, the KIC genotypes have two distinct phenotypes - mesenchymal or epithelial. The tumor model mimics the ITH of human PDAC to study the effects of ITH on the gemcitabine response. The results show gemcitabine resistance induced by ITH. Remarkably, it shows that cancer cell-cell interactions induce the gemcitabine resistance potentially through epithelial-mesenchymal-transition. The tumor model can provide a useful testbed to study interaction mechanisms between heterogeneous cancer cell subpopulations.
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http://dx.doi.org/10.1039/d0lc00707bDOI Listing
October 2020

Subtype-specific characterization of breast cancer invasion using a microfluidic tumor platform.

PLoS One 2020 16;15(6):e0234012. Epub 2020 Jun 16.

School of Mechanical Engineering, Purdue University, West Lafayette, IN, United States of America.

Understanding progression of breast cancers to invasive ductal carcinoma (IDC) can significantly improve breast cancer treatments. However, it is still difficult to identify genetic signatures and the role of tumor microenvironment to distinguish pathological stages of pre-invasive lesion and IDC. Presence of multiple subtypes of breast cancers makes the assessment more challenging. In this study, an in-vitro microfluidic assay was developed to quantitatively assess the subtype-specific invasion potential of breast cancers. The developed assay is a microfluidic platform in which a ductal structure of epithelial cancer cells is surrounded with a three-dimensional (3D) collagen matrix. In the developed platform, two triple negative cancer subtypes (MDA-MB-231 and SUM-159PT) invaded into the surrounding matrix but the luminal A subtype, MCF-7, did not. Among invasive subtypes, SUM-159PT cells showed significantly higher invasion and degradation of the surrounding matrix than MDA-MB-231. Interestingly, the cells cultured on the platform expressed higher levels of CD24 than in their conventional 2D cultures. This microfluidic platform may be a useful tool to characterize and predict invasive potential of breast cancer subtypes or patient-derived cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0234012PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7297326PMC
August 2020

A Biomimetic Tumor Model of Heterogeneous Invasion in Pancreatic Ductal Adenocarcinoma.

Small 2020 03 30;16(10):e1905500. Epub 2020 Jan 30.

School of Mechanical Engineering, Purdue University, West Lafayette, IN, 47907, USA.

Pancreatic ductal adenocarcinoma (PDAC) is a complex, heterogeneous, and genetically unstable disease. Its tumor microenvironment (TME) is complicated by heterogeneous cancer cell populations and strong desmoplastic stroma. This complex and heterogeneous environment makes it challenging to discover and validate unique therapeutic targets. Reliable and relevant in vitro PDAC tumor models can significantly advance the understanding of the PDAC TME and may enable the discovery and validation of novel drug targets. In this study, an engineered tumor model is developed to mimic the PDAC TME. This biomimetic model, named ductal tumor-microenvironment-on-chip (dT-MOC), permits analysis and experimentation on the epithelial-mesenchymal transition (EMT) and local invasion with intratumoral heterogeneity. This dT-MOC is a microfluidic platform where a duct of murine genetically engineered pancreatic cancer cells is embedded within a collagen matrix. The cancer cells used carry two of the three mutations of KRAS, CDKN2A, and TP53, which are key driver mutations of human PDAC. The intratumoral heterogeneity is mimicked by co-culturing these cancer cells. Using the dT-MOC model, heterogeneous invasion characteristics, and response to transforming growth factor-beta1 are studied. A mechanism of EMT and local invasion caused by the interaction between heterogeneous cancer cell populations is proposed.
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http://dx.doi.org/10.1002/smll.201905500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7069790PMC
March 2020

Thrombin Signaling Promotes Pancreatic Adenocarcinoma through PAR-1-Dependent Immune Evasion.

Cancer Res 2019 07 2;79(13):3417-3430. Epub 2019 May 2.

Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital, Cincinnati, Ohio.

Pancreatic ductal adenocarcinoma (PDAC) is associated with robust activity of the coagulation system. To determine mechanisms by which clotting factors influence PDAC tumor progression, we generated and characterized C57Bl/6-derived KPC ( ) cell lines. Tissue factor (TF) and protease-activated receptor-1 (PAR-1) were highly expressed in primary KPC pancreatic lesions and KPC cell lines similar to expression profiles observed in biopsies of patients with PDAC. In allograft studies, tumor growth and metastatic potential were significantly diminished by depletion of or in cancer cells or by genetic or pharmacologic reduction of the coagulation zymogen prothrombin in mice. Notably, PAR-1-deleted KPC cells (KPC-Par-1) failed to generate sizable tumors, a phenotype completely rescued by restoration of expression. Expression profiling of KPC and KPC-Par-1 cells indicated that thrombin-PAR-1 signaling significantly altered immune regulation pathways. Accordingly, KPC-Par-1 cells failed to form tumors in immune-competent mice but displayed robust tumor growth comparable to that observed with control KPC cells in immune-compromised mice. Immune cell depletion studies indicated that CD8 T cells, but not CD4 cells or natural killer cells, mediated elimination of KPC-Par-1 tumor cells in C57Bl/6 mice. These results demonstrate that PDAC is driven by activation of the coagulation system through tumor cell-derived TF, circulating prothrombin, and tumor cell-derived PAR-1 and further indicate that one key mechanism of thrombin/PAR-1-mediated tumor growth is suppression of antitumor immunity in the tumor microenvironment. SIGNIFICANCE: The tissue factor-thrombin-PAR-1 signaling axis in tumor cells promotes PDAC growth and disease progression with one key mechanism being suppression of antitumor immunity in the microenvironment.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-3206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6699516PMC
July 2019

Identification and Characterization of AES-135, a Hydroxamic Acid-Based HDAC Inhibitor That Prolongs Survival in an Orthotopic Mouse Model of Pancreatic Cancer.

