Human Pancreatic Intraepithelial Neoplasia Research Articles

ATDC induces an invasive switch in KRAS-induced pancreatic tumorigenesis

The initiation of pancreatic ductal adenocarcinoma (PDA) is linked to activating mutations in KRAS. However, in PDA mouse models, expression of oncogenic mutant KRAS during development gives rise to tumors only after a prolonged latency or following induction of pancreatitis. Here we describe a novel mouse model expressing ataxia telangiectasia group D complementing gene (ATDC, also known as TRIM29 [tripartite motif 29]) that, in the presence of oncogenic KRAS, accelerates pancreatic intraepithelial neoplasia (PanIN) formation and the development of invasive and metastatic cancers. We found that ATDC up-regulates CD44 in mouse and human PanIN lesions via activation of β-catenin signaling, leading to the induction of an epithelial-to-mesenchymal transition (EMT) phenotype characterized by expression of Zeb1 and Snail1. We show that ATDC is up-regulated by oncogenic Kras in a subset of PanIN cells that are capable of invading the surrounding stroma. These results delineate a novel molecular pathway for EMT in pancreatic tumorigenesis, showing that ATDC is a proximal regulator of EMT.

Covering the Cover

Covering the Cover

The biological features of PanIN initiated from oncogenic Kras mutation in genetically engineered mouse models

Pancreatic intraepithelial neoplasia (PanIN) is the most common premalignant lesion of the pancreas. Further understanding of the biological behavior and molecular genetic alterations in the stepwise progression of PanINs is necessary toward the development of pancreatic ductal adenocarcinoma (PDAC) interventions. In this study, we analyzed the morphological characteristics, molecular alterations, and biological behavior of pancreatic wild-type and neoplasia tissues, including analysis of PanIN cell line SH-PAN (isolated from Pdx-1-Cre; LSL-KrasG12D/+ mouse) and PDAC cell line DT-PCa (isolated from Pdx1-Cre; LSL-KrasG12D/+; LSL-Tp53R172H/+ mouse). Results show that KrasG12D induces ductal lesion PanINs. Increased expression of EGFR, Her-2/Neu, p-MAPK and β-Catenin was observed in low-grade PanINs. Tp53 was not expressed in wild-type and low-grade PanINs, however, increased expression was observed in high-grade PanINs. Furthermore, SH-PAN cells did not exhibit any colony formation and showed significantly lower migration and invasion ability compared with DT-PCa cells. Notably, we first found PPP2R2A (protein phosphatase 2, regulatory subunit B, alpha) expression was significantly higher in SH-PAN cells than DT-PCa cells, and was high in 96 of 172 peritumoral normal human pancreatic tissues and 20 of 36 human low- or middle-grade PanIN tissues, whereas, was weak or negligible in 12 of 20 human high-grade PanIN tissues and 124 of 172 human PDAC tissues post-operation. The expression of PPP2R2A appears to be correlated with clinical survival. Taken together, KrasG12D – driven PanIN showed the tumorigenic ability, however, did not undergo a malignant transformation, and decreased expression of PPP2R2A in PDACs may provided a new target for pancreatic carcinoma intervention.

Abstract ED02-03: Animal models for human pancreatic cancer prevention

Abstract Pancreatic cancer has the worst prognosis and is the fourth leading cause of cancer-related deaths in the United States. The five-year survival for this disease is <6% and it is lethal to almost all of the patients diagnosed. As with many epithelial cancers, pre-invasive precursors in the pancreas progress slowly over many years to decades to development of invasive pancreatic cancers in humans. Lack of early detection and effective interventions are major factors contributing to the poor prognosis and dismal survival rates of pancreatic cancer patients for more than sixty years. Detection of pancreatic cancer at an early stage might permit life-saving interventions. Use of tumor xenografts and/or orthotopic models to predict valid targets and for design and development of drugs for pancreatic cancer patients has been disappointing. Animal models with etiological relevance that recapitulate the molecular pathology of human pancreatic intraepithelial neoplasia (PanINs) and their progression to pancreatic ductal adenocarcinoma (PDAC) are ideal models for establishing pancreatic cancer prevention and treatment strategies. Recent advent in genetically engineered mouse (GEM) models, such as development of transgenic KrasG12D-dependent mouse models of PDAC, that mimic human PDAC offer opportunities to identify putative targets of pancreatic lesion development and targeted drug strategies for PDAC prevention. We will present recent advances on i) selection of optimal Kras mouse models for prevention and early diagnosis, and ii) application of Whole Transcriptome (SOLiD) approaches to identify druggable targets for PanINs and PDAC. Then, iii) we will provide a few examples of development of molecularly targeted chemopreventive drugs using the krasG12D mouse model. Citation Format: Chinthalapally V. Rao. Animal models for human pancreatic cancer prevention. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr ED02-03.

