Audrey M. Hendley

p53 mutations cooperate with oncogenic Kras to promote adenocarcinoma from pancreatic ductal cells

Pancreatic cancer is one of the most lethal malignancies, with virtually all patients eventually succumbing to their disease. Mutations in p53 have been documented in >50% of pancreatic cancers. Owing to the high incidence of p53 mutations in PanIN 3 lesions and pancreatic tumors, we interrogated the comparative ability of adult pancreatic acinar and ductal cells to respond to oncogenic Kras and mutant Tp53R172H using Hnf1b:CreERT2 and Mist1:CreERT2 mice. These studies involved co-activation of a membrane-tethered GFP lineage label, allowing for direct visualization and isolation of cells undergoing Kras and mutant p53 activation. Kras activation in Mist1+ adult acinar cells resulted in brisk PanIN formation, whereas no evidence of pancreatic neoplasia was observed for up to 6 months following Kras activation in Hnf1beta+ adult ductal cells. In contrast to the lack of response to oncogenic Kras alone, simultaneous activation of Kras and mutant p53 in adult ductal epithelium generated invasive PDAC in 75% of mice as early as 2.5 months after tamoxifen administration. These data demonstrate that pancreatic ductal cells, whereas exhibiting relative resistance to oncogenic Kras alone, can serve as an effective cell of origin for pancreatic ductal adenocarcinoma in the setting of gain-of-function mutations in p53.

p120 Catenin is required for normal tubulogenesis but not epithelial integrity in developing mouse pancreas

The intracellular protein p120 catenin aids in maintenance of cell-cell adhesion by regulating E-cadherin stability in epithelial cells. In an effort to understand the biology of p120 catenin in pancreas development, we ablated p120 catenin in mouse pancreatic progenitor cells, which resulted in deletion of p120 catenin in all epithelial lineages of the developing mouse pancreas: islet, acinar, centroacinar, and ductal. Loss of p120 catenin resulted in formation of dilated epithelial tubules, expansion of ductal epithelia, loss of acinar cells, and the induction of pancreatic inflammation. Aberrant branching morphogenesis and tubulogenesis were also observed. Throughout development, the phenotype became more severe, ultimately resulting in an abnormal pancreas comprised primarily of duct-like epithelium expressing early progenitor markers. In pancreatic tissue lacking p120 catenin, overall epithelial architecture remained intact; however, actin cytoskeleton organization was disrupted, an observation associated with increased cytoplasmic PKCζ. Although we observed reduced expression of adherens junction proteins E-cadherin, β-catenin, and α-catenin, p120 catenin family members p0071, ARVCF, and δ-catenin remained present at cell membranes in homozygous p120(f/f) pancreases, potentially providing stability for maintenance of epithelial integrity during development. Adult mice homozygous for deletion of p120 catenin displayed dilated main pancreatic ducts, chronic pancreatitis, acinar to ductal metaplasia (ADM), and mucinous metaplasia that resembles PanIN1a. Taken together, our data demonstrate an essential role for p120 catenin in pancreas development.

Dicer is required for maintenance of adult pancreatic acinar cell identity and plays a role in Kras-driven pancreatic neoplasia

The role of miRNA processing in the maintenance of adult pancreatic acinar cell identity and during the initiation and progression of pancreatic neoplasia has not been studied in detail. In this work, we deleted Dicer specifically in adult pancreatic acinar cells, with or without simultaneous activation of oncogenic Kras. We found that Dicer is essential for the maintenance of acinar cell identity. Acinar cells lacking Dicer showed increased plasticity, as evidenced by loss of polarity, initiation of epithelial-to-mesenchymal transition (EMT) and acinar-to-ductal metaplasia (ADM). In the context of oncogenic Kras activation, the initiation of ADM and pancreatic intraepithelial neoplasia (PanIN) were both highly sensitive to Dicer gene dosage. Homozygous Dicer deletion accelerated the formation of ADM but not PanIN. In contrast, heterozygous Dicer deletion accelerated PanIN initiation, revealing complex roles for Dicer in the regulation of both normal and neoplastic pancreatic epithelial identity.

p120 Catenin suppresses basal epithelial cell extrusion in invasive pancreatic neoplasia

Aberrant regulation of cellular extrusion can promote invasion and metastasis. Here, we identify molecular requirements for early cellular invasion using a premalignant mouse model of pancreatic cancer with conditional knockout of p120 catenin (Ctnnd1). Mice with biallelic loss of p120 catenin progressively develop high-grade pancreatic intraepithelial neoplasia (PanIN) lesions and neoplasia accompanied by prominent acute and chronic inflammatory processes, which is mediated, in part, through NF-κB signaling. Loss of p120 catenin in the context of oncogenic Kras also promotes remarkable apical and basal epithelial cell extrusion. Abundant single epithelial cells exit PanIN epithelium basally, retain epithelial morphology, survive, and display features of malignancy. Similar extrusion defects are observed following p120 catenin knockdown in vitro, and these effects are completely abrogated by the activation of S1P/S1pr2 signaling. In the context of oncogenic Kras, p120 catenin loss significantly reduces expression of genes mediating S1P/S1pr2 signaling in vivo and in vitro, and this effect is mediated at least, in part, through activation of NF-κB. These results provide insight into mechanisms controlling early events in the metastatic process and suggest that p120 catenin and S1P/S1pr2 signaling enhance cancer progression by regulating epithelial cell invasion. Cancer Res; 76(11); 3351-63. ©2016 AACR.