J Med Chem 2019 03 6;62(5):2651-2665. Epub 2019 Mar 6.

Department of Chemical and Physical Sciences , University of Toronto Mississauga , 3359 Mississauga Road , Mississauga , Ontario L5L 1C6 , Canada.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, incurable cancer with a 20% 1 year survival rate. While standard-of-care therapy can prolong life in a small fraction of cases, PDAC is inherently resistant to current treatments, and novel therapies are urgently required. Histone deacetylase (HDAC) inhibitors are effective in killing pancreatic cancer cells in in vitro PDAC studies, and although there are a few clinical studies investigating combination therapy including HDAC inhibitors, no HDAC drug or combination therapy with an HDAC drug has been approved for the treatment of PDAC. We developed an inhibitor of HDACs, AES-135, that exhibits nanomolar inhibitory activity against HDAC3, HDAC6, and HDAC11 in biochemical assays. In a three-dimensional coculture model, AES-135 kills low-passage patient-derived tumor spheroids selectively over surrounding cancer-associated fibroblasts and has excellent pharmacokinetic properties in vivo. In an orthotopic murine model of pancreatic cancer, AES-135 prolongs survival significantly, therefore representing a candidate for further preclinical testing.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190078PMC
March 2019

Disruption of stromal hedgehog signaling initiates RNF5-mediated proteasomal degradation of PTEN and accelerates pancreatic tumor growth.

Life Sci Alliance 2018 Oct 26;1(5):e201800190. Epub 2018 Oct 26.

Hollings Cancer Center and Department of Biochemistry & Molecular Biology, Medical University of South Carolina, Charleston, SC, USA.

The contribution of the tumor microenvironment to pancreatic ductal adenocarcinoma (PDAC) development is currently unclear. We therefore examined the consequences of disrupting paracrine Hedgehog (HH) signaling in PDAC stroma. Herein, we show that ablation of the key HH signaling gene () in stromal fibroblasts led to increased proliferation of pancreatic tumor cells. Furthermore, deletion resulted in proteasomal degradation of the tumor suppressor PTEN and activation of oncogenic protein kinase B (AKT) in fibroblasts. An unbiased proteomic screen identified RNF5 as a novel E3 ubiquitin ligase responsible for degradation of phosphatase and tensin homolog (PTEN) in -null fibroblasts. () knockdown or pharmacological inhibition of glycogen synthase kinase 3β (GSKβ), the kinase that marks PTEN for ubiquitination, rescued PTEN levels and reversed the oncogenic phenotype, identifying a new node of PTEN regulation. In PDAC patients, low stromal PTEN correlated with reduced overall survival. Mechanistically, PTEN loss decreased hydraulic permeability of the extracellular matrix, which was reversed by hyaluronidase treatment. These results define non-cell autonomous tumor-promoting mechanisms activated by disruption of the HH/PTEN axis and identifies new targets for restoring stromal tumor-suppressive functions.
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http://dx.doi.org/10.26508/lsa.201800190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6238420PMC
October 2018

BATF regulates the expression of Nfil3, Wnt10a and miR155hg for efficient induction of antibody class switch recombination in mice.

Eur J Immunol 2018 09 26;48(9):1492-1505. Epub 2018 Jun 26.

Department of Biological Sciences and Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN, USA.

BATF functions in T cells and B cells to control the host response to antigen and promote the production of class switched immunoglobulins. In this study, we demonstrate that BATF expression increases rapidly, and transiently, following B cell stimulation and use an inducible murine model of BATF deletion to show that this induction is necessary, and sufficient, for immunoglobulin (Ig) class switch recombination (CSR). We examine two genes (Nfil3 and miR155gh) that are positively regulated, and one gene (Wnt10a) that is negatively regulated by BATF during CSR. These genes play essential roles in CSR and each impacts the expression and/or function of the others. Our observations allow these targets of BATF regulation to be positioned in a network upstream of the activation of germline transcripts (GLT) from the IgH locus and of transcriptional activation of Aicda - the gene encoding the enzyme directing Ig gene rearrangements. This work extends the knowledge of the molecular control of CSR and, importantly, positions the induction and function of BATF as an early event in this process.
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http://dx.doi.org/10.1002/eji.201747360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357966PMC
September 2018

Induced PTF1a expression in pancreatic ductal adenocarcinoma cells activates acinar gene networks, reduces tumorigenic properties, and sensitizes cells to gemcitabine treatment.

Mol Oncol 2018 06 21;12(7):1104-1124. Epub 2018 May 21.

Department of Biological Sciences, Purdue University, West Lafayette, IN, USA.