Early Detection and Prevention of Pancreatic Cancer: Use of Genetically Engineered Mouse Models and advanced Imaging Technologies

Lack of early detection and effective interventions are major factors contributing to the poor prognosis and dismal survival rates of pancreatic cancer patients for more than sixty years. Detection of pancreatic cancer at an early stage might permit life-saving intervention. Clinical and preclinical diagnosis and evaluation of pancreatic cancers involve several imaging technologies including magnetic resonance imaging (MRI), Positron emission tomography (PET), Computed tomography (CT), Ultrasound (US), bioluminescent imaging and single photon emission computed tomography (SPECT). The advent of genetically engineered animal models that recapitulate the cellular and molecular pathology of human pancreatic intraepithelial neoplasia (PanINs) and pancreatic ductal adenocarcinoma (PDAC) has not yet yielded translational implications. Although the use of tumor xenografts to predict drug efficacy in patients has been disappointing, use of novel transgenic mice models should permit improved early detection and development of drug regimens through integration of appropriate imaging modalities. This review will consider issues that are unique to working with transgenic mouse models, such as the biology of genetically engineered mouse (GEM) models, stage- tumor-specific detection using imaging technologies, use of monoclonal antibodies, nanoparticles, and biomarkers, and development of chemopreventive and chemotherapeutic drugs for PDAC. These issues will be considered in the context of recently developed preclinical models of pancreatic cancer.

GATA6 Activates Wnt Signaling in Pancreatic Cancer by Negatively Regulating the Wnt Antagonist Dickkopf-1

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease characterized by late diagnosis and treatment resistance. Recurrent genetic alterations in defined genes in association with perturbations of developmental cell signaling pathways have been associated with PDAC development and progression. Here, we show that GATA6 contributes to pancreatic carcinogenesis during the temporal progression of pancreatic intraepithelial neoplasia by virtue of Wnt pathway activation. GATA6 is recurrently amplified by both quantitative-PCR and fluorescent in-situ hybridization in human pancreatic intraepithelial neoplasia and in PDAC tissues, and GATA6 copy number is significantly correlated with overall patient survival. Forced overexpression of GATA6 in cancer cell lines enhanced cell proliferation and colony formation in soft agar in vitro and growth in vivo, as well as increased Wnt signaling. By contrast siRNA mediated knockdown of GATA6 led to corresponding decreases in these same parameters. The effects of GATA6 were found to be due to its ability to bind DNA, as forced overexpression of a DNA-binding mutant of GATA6 had no effects on cell growth in vitro or in vivo, nor did they affect Wnt signaling levels in these same cells. A microarray analysis revealed the Wnt antagonist Dickopf-1 (DKK1) as a dysregulated gene in association with GATA6 knockdown, and direct binding of GATA6 to the DKK1 promoter was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift assays. Transient transfection of GATA6, but not mutant GATA6, into cancer cell lines led to decreased DKK1 mRNA expression and secretion of DKK1 protein into culture media. Forced overexpression of DKK1 antagonized the effects of GATA6 on Wnt signaling in pancreatic cancer cells. These findings illustrate that one mechanism by which GATA6 promotes pancreatic carcinogenesis is by virtue of its activation of canonical Wnt signaling via regulation of DKK1.

Abstract 2449: Pancreatic cancer tumor initiating cells are marked by the presence of a primary cilium

Abstract Primary cilia are microtubule rich projections eminating from the apical surface of epithelial cells. Primary cilia are responsible for the transduction of growth factor and morphogenetic signaling pathways, including the hedgehog, wnt and platelet-derived growth factor pathways. Initial observations in the field of pancreatic cancer have suggested the absence of primary cilia in human pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma. While absent in the majority of pancreatic cancer cells, primary cilia have been identified in differentiated acinar cells undergoing an acinar to ductal metaplasia. This metaplasia is well characterized in pancreatic injury and regeneration models and accelerates tumor formation in the presence of acinar specific KrasG12D mutations. Thus, we are addressing the hypothesis that primary cilia, while not expressed in bulk tumor populations, are maintained in pancreatic tumor-initiating cells. To address the hypothesis, we have used confocal microscopy to quantify the percentage of cells within low grade human PanIN lesions that express a primary cilium. Performing confocal microscopy on a human PanIN tissue array (n=23), we have determined that in 15% of PanIN lesions, 13-30% of the cells are expressing a primary cilium, as is evidenced by staining for acetylated alpha tubulin. We have also developed a sorting strategy using an acetylated alpha tubulin antibody to isolate ciliated cells by Fluorescence Activated Cell Sorting (FACS) analysis. Using this protocol, we have discovered that human pancreatic cancer cells bearing a primary cilium are markedly enriched in cells also positive for either CD133 or CD24/CD44, representing putative pancreatic cancer stem cell populations. We have functionally analyzed the tumor-initiating capacity of ciliated vs non-ciliated cells using tumor sphere experiments. In this assay, ciliated cells show a significant increase (100 fold) in tumor sphere-forming capacity, suggesting that the presence of a primary cilium may mark a pancreatic cancer tumor initiating population. These data are informative regarding the earliest initiating events in pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2449. doi:10.1158/1538-7445.AM2011-2449