11 Inhibition of KRAS Activation Ameliorates Pre-Cancerous Metaplastic Conversion in the Mouse Stomach

Background: Approximately 35% of patients with ulcerative colitis (UC) will require surgery some time during the course of their disease. However, this past decade has seen major changes in the medical management of UC with FDA-approval of anti tumor necrosis factor alpha (Anti-TNF), for moderate to severe UC. Anti-TNF has demonstrated beneficial impact on the short-term colectomy rates in patients with UC. However, limited data are available regarding the long-term efficacy. The aim of this study was to determine the trends in the rate of colectomy in pre and post biologics era in hospitalized patients with UC. Methods: Nationwide Inpatient Sample (NIS), maintained by the Healthcare Cost and Utilization Project of the Agency for Healthcare Quality and Research, is the largest all-payer inpatient hospitalization database in the US. Hospitalizations from prior to the approval of biologics for ulcerative colitis (the pre-biologic era (1998-2005) and for post-biologic period from 2006-2011 were used. Our study included all patients hospitalized for primary diagnosis of Ulcerative Colitis (ICD-9-CM 556.x) and secondary diagnosis of UC related comorbidities and procedures. Surgeries were classified as elective if the admission was coded as being elective, the source of admission was not the emergency room and surgery occurred on day 0 or 1 of hospitalization. It was defined as emergent if the patient was admitted from the emergency room, the admission was characterized as emergent, and the procedure was performed within 0, 1 day of admission. A previously published risk score was used to adjust for severity of ulcerative colitis hospitalization. Results: A total of 399,579 admissions with primary diagnosis of UC and 38,505 (10%) colectomies were recorded during this period. 212,951 (53%) were females and average age was 46 years. Descriptive data of the group are shown in table 1. There was no difference in colectomy rate in pre and post biologic periods (figures 1). Females, whites and teaching hospital had significantly high rates of colectomies (table 1). Rate of colectomy is significantly increased up to age 70 with lowest rate among the age-group 10-20 yrs (p <.0001). Conclusions: No difference in colectomy rate in UC was noted in post and pre biologic era. Females, whites and admission to teaching hospital are predictors of colectomy. Multivariate logistic regression on colectomy

Abstract PR04: Clonal composition and clonal selection during PanIN progression

Pancreatic cancer is the fourth most common cause of cancer death in the United States. It remains a highly lethal malignancy despite surgical and chemotherapeutic advances. In spite of progress made delineating the mechanisms underlying pancreatic cancer, there has been little improvement in survival rates over the past fifty years. Copy number analysis of human invasive pancreatic ductal adenocarcinoma (PDAC) revealed massively rearranged cellular karyotypes as well as significant clonal complexity. Given the genetic complexity of already invasive PDAC, we believe it will be important to understand earlier events in pancreatic cancer, i.e. PanIN formation and progression. We hypothesized that stage-specific bottlenecks may lead to clonal selection during pancreatic intraepithelial neoplasia (PanIN) formation and progression. In order to test this hypothesis, we examined evolving clonal complexity during the progression of pancreatic intraepithelial neoplasia in the Mist1Tg/wt; LSL-KrasG12D (KCiMist1) mouse model of pancreatic cancer. Following KrasG12D activation in adult acinar cells, these mice develop acinar to ductal metaplasia (ADM) and preinvasive PanIN 1 - PanIN 3 lesions in a manner that faithfully recapitulates the human disease. Our strategy involves crossing MistTg/wt; LSL-KrasG12D to Brainbow2.1Tg/Tg “Confetti” mice, in which individual cells undergo stochastic recombination of the Confetti reporter to activate expression of either nuclear GFP, membrane-associated CFP, cytoplasmic RFP or cytoplasmic YFP. Following tamoxifen induction, mice were given cerulein injections to increase PanIN density, and sacrificed at four and nine weeks post-tamoxifen. Following additional immunofluorescent labeling with EpCAM to demarcate individual cells, confocal imaging was performed. Lesions were characterized by grade (ADM, early PanIN and late PanIN), and the number of clones, defined as adjacent cells sharing an identical fluorescent protein signature, was compared at 4 and 9 weeks. For AMDs, early PanIN and late PanIN, we observe both monoclonal and polyclonal lesions. Our analysis of evolving clonal complexity over time is ongoing, but preliminary data suggests a time-dependent decrease in clonal complexity. The average number of clones seen in individual ADM and late PanIN lesions decreased in the mice sacrificed at nine week post tamoxifen induction compared to those sacrificed at four weeks post tamoxifen induction (1.5 +/- 0.52 clones per lesion vs. 2.29 +/- 1.14 clones per lesion for ADM and 2.5 +/- 1.29 vs. 3.75 +/- 1.89 clones per lesion for late PanIN). In contrast, there was little difference in clonal complexity noted for early PanINs at 4 vs. 9 weeks (2.17 +/- 1.19 clones per lesion in the four week cohort vs. 2.42 +/- 1.24 clones per lesion in the nine week time point). These preliminary data suggest an overall trend towards decreased clonal complexity of PanIN lesions over time, suggesting significant selective pressure at even the earliest stages of pancreatic cancer. This abstract is also presented as Poster A62. Citation Format: Kelly J. Lafaro, Audrey Hendley, Jennifer Bailey, Steven Leach. Clonal composition and clonal selection during PanIN progression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr PR04.