Pancreatic acinar cells synthesize, package, and secrete digestive enzymes into the duodenum to aid in nutrient absorption and meet metabolic demands. When exposed to cellular stresses and insults, acinar cells undergo a dedifferentiation process termed acinar-ductal metaplasia (ADM). ADM lesions with oncogenic mutations eventually give rise to pancreatic ductal adenocarcinoma (PDAC). In healthy pancreata, the basic helix-loop-helix (bHLH) factors MIST1 and PTF1a coordinate an acinar-specific transcription network that maintains the highly developed differentiation status of the cells, protecting the pancreas from undergoing a transformative process. However, when MIST1 and PTF1a gene expression is silenced, cells are more prone to progress to PDAC. In this study, we tested whether induced MIST1 or PTF1a expression in PDAC cells could (i) re-establish the transcriptional program of differentiated acinar cells and (ii) simultaneously reduce tumor cell properties. As predicted, PTF1a induced gene expression of digestive enzymes and acinar-specific transcription factors, while MIST1 induced gene expression of vesicle trafficking molecules as well as activation of unfolded protein response components, all of which are essential to handle the high protein production load that is characteristic of acinar cells. Importantly, induction of PTF1a in PDAC also influenced cancer-associated properties, leading to a decrease in cell proliferation, cancer stem cell numbers, and repression of key ATP-binding cassette efflux transporters resulting in heightened sensitivity to gemcitabine. Thus, activation of pancreatic bHLH transcription factors rescues the acinar gene program and decreases tumorigenic properties in pancreatic cancer cells, offering unique opportunities to develop novel therapeutic intervention strategies for this deadly disease.
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http://dx.doi.org/10.1002/1878-0261.12314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6026875PMC
June 2018

CXCR4-expressing progenitors in the gastric antrum contribute to gastric cancer development.

Oncotarget 2017 Dec 10;8(67):111012-111025. Epub 2017 Nov 10.

Division of Digestive and Liver Disease, Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, NY, USA.

was recently shown to identify a discrete population of stem cells within the isthmus of the oxyntic gland within the gastric corpus. Chief cells at the base of the gastric corpus also express . The relevance of expression as a marker of specific cell populations within the antral glands of the distal stomach, however, is unknown. Using -CreERT mice, we revealed that antral cells, distinct from the population in the corpus, comprise long-lived progenitors that reside within the antral isthmus above or CCK2R cells. antral progenitors can serve as an origin of antral tumors induced by loss of Apc or MNU treatment. antral progenitors, as well as other antral stem/progenitor population, express Cxcr4, and are located in close proximity to Cxcl12 (the Cxcr4 ligand)-expressing endothelium. During antral carcinogenesis, there is an expansion of Cxcr4 epithelial cells as well as the Cxcl12 perivascular niche. Deletion of Cxcl12 in endothelial cells or pharmacological blockade of Cxcr4 inhibits antral tumor growth. Cxcl12/Cxcr4 signaling may be a potential therapeutic target.
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http://dx.doi.org/10.18632/oncotarget.22451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762301PMC
December 2017

A single transcription factor is sufficient to induce and maintain secretory cell architecture.

Genes Dev 2017 01 7;31(2):154-171. Epub 2017 Feb 7.

Division of Gastroenterology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

We hypothesized that basic helix-loop-helix (bHLH) MIST1 (BHLHA15) is a "scaling factor" that universally establishes secretory morphology in cells that perform regulated secretion. Here, we show that targeted deletion of MIST1 caused dismantling of the secretory apparatus of diverse exocrine cells. Parietal cells (PCs), whose function is to pump acid into the stomach, normally lack MIST1 and do not perform regulated secretion. Forced expression of MIST1 in PCs caused them to expand their apical cytoplasm, rearrange mitochondrial/lysosome trafficking, and generate large secretory granules. induced a cohort of genes regulated by MIST1 in multiple organs but did not affect PC function. MIST1 bound CATATG/CAGCTG E boxes in the first intron of genes that regulate autophagosome/lysosomal degradation, mitochondrial trafficking, and amino acid metabolism. Similar alterations in cell architecture and gene expression were also caused by ectopically inducing MIST1 in vivo in hepatocytes. Thus, MIST1 is a scaling factor necessary and sufficient by itself to induce and maintain secretory cell architecture. Our results indicate that, whereas mature cell types in each organ may have unique developmental origins, cells performing similar physiological functions throughout the body share similar transcription factor-mediated architectural "blueprints."
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http://dx.doi.org/10.1101/gad.285684.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5322730PMC
January 2017

Stromal ETS2 Regulates Chemokine Production and Immune Cell Recruitment during Acinar-to-Ductal Metaplasia.

Neoplasia 2016 09;18(9):541-52

Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA; Department of Cancer Biology & Genetics, The Ohio State University, Columbus, OH 43210, USA. Electronic address:

Preclinical studies have suggested that the pancreatic tumor microenvironment both inhibits and promotes tumor development and growth. Here we establish the role of stromal fibroblasts during acinar-to-ductal metaplasia (ADM), an initiating event in pancreatic cancer formation. The transcription factor V-Ets avian erythroblastosis virus E26 oncogene homolog 2 (ETS2) was elevated in smooth muscle actin-positive fibroblasts in the stroma of pancreatic ductal adenocarcinoma (PDAC) patient tissue samples relative to normal pancreatic controls. LSL-Kras(G12D/+); LSL-Trp53(R172H/+); Pdx-1-Cre (KPC) mice showed that ETS2 expression initially increased in fibroblasts during ADM and remained elevated through progression to PDAC. Conditional ablation of Ets-2 in pancreatic fibroblasts in a Kras(G12D)-driven mouse ADM model decreased the amount of ADM events. ADMs from fibroblast Ets-2-deleted animals had reduced epithelial cell proliferation and increased apoptosis. Surprisingly, fibroblast Ets-2 deletion significantly altered immune cell infiltration into the stroma, with an increased CD8+ T-cell population, and decreased presence of regulatory T cells (Tregs), myeloid-derived suppressor cells, and mature macrophages. The mechanism involved ETS2-dependent chemokine ligand production in fibroblasts. ETS2 directly bound to regulatory sequences for Ccl3, Ccl4, Cxcl4, Cxcl5, and Cxcl10, a group of chemokines that act as potent mediators of immune cell recruitment. These results suggest an unappreciated role for ETS2 in fibroblasts in establishing an immune-suppressive microenvironment in response to oncogenic Kras(G12D) signaling during the initial stages of tumor development.
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http://dx.doi.org/10.1016/j.neo.2016.07.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5031867PMC
September 2016

MIST1 and PTF1 Collaborate in Feed-Forward Regulatory Loops That Maintain the Pancreatic Acinar Phenotype in Adult Mice.