Abstract 2531: The selective CCR9 antagonist CCX8037 inhibits CCL25-CCR9-STAT3 mediated proliferation of pancreatic cancer cells

Abstract Introduction: We previously reported the expression of CCR9 in pancreatic intraepithelial neoplasia (PanIN) and invasive pancreatic cancer tissues. Here, our objective was to investigate signaling downstream of CCR9 and to determine the anti-proliferative effects of the candidate therapeutic CCR9 antagonist CCX8037. Methods: PanIN and corresponding invasive pancreatic cancer (5143PDA) and liver metastasis (5143LM) cell lines were derived from Pdx-1-Cre;LSL-K-rasG12D and Pdx-1-Cre;LSL-K-rasG12D/p53R172H mice. In culture, murine and human pancreatic cancer cells (PANC-1) were exposed to CCL25, the specific CCR9 ligand. The phosphorylation of STAT3, an established regulator of pancreatic cancer proliferation, was measured by Western blot assay and proliferation assays were performed with the small molecule CCX8037 and the JAK/STAT inhibitor AG490. Results: First, we verified that exposure to CCL25 enhanced cell proliferation (20%-40%) in both murine and human pancreatic cancer cells. To investigate CCL25/CCR9 signaling, we exposed cells to CCL25 and observed increased phosphorylation of STAT3 in the cells. Blockade of STAT3 signaling with AG490 abrogated CCL25/CCR9-mediated proliferation in both murine and human pancreatic cancer cells. Pre-treatment of PanIN and pancreatic cancer cells with CCX8037 blocked CCL25-mediated phosphorylation of STAT3 and inhibited CCL25-mediated cell proliferation. Conclusion: Our study demonstrates for the first time that the CCL25/CCR9 axis activates downstream STAT3 signaling to mediate proliferation in murine and human PanIN and pancreatic cancer cells. Additionally, our data shows that the candidate therapeutic agent CCX8037 effectively blocks CCL25/CCR9-mediated proliferation in pancreatic cancer and warrants investigation in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2531.

Widespread activation of the DNA damage response in human pancreatic intraepithelial neoplasia

Pancreatic intraepithelial neoplasia (PanIN) lesions are the most common non-invasive precursors of pancreatic adenocarcinoma. We postulated that accumulating DNA damage within the PanIN epithelium activates checkpoint mechanisms. Tissue microarrays were constructed from 81 surgically resected primary pancreatic adenocarcinomas and an independent set of 58 PanIN lesions (31 PanIN-1, 14 PanIN-2, and 13 PanIN-3). Immunohistochemical labeling was carried out using anti-γH2AXSer139, anti-phosphoATMSer1981, anti-phosphoChk2Thr68, and anti-p53. A ‘histologic score’ combining area and intensity of labeling in the nuclear compartment was determined for each lesion. A progressive increase in γH2AXSer139 labeling, consistent with escalating DNA damage, was observed in the non-invasive precursor lesions (scores of 4.34, 6.21, and 7.50, respectively, for PanIN-1, -2, and -3), compared with the pancreatic ductal epithelium (score 2.36) (ANOVA, P<0.0001). In conjunction, activation of the ataxia telangiectasia mutated (ATM)–Chk2 checkpoint pathway was observed in all histological grades of PanIN lesions. Specifically, pATMSer1981 histologic scores for PanIN-1, PanIN-2, and PanIN-3 were 4.83, 5.14, and 7.17, respectively, versus 2.33 for the ductal epithelium (ANOVA, P<0.0001); the corresponding scores for pChk2Thr68 were 5.43, 7.64, and 5.44 in PanINs-1, -2, and -3, respectively, versus 2.75 in the ductal epithelium (ANOVA, P<0.0001). In contrast, absent to minimal nuclear p53 was observed in the ductal epithelium, and in PanINs-1 and -2 (a histologic score of 0–1.86), with a significant upregulation (corresponding to mutational inactivation) seen only at the stage of PanIN-3 and invasive neoplasia (histologic scores of 4.00 and 4.22). Nuclear p53 accumulation in cancers was associated with attenuation of the ATM–Chk2 checkpoint and a restitution to ‘baseline’ levels. To conclude, activation of the ATM–Chk2 checkpoint pathway is commonly observed in PanINs, likely in response to the accumulating DNA damage from events such as oncogene mutations and telomere dysfunction. Loss of p53 function appears to be a critical determinant for bypassing this checkpoint and the subsequent progression to invasive adenocarcinoma.