Abstract A10: In search of Kras resistance genes: Whole transcriptome analysis identifies critical pathways mediating resistance and sensitivity to oncogenic Kras

In human tumorigenesis, mutational oncogene activation is postulated to occur in either single cells or small numbers of cells, followed by clonal expansion and tumor initiation. In the field of pancreatic cancer research, transgenic animal models mimicking the human disease have contributed to major advancements in our understanding of pancreatic cancer biology. These models have allowed the effects of oncogenic Kras (Kras G12D ) activation to be evaluated in different pancreatic compartments using Cre/Lox technology. When oncogenic Kras is expressed under the control of endogenous Kras regulatory elements and selectively activated in embryonic pancreatic epithelium using either Pdx-1:Cre, Ptf1a:Cre or Nestin:Cre driver lines, the Kras G12D allele induces murine pancreatic intraepithelial neoplastic lesions (mPanIN) at as early as two weeks of age. In adult pancreas, selective activation of oncogenic Kras in the acinar cell compartment using either tamoxifen-regulated Elastase (Ela):CreERT2, Mist1:CreERT2 or Ptf1:CreER driver lines also leads to effective mPanIN formation, supporting the possibility that adult acinar cells may serve as the cell type of origin for “ductal” neoplasia. In comparison, activation of oncogenic Kras in Sox9-expressing ductal epithelium leads to only low frequency mPanIN formation (Kopp et al, 2012), indicating that Sox9-expressing duct cells may be more resistant to transformation by oncogenic Kras. We have thoroughly interrogated the ability of the pancreatic ductal epithelium to respond to oncogenic Kras using the HNF1β:CreERT2 transgenic mouse. Our unpublished data support the findings of Kopp et al., as activation of Kras fails to induce mPanIN up to six months after the induction of recombination. Furthermore, combined KrasG12D activation and loss of the potent tumor suppressor p53 also fails to induce mPanIN formation from the HNF1β ductal compartment. In order to better understand the differential responsiveness of adult acinar and ductal cells to oncogenic Kras, we have FACS sorted acinar cells from PanIN-forming Mist1:CreERT2;LsL-Kras;mTmG mice and duct cells from non-PanIN forming HNF1β:CreERT2;LsL-Kras;mTmG mice at 0, 1 and 3 weeks following tamoxifen administration, and prior to the onset of any morphologic change. Following FACS-based cell isolation, we have performed whole genome transcriptional profiling to identify candidate genes mediating differential responsiveness to oncogenic Kras. This approach has identified a panel of candidate genes mediating Kras resistance in ductal cells, and a corresponding panel of candidate genes mediating Kras sensitivity in acinar cells. Genes potentially mediating Kras resistance in the ductal compartment include Plkha1, Slc9a9 and Pdcd10, while the loss of Gmnn and Med6 in the acinar cells appears to be associated with cell type-specific sensitivity to oncogenic Kras. Functional interrogation of identified genes is currently underway. Citation Format: Jennifer M. Bailey, Janivette Alsina, Florencia McAllister, Audrey Hendley, Kelly Lafaro, Anzer Habibulla, Luigi Marchionni, Anirban Maitra, Steven Leach. In search of Kras resistance genes: Whole transcriptome analysis identifies critical pathways mediating resistance and sensitivity to oncogenic Kras. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A10.

Abstract 4186: p120 catenin: A novel regulator of epithelial cell delamination in early Kras-driven pancreatic cancer

By 2020, pancreatic cancer is estimated to climb from the 4th to the 2nd most common cause of cancer-related deaths in the United States. This deadly disease has continued to remain largely refractory to chemotherapeutic and treatment regimens, and patients often experience a heavy metastatic burden. A study combining the Sleeping beauty transposon random insertion mutagenesis system with an oncogenic KrasG12D allele in mice as a screen to identify candidate pancreatic cancer genes identified genes enriched in adherens and tight junctions as significantly enriched in Kras-driven neoplasia with Ctnnd1 as a locus commonly mutated in mice developing metastatic progression of