Mol Cell Biol 2016 Dec 14;36(23):2945-2955. Epub 2016 Nov 14.

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA

Much remains unknown regarding the regulatory networks formed by transcription factors in mature, differentiated mammalian cells , despite many studies of individual DNA-binding transcription factors. We report a constellation of feed-forward loops formed by the pancreatic transcription factors MIST1 and PTF1 that govern the differentiated phenotype of the adult pancreatic acinar cell. PTF1 is an atypical basic helix-loop-helix transcription factor complex of pancreatic acinar cells and is critical to acinar cell fate specification and differentiation. MIST1, also a basic helix-loop-helix transcription factor, enhances the formation and maintenance of the specialized phenotype of professional secretory cells. The MIST1 and PTF1 collaboration controls a wide range of specialized cellular processes, including secretory protein synthesis and processing, exocytosis, and homeostasis of the endoplasmic reticulum. PTF1 drives transcription, and MIST1 and PTF1 bind and drive the transcription of over 100 downstream acinar genes. PTF1 binds two canonical bipartite sites within a 0.7-kb transcriptional enhancer upstream of that are essential for the activity of the enhancer MIST1 and PTF1 coregulate target genes synergistically or additively, depending on the target transcriptional enhancer. The frequent close binding proximity of PTF1 and MIST1 in pancreatic acinar cell chromatin implies extensive collaboration although the collaboration is not dependent on a stable physical interaction.
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http://dx.doi.org/10.1128/MCB.00370-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5108882PMC
December 2016

MIST1 Links Secretion and Stress as both Target and Regulator of the Unfolded Protein Response.

Mol Cell Biol 2016 Dec 14;36(23):2931-2944. Epub 2016 Nov 14.

Department of Biological Sciences, Bindley Bioscience Center and Purdue University Center for Cancer Research, Purdue University, West Lafayette, Indiana, USA

Transcriptional networks that govern secretory cell specialization, including instructing cells to develop a unique cytoarchitecture, amass extensive protein synthesis machinery, and be embodied to respond to endoplasmic reticulum (ER) stress, remain largely uncharacterized. In this study, we discovered that the secretory cell transcription factor MIST1 (), previously shown to be essential for cytoskeletal organization and secretory activity, also functions as a potent ER stress-inducible transcriptional regulator. Genome-wide DNA binding studies, coupled with genetic mouse models, revealed MIST1 gene targets that function along the entire breadth of the protein synthesis, processing, transport, and exocytosis networks. Additionally, key MIST1 targets are essential for alleviating ER stress in these highly specialized cells. Indeed, MIST1 functions as a coregulator of the unfolded protein response (UPR) master transcription factor XBP1 for a portion of target genes that contain adjacent MIST1 and XBP1 binding sites. Interestingly, gene expression is induced during ER stress by XBP1, but as ER stress subsides, MIST1 serves as a feedback inhibitor, directly binding the promoter and repressing transcript production. Together, our findings provide a new paradigm for XBP1-dependent UPR regulation and position MIST1 as a potential biotherapeutic for numerous human diseases.
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http://dx.doi.org/10.1128/MCB.00366-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5108881PMC
December 2016

Genetic ablation of Smoothened in pancreatic fibroblasts increases acinar-ductal metaplasia.

Genes Dev 2016 09 15;30(17):1943-55. Epub 2016 Sep 15.

Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA; Cancer Biology and Genetics Department, The Ohio State University, Columbus, Ohio 43210, USA;

The contribution of the microenvironment to pancreatic acinar-to-ductal metaplasia (ADM), a preneoplastic transition in oncogenic Kras-driven pancreatic cancer progression, is currently unclear. Here we show that disruption of paracrine Hedgehog signaling via genetic ablation of Smoothened (Smo) in stromal fibroblasts in a Kras(G12D) mouse model increased ADM. Smo-deleted fibroblasts had higher expression of transforming growth factor-α (Tgfa) mRNA and secreted higher levels of TGFα, leading to activation of EGFR signaling in acinar cells and increased ADM. The mechanism involved activation of AKT and noncanonical activation of the GLI family transcription factor GLI2. GLI2 was phosphorylated at Ser230 in an AKT-dependent fashion and directly regulated Tgfa expression in fibroblasts lacking Smo Additionally, Smo-deleted fibroblasts stimulated the growth of Kras(G12D)/Tp53(R172H) pancreatic tumor cells in vivo and in vitro. These results define a non-cell-autonomous mechanism modulating Kras(G12D)-driven ADM that is balanced by cross-talk between Hedgehog/SMO and AKT/GLI2 pathways in stromal fibroblasts.
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http://dx.doi.org/10.1101/gad.283499.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066238PMC
September 2016

Notch activation drives adipocyte dedifferentiation and tumorigenic transformation in mice.

J Exp Med 2016 09 29;213(10):2019-37. Epub 2016 Aug 29.

Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 Center for Cancer Research, Purdue University, West Lafayette, IN 47907

Liposarcomas (LPSs) are the most common soft-tissue cancer. Because of the lack of animal models, the cellular origin and molecular regulation of LPS remain unclear. Here, we report that mice with adipocyte-specific activation of Notch signaling (Ad/N1ICD) develop LPS with complete penetrance. Lineage tracing confirms the adipocyte origin of Ad/N1ICD LPS. The Ad/N1ICD LPS resembles human dedifferentiated LPS in histological appearance, anatomical localization, and gene expression signature. Before transformation, Ad/N1ICD adipocytes undergo dedifferentiation that leads to lipodystrophy and metabolic dysfunction. Although concomitant Pten deletion normalizes the glucose metabolism of Ad/N1ICD mice, it dramatically accelerates the LPS prognosis and malignancy. Transcriptomes and lipidomics analyses indicate that Notch activation suppresses lipid metabolism pathways that supply ligands to Pparγ, the master regulator of adipocyte homeostasis. Accordingly, synthetic Pparγ ligand supplementation induces redifferentiation of Ad/N1ICD adipocytes and tumor cells, and prevents LPS development in Ad/N1ICD mice. Importantly, the Notch target HES1 is abundantly expressed in human LPS, and Notch inhibition suppresses the growth of human dedifferentiated LPS xenografts. Collectively, ectopic Notch activation is sufficient to induce dedifferentiation and tumorigenic transformation of mature adipocytes in mouse.
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http://dx.doi.org/10.1084/jem.20160157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5030803PMC
September 2016

Plk1 inhibition enhances the efficacy of gemcitabine in human pancreatic cancer.

Cell Cycle 2016 ;15(5):711-9

a Department of Biochemistry , Purdue University , West Lafayette , IN , USA.

Gemcitabine is the standard-of-care for chemotherapy in patients with pancreatic adenocarcinoma and it can directly incorporate into DNA or inhibit ribonucleotide reductase to prevent DNA replication and, thus, tumor cell growth. Most pancreatic tumors, however, develop resistance to gemcitabine. Polo-like kinase 1 (Plk1), a critical regulator in many cell cycle events, is significantly elevated in human pancreatic cancer. In this study, we show that Plk1 is required for the G1/S transition and that inhibition of Plk1 significantly reduces the DNA synthesis rate in human pancreatic cancer cells. Furthermore, the combined effect of a specific Plk1 inhibitor GSK461364A with gemcitabine was examined. We show that inhibition of Plk1 significantly potentiates the anti-neoplastic activity of gemcitabine in both cultured pancreatic cancer cells and Panc1-derived orthotopic pancreatic cancer xenograft tumors. Overall, our study demonstrates that co-targeting Plk1 can significantly enhance the efficacy of gemcitabine, offering a promising new therapeutic option for the treatment of gemcitabine-resistant human pancreatic cancer.
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http://dx.doi.org/10.1080/15384101.2016.1148838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4845940PMC
December 2016

Silencing Mist1 Gene Expression Is Essential for Recovery from Acute Pancreatitis.

PLoS One 2015 30;10(12):e0145724. Epub 2015 Dec 30.

Department of Biological Sciences, the Purdue Center for Cancer Research, and the Bindley Bioscience Center, Purdue University, West Lafayette, IN, 47907-2057, United States of America.

Acinar cells of the exocrine pancreas are tasked with synthesizing, packaging and secreting vast quantities of pro-digestive enzymes to maintain proper metabolic homeostasis for the organism. Because the synthesis of high levels of hydrolases is potentially dangerous, the pancreas is prone to acute pancreatitis (AP), a disease that targets acinar cells, leading to acinar-ductal metaplasia (ADM), inflammation and fibrosis-events that can transition into the earliest stages of pancreatic ductal adenocarcinoma. Despite a wealth of information concerning the broad phenotype associated with pancreatitis, little is understood regarding specific transcriptional regulatory networks that are susceptible to AP and the role these networks play in acinar cell and exocrine pancreas responses. In this study, we examined the importance of the acinar-specific maturation transcription factor MIST1 to AP damage and organ recovery. Analysis of wild-type and Mist1 conditional null mice revealed that Mist1 gene transcription and protein accumulation were dramatically reduced as acinar cells underwent ADM alterations during AP episodes. To test if loss of MIST1 function was primarily responsible for the damaged status of the organ, mice harboring a Cre-inducible Mist1 transgene (iMist1) were utilized to determine if sustained MIST1 activity could alleviate AP damage responses. Unexpectedly, constitutive iMist1 expression during AP led to a dramatic increase in organ damage followed by acinar cell death. We conclude that the transient silencing of Mist1 expression is critical for acinar cells to survive an AP episode, providing cells an opportunity to suppress their secretory function and regenerate damaged cells. The importance of MIST1 to these events suggests that modulating key pancreas transcription networks could ease clinical symptoms in patients diagnosed with pancreatitis and pancreatic cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0145724PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4696804PMC
June 2016

Recapitulation of complex transport and action of drugs at the tumor microenvironment using tumor-microenvironment-on-chip.

Cancer Lett 2016 09 10;380(1):319-29. Epub 2015 Dec 10.

Departments of Medicine, Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Pancreatic Cancer Signature Center, Indiana University Simon Cancer Center, Indianapolis, IN 46202, USA.

Targeted delivery aims to selectively distribute drugs to targeted tumor tissues but not to healthy tissues. This can address many clinical challenges by maximizing the efficacy but minimizing the toxicity of anti-cancer drugs. However, a complex tumor microenvironment poses various barriers hindering the transport of drugs and drug delivery systems. New tumor models that allow for the systematic study of these complex environments are highly desired to provide reliable test beds to develop drug delivery systems for targeted delivery. Recently, research efforts have yielded new in vitro tumor models, the so called tumor-microenvironment-on-chip, that recapitulate certain characteristics of the tumor microenvironment. These new models show benefits over other conventional tumor models, and have the potential to accelerate drug discovery and enable precision medicine. However, further research is warranted to overcome their limitations and to properly interpret the data obtained from these models. In this article, key features of the in vivo tumor microenvironment that are relevant to drug transport processes for targeted delivery were discussed, and the current status and challenges for developing in vitro transport model systems were reviewed.
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http://dx.doi.org/10.1016/j.canlet.2015.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4902790PMC
September 2016

Rapid and unbiased extraction of chromatin associated RNAs from purified native chromatin.