CC Chemokine Receptor 9 Enhances Proliferation in Pancreatic Intraepithelial Neoplasia and Pancreatic Cancer Cells

IntroductionChemokine receptors may regulate the progression and metastasis of invasive malignancies. There are little data, however, regarding their role in premalignant lesions. Our objective was to determine the role of CC chemokine receptor 9 (CCR9) in pancreatic intraepithelial neoplasia (PanIN). MethodsHuman and murine formalin-fixed paraffin-embedded (FFPE) PanIN specimens were assessed for CCR9 expression. The established murine PanIN, invasive pancreatic cancer (5143PDA) and liver metastasis (5143LM) cell lines, and human pancreatic cancer cell line (PANC-1) were obtained to verify CCR9 expression and function. ResultsImmunohistochemistry of FFPE specimens demonstrated CCR9 expression in both murine and human PanIN lesions. CCR9 expression in murine and human cell lines was verified by Western blot assay, immunofluorescence, and flow cytometry. CCR9 function was demonstrated by in vitro exposure to CCL25, the selective CCR9 ligand, which resulted in significantly increased cell proliferation in PanIN and pancreatic cancer cell lines. ConclusionsThis is the first report of chemokine receptor CCR9 expression in murine and human PanIN tissues. Our results demonstrate enhanced PanIN and pancreatic cancer cell proliferation with activation of CCR9 by its selective ligand CCL25. CCR9 may prove to be a novel therapeutic target for PanIN and its progression to invasive cancer.

Molecular genetics of pancreatic intraepithelial neoplasia

Recent evidence suggests that noninvasive precursor lesions, classified as pancreatic intraepithelial neoplasia (PanIN), can progress to invasive pancreatic cancer. This review will discuss the major genetic alterations in PanIN lesions. A comprehensive review of the literature was performed in order to find studies on the molecular profile of human PanIN lesions. In addition, recent publications on genetically engineered mouse models of preinvasive neoplasia and pancreatic cancers were reviewed. PanINs demonstrate abnormalities at the genomic (DNA), transcriptomic (RNA), and proteomic levels, and there is a progressive accumulation of molecular alterations that accompany the histological progression from low-grade PanIN-1A to high-grade PanIN-3 lesions. Molecular changes in PanINs can be classified as "early" (KRAS2 mutations, telomere shortening, p21(WAF1/CIP1) up-regulation, etc.), "intermediate" (cyclin D1 up-regulation, expression of proliferation antigens, etc.), or "late" (BRCA2 and TP53 mutations, DPC4/SMAD4/MADH4 inactivation, etc.). All the genetic changes observed in PanINs are also found in invasive ductal adenocarcinomas, where they usually occur at a higher frequency. Genetically engineered mice expressing mutant Kras in the pancreas, with or without additional genetic alterations, provide a unique in vivo platform to study the pancreatic cancer progression model. Molecular studies have been instrumental in establishing that PanIN lesions are the noninvasive precursors for invasive ductal adenocarcinomas. The availability of molecular date provides the basis for designing rational early detection strategies and therapeutic intervention trials before pancreatic neoplasms invade, with the intention of alleviating the dismal prognosis associated with this disease.