J Chromatogr A 2015 Dec 22;1426:64-8. Epub 2015 Nov 22.

Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, USA; Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN 47907, USA; Purdue Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA. Electronic address:

An ultra fast and unbiased method that uses salicylic acid coated magnetic nanoparticles (SAMNPs) and magnetophoretic chromatography is developed to extract chromatin associated RNAs (CARs). The SAMNPs were first used for enriching cells from the cell culture media and further used for capturing chromatin after cells were lysed. The formed SAMNPs-chromatin complexes were transferred to a viscous polyethylene glycol (PEG) solution stored in a 200-μl pipette tip. Due to the difference in viscosities, a bi-layer liquid was formed inside the pipette tip. The SAMNPs-chromatin complexes were separated from the free SAMNPs and free RNA-SAMNPs complexes by applying an external magnetic field. The CARs were further extracted from the SAMNP-chromatin complexes directly. The extracted CARs were reverse transcribed as cDNA and further characterized by real-time qPCR. The total assay time taken for cell separation, chromatin purification and chromatin associated RNAs extraction can be accomplished in less than 2h.
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http://dx.doi.org/10.1016/j.chroma.2015.11.067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4766097PMC
December 2015

Mist1 Expressing Gastric Stem Cells Maintain the Normal and Neoplastic Gastric Epithelium and Are Supported by a Perivascular Stem Cell Niche.

Cancer Cell 2015 Dec 12;28(6):800-814. Epub 2015 Nov 12.

Division of Digestive and Liver Disease, Department of Medicine, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA. Electronic address:

The regulation and stem cell origin of normal and neoplastic gastric glands are uncertain. Here, we show that Mist1 expression marks quiescent stem cells in the gastric corpus isthmus. Mist1(+) stem cells serve as a cell-of-origin for intestinal-type cancer with the combination of Kras and Apc mutation and for diffuse-type cancer with the loss of E-cadherin. Diffuse-type cancer development is dependent on inflammation mediated by Cxcl12(+) endothelial cells and Cxcr4(+) gastric innate lymphoid cells (ILCs). These cells form the perivascular gastric stem cell niche, and Wnt5a produced from ILCs activates RhoA to inhibit anoikis in the E-cadherin-depleted cells. Targeting Cxcr4, ILCs, or Wnt5a inhibits diffuse-type gastric carcinogenesis, providing targets within the neoplastic gastric stem cell niche.
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http://dx.doi.org/10.1016/j.ccell.2015.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684751PMC
December 2015

Cotargeting Polo-Like Kinase 1 and the Wnt/β-Catenin Signaling Pathway in Castration-Resistant Prostate Cancer.

Mol Cell Biol 2015 Dec 5;35(24):4185-98. Epub 2015 Oct 5.

Department of Biochemistry, Purdue University, West Lafayette, Indiana, USA Center for Cancer Research, Purdue University, West Lafayette, Indiana, USA

The Wnt/β-catenin signaling pathway has been identified as one of the predominantly upregulated pathways in castration-resistant prostate cancer (CRPC). However, whether targeting the β-catenin pathway will prove effective as a CRPC treatment remains unknown. Polo-like kinase 1 (Plk1) is a critical regulator in many cell cycle events, and its level is significantly elevated upon castration of mice carrying xenograft prostate tumors. Indeed, inhibition of Plk1 has been shown to inhibit tumor growth in several in vivo studies. Here, we show that Plk1 is a negative regulator of Wnt/β-catenin signaling. Plk1 inhibition or depletion enhances the level of cytosolic and nuclear β-catenin in human prostate cancer cells. Furthermore, inhibition of Wnt/β-catenin signaling significantly potentiates the antineoplastic activity of the Plk1 inhibitor BI2536 in both cultured prostate cancer cells and CRPC xenograft tumors. Mechanistically, axin2, a negative regulator of the β-catenin pathway, serves as a substrate of Plk1, and Plk1 phosphorylation of axin2 facilitates the degradation of β-catenin by enhancing binding between glycogen synthase kinase 3β (GSK3β) and β-catenin. Plk1-phosphorylated axin2 also exhibits resistance to Cdc20-mediated degradation. Overall, this study identifies a novel Plk1-Wnt signaling axis in prostate cancer, offering a promising new therapeutic option to treat CRPC.
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http://dx.doi.org/10.1128/MCB.00825-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4648817PMC
December 2015

The basic helix-loop-helix transcription factor E47 reprograms human pancreatic cancer cells to a quiescent acinar state with reduced tumorigenic potential.

Pancreas 2015 Jul;44(5):718-27

From the *Development and Aging Program, Neuroscience, Aging, and Stem Cell Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA; †Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Institute of Women's Life Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea; ‡Department of Pediatrics, University of California, San Diego, La Jolla, CA; §Department of Biological Sciences, Center for Cancer Research, the Bindley Bioscience Center, Purdue University, West Lafayette, IN; and ∥Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA.

Objectives: Pancreatic ductal adenocarcinoma (PDA) initiates from quiescent acinar cells that attain a Kras mutation, lose signaling from basic helix-loop-helix (bHLH) transcription factors, undergo acinar-ductal metaplasia, and rapidly acquire increased growth potential. We queried whether PDA cells can be reprogrammed to revert to their original quiescent acinar cell state by shifting key transcription programs.

Methods: Human PDA cell lines were engineered to express an inducible form of the bHLH protein E47. Gene expression, growth, and functional studies were investigated using microarray, quantitative polymerase chain reaction, immunoblots, immunohistochemistry, small interfering RNA, chromatin immunoprecipitation analyses, and cell transplantation into mice.

Results: In human PDA cells, E47 activity triggers stable G0/G1 arrest, which requires the cyclin-dependent kinase inhibitor p21 and the stress response protein TP53INP1. Concurrently, E47 induces high level expression of acinar digestive enzymes and feed forward activation of the acinar maturation network regulated by the bHLH factor MIST1. Moreover, induction of E47 in human PDA cells in vitro is sufficient to inhibit tumorigenesis.

Conclusions: Human PDA cells retain a high degree of plasticity, which can be exploited to induce a quiescent acinar cell state with reduced tumorigenic potential. Moreover, bHLH activity is a critical node coordinately regulating human PDA cell growth versus cell fate.
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http://dx.doi.org/10.1097/MPA.0000000000000328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4464938PMC
July 2015

Salivary gland homeostasis is maintained through acinar cell self-duplication.

Dev Cell 2015 Apr 2;33(2):231-7. Epub 2015 Apr 2.

Center for Oral Biology, Department of Biomedical Genetics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA. Electronic address:

Current dogma suggests that salivary gland homeostasis is stem cell dependent. However, the extent of stem cell contribution to salivary gland maintenance has not been determined. We investigated acinar cell replacement during homeostasis, growth, and regeneration, using an inducible CreER(T2) expressed under the control of the Mist1 gene locus. Genetic labeling, followed by a chase period, showed that acinar cell replacement is not driven by the differentiation of unlabeled stem cells. Analysis using R26(Brainbow2.1) reporter revealed continued proliferation and clonal expansion of terminally differentiated acinar cells in all major salivary glands. Induced injury also demonstrated the regenerative potential of pre-labeled acinar cells. Our results support a revised model for salivary gland homeostasis based predominantly on self-duplication of acinar cells, rather than on differentiation of stem cells. The proliferative capacity of differentiated acinar cells may prove critical in the implementation of cell-based strategies to restore the salivary glands.
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http://dx.doi.org/10.1016/j.devcel.2015.02.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4406828PMC
April 2015

Plk1 phosphorylation of PTEN causes a tumor-promoting metabolic state.

Mol Cell Biol 2014 Oct 21;34(19):3642-61. Epub 2014 Jul 21.

Department of Biochemistry, Purdue University, West Lafayette, Indiana, USA Purdue Center for Cancer Research, Purdue University, West Lafayette, Indiana, USA

One outcome of activation of the phosphatidylinositol 3-kinase (PI3K) pathway is increased aerobic glycolysis, but the upstream signaling events that regulate the PI3K pathway, and thus the Warburg effect, are elusive. Increasing evidence suggests that Plk1, a cell cycle regulator, is also involved in cellular events in addition to mitosis. To test whether Plk1 contributes to activation of the PI3K pathway, and thus aerobic glycolysis, we examined potential targets of Plk1 and identified PTEN as a Plk1 substrate. We hypothesize that Plk1 phosphorylation of PTEN leads to its inactivation, activation of the PI3K pathway, and the Warburg effect. Our data show that overexpression of Plk1 leads to activation of the PI3K pathway and enhanced aerobic glycolysis. In contrast, inhibition of Plk1 causes markedly reduced glucose metabolism in mice. Mechanistically, we show that Plk1 phosphorylation of PTEN and Nedd4-1, an E3 ubiquitin ligase of PTEN, results in PTEN inactivation. Finally, we show that Plk1 phosphorylation of PTEN promotes tumorigenesis in both its phosphatase-dependent and -independent pathways, revealing potentially new drug targets to arrest tumor cell growth.
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http://dx.doi.org/10.1128/MCB.00814-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4187734PMC
October 2014

A photo-degradable gene delivery system for enhanced nuclear gene transcription.

Biomaterials 2014 Jan 27;35(3):1040-9. Epub 2013 Oct 27.

School of Chemical Engineering, Purdue University, West Lafayette, IN 47907, United States.

There currently exists a significant gap in our understanding of how the detailed chemical characteristics of polycation gene carriers influence their delivery performances in overcoming an important cellular-level transport barrier, i.e., intranuclear gene transcription. In this study, a UV-degradable gene carrier material (ENE4-1) was synthesized by crosslinking low molecular weight branched polyethylenimine (bPEI-2k) molecules using UV-cleavable o-nitrobenzyl urethane (NBU) as the linker molecule. NBU degrades upon exposure to mild UV irradiation. Therefore, this UV-degradable carrier allows us to control the chemical characteristics of the polymer/DNA complex (polyplex) particles at desired locations within the intracellular environment. By using this photolytic DNA carrier, we found that the exact timing of the UV degradation significantly influences the gene transfection efficiencies of ENE4-1/DNA(pGL2) polyplexes in HeLa cells. Interestingly, even if the polyplexes were UV-degraded at different intracellular locations/times, their nuclear entry efficiency was not influenced by the location/timing of UV degradation. The UV treatment did not influence the size or binding strength of the polyplexes. However, we confirmed that the degradation of the carrier molecules impacts the chemical characteristics of the polyplexes (it produces carbamic acid and nitrosobenzyl aldehyde groups on ENE4-1). We believe that these anionic acid groups enhance the interaction of the polyplexes with nuclear transcription proteins and thus the final gene expression levels; this effect was found to occur, even though UV irradiation itself has a general effect of reducing transfection efficiencies. Excess (uncomplexed) ENE4-1 polymers appear to not play any role in the UV-enhanced gene transcription phenomenon.
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http://dx.doi.org/10.1016/j.biomaterials.2013.10.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3849696PMC
January 2014

Pancreatic Acinar Cell 3-Dimensional Culture.