Gene Expression Profiles in Pancreatic Intraepithelial Neoplasia Reflect the Effects of Hedgehog Signaling on Pancreatic Ductal Epithelial Cells

Invasive pancreatic cancer is thought to develop through a series of noninvasive duct lesions known as pancreatic intraepithelial neoplasia (PanIN). We used cDNA microarrays interrogating 15,000 transcripts to identify 49 genes that were differentially expressed in microdissected early PanIN lesions (PanIN-1B/2) compared with microdissected normal duct epithelium. In this analysis, a cluster of extrapancreatic foregut markers, including pepsinogen C, MUC6, KLF4, and TFF1, was found to be up-regulated in PanIN. Up-regulation of these genes was further validated using combinations of real-time reverse transcription-PCR, in situ hybridization, and immunohistochemistry in a total of 150 early PanIN lesions from 81 patients. Identification of these gastrointestinal transcripts in human PanIN prompted assessment of other foregut markers by both semiquantitative and real-time reverse transcription-PCR, revealing similar up-regulation of Sox-2, Gastrin, HoxA5, GATA4/5/6, Villin and Forkhead 6 (Foxl1). In contrast to frequent expression of multiple gastric epithelial markers, the intestinal markers intestinal fatty acid binding protein, CDX1 and CDX2 were rarely expressed either in PanIN lesions or in invasive pancreatic cancer. Hedgehog pathway activation induced by transfection of immortalized human pancreatic ductal epithelial cells with Gli1 resulted in up-regulation of the majority of foregut markers seen in early PanIN lesions. These data show frequent up-regulation of foregut markers in early PanIN lesions and suggest that PanIN development may involve Hedgehog-mediated conversion to a gastric epithelial differentiation program.

Matrix metalloproteinase-7 is expressed by pancreatic cancer precursors and regulates acinar-to-ductal metaplasia in exocrine pancreas.

In gastrointestinal epithelium, metaplastic conversion between predominant cell types is associated with an increased risk of neoplasia. However, the mechanisms regulating metaplastic transitions in adult epithelia are largely undefined. Here we show that matrix metalloproteinase-7 (MMP-7) is expressed not only in the majority of human pancreatic ductal adenocarcinoma specimens, but also in human pancreatic intraepithelial neoplasia and metaplastic duct lesions in human and mouse. In a mouse model of pancreatic acinar-to-ductal metaplasia, MMP-7 progressively accumulates during the metaplastic transition, resulting in a concomitant increase in solubilization of Fas ligand (FasL). Under identical conditions, mice either deficient in MMP-7 or carrying an inactive FasL gene are severely inhibited in development of progressive metaplasia and acinar cell apoptosis. Thus, MMP-7 and FasL influence the initiation and maintenance of metaplastic events in pancreatic epithelium, explaining the observed link between metaplasia and apoptosis in pancreas and other gastrointestinal tissues.

Matrix metalloproteinase-7 is expressed by pancreatic cancer precursors and regulates acinar-to-ductal metaplasia in exocrine pancreas

In gastrointestinal epithelium, metaplastic conversion between predominant cell types is associated with an increased risk of neoplasia. However, the mechanisms regulating metaplastic transitions in adult epithelia are largely undefined. Here we show that matrix metalloproteinase-7 (MMP-7) is expressed not only in the majority of human pancreatic ductal adenocarcinoma specimens, but also in human pancreatic intraepithelial neoplasia and metaplastic duct lesions in human and mouse. In a mouse model of pancreatic acinar-to-ductal metaplasia, MMP-7 progressively accumulates during the metaplastic transition, resulting in a concomitant increase in solubilization of Fas ligand (FasL). Under identical conditions, mice either deficient in MMP-7 or carrying an inactive FasL gene are severely inhibited in development of progressive metaplasia and acinar cell apoptosis. Thus, MMP-7 and FasL influence the initiation and maintenance of metaplastic events in pancreatic epithelium, explaining the observed link between metaplasia and apoptosis in pancreas and other gastrointestinal tissues.

Matrix metalloproteinase-7 is expressed by pancreatic cancer precursors and regulates acinar-to-ductal metaplasia in exocrine pancreas

In gastrointestinal epithelium, metaplastic conversion between predominant cell types is associated with an increased risk of neoplasia. However, the mechanisms regulating metaplastic transitions in adult epithelia are largely undefined. Here we show that matrix metalloproteinase-7 (MMP-7) is expressed not only in the majority of human pancreatic ductal adenocarcinoma specimens, but also in human pancreatic intraepithelial neoplasia and metaplastic duct lesions in human and mouse. In a mouse model of pancreatic acinar-to-ductal metaplasia, MMP-7 progressively accumulates during the metaplastic transition, resulting in a concomitant increase in solubilization of Fas ligand (FasL). Under identical conditions, mice either deficient in MMP-7 or carrying an inactive FasL gene are severely inhibited in development of progressive metaplasia and acinar cell apoptosis. Thus, MMP-7 and FasL influence the initiation and maintenance of metaplastic events in pancreatic epithelium, explaining the observed link between metaplasia and apoptosis in pancreas and other gastrointestinal tissues.