Bio Protoc 2013 Oct;3(19)

Biological Sciences Department, Purdue University, West Lafayette, USA.

Normal pancreatic acinar cells are difficult to maintain on traditional plastic culture surfaces due to their physical properties of housing large quantities of digestive enzymes and the formation of intercellular tight junctions and gap junctions (Apte and Wilson 2005; Rukstalis ., 2003). However, placing primary acinar cells within a 3-dimensional matrix (3D-culture) maintains the cells for sufficient time so that they can be monitored for physiological changes to different stimuli. We have used a modified collagen 3D-culture system that has been adapted from Means . (2005) to model the very early events associated with pancreatic cancer development. In this model, Kras-expressing pancreatic acinar cells, or wildtype acinar cells treated with EGFR-dependent growth factors (, TGFα), convert to ductal cysts that mimic the acinar-to-ductal metaplasia (ADM) stage that precedes formation of Pancreatic Intraepithelial Neoplasia (PanIN) and Pancreatic Ductal Adenocarcinoma (PDAC) (Means ., 2005; Shi ., 2013).
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4957951PMC
http://dx.doi.org/10.21769/bioprotoc.930DOI Listing
October 2013

Plk1 phosphorylation of orc2 and hbo1 contributes to gemcitabine resistance in pancreatic cancer.

Mol Cancer Ther 2013 Jan 27;12(1):58-68. Epub 2012 Nov 27.

Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.

Although gemcitabine is the standard chemotherapeutic drug for treatment of pancreatic cancer, almost all patients eventually develop resistance to this agent. Previous studies identified Polo-like kinase 1 (Plk1) as the mediator of gemcitabine resistance, but the molecular mechanism remains unknown. In this study, we show that Plk1 phosphorylation of Orc2 and Hbo1 mediates the resistance to gemcitabine. We show that the level of Plk1 expression positively correlates with gemcitabine resistance, both in pancreatic cancer cells and xenograft tumors. Overexpression of Plk1 increases gemcitabine resistance, while inhibition of Plk1 sensitizes pancreatic cancer cells to gemcitabine treatment. To validate our findings, we show that inhibition of Plk1 sensitizes tumors to gemcitabine treatment in a mouse xenograft study. Mechanistically, we find that Plk1 phosphorylation of Orc2 maintains DNA replication on gemcitabine treatment. Furthermore, Plk1 phosphorylation of Hbo1 transcriptionally increases cFos expression and consequently elevates its target multidrug resistance 1 (MDR1), which was previously reported to confer chemotherapeutic drug resistance. Knockdown of cFos or MDR1 sensitizes gemcitabine-resistant cells to gemcitabine treatment. Finally, pancreatic cancer cells expressing Plk1-unphosphorylatable mutants of Orc2 or Hbo1 are more sensitive to gemcitabine than cells expressing wild-type Orc2 or Hbo1. In short, our study provides a mechanism for Plk1-mediated gemcitabine resistance, suggesting that Plk1 is a promising target for treatment of gemcitabine-resistant pancreatic cancer.
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http://dx.doi.org/10.1158/1535-7163.MCT-12-0632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3732037PMC
January 2013

Induced Mist1 expression promotes remodeling of mouse pancreatic acinar cells.

Gastroenterology 2012 Aug 14;143(2):469-80. Epub 2012 Apr 14.

Department of Biological Sciences, Purdue University, West Lafayette, IN, USA.

Background & Aims: Early embryogenesis involves cell fate decisions that define the body axes and establish pools of progenitor cells. Development does not stop once lineages are specified; cells continue to undergo specific maturation events, and changes in gene expression patterns lead to their unique physiological functions. Secretory pancreatic acinar cells mature postnatally to synthesize large amounts of protein, polarize, and communicate with other cells. The transcription factor MIST1 is expressed by only secretory cells and regulates maturation events. MIST1-deficient acinar cells in mice do not establish apical-basal polarity, properly position zymogen granules, or communicate with adjacent cells, disrupting pancreatic function. We investigated whether MIST1 directly induces and maintains the mature phenotype of acinar cells.

Methods: We analyzed the effects of Cre-mediated expression of Mist1 in adult Mist1-deficient (Mist1(KO)) mice. Pancreatic tissues were collected and analyzed by light and electron microscopy, immunohistochemistry, real-time polymerase chain reaction analysis, and chromatin immunoprecipitation. Primary acini were isolated from mice and analyzed in amylase secretion assays.

Results: Induced expression of Mist1 in adult Mist1(KO) mice restored wild-type gene expression patterns in acinar cells. The acinar cells changed phenotypes, establishing apical-basal polarity, increasing the size of zymogen granules, reorganizing the cytoskeletal network, communicating intercellularly (by synthesizing gap junctions), and undergoing exocytosis.

Conclusions: The exocrine pancreas of adult mice can be remodeled by re-expression of the transcription factor MIST1. MIST1 regulates acinar cell maturation and might be used to repair damaged pancreata in patients with pancreatic disorders.
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http://dx.doi.org/10.1053/j.gastro.2012.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3664941PMC
August 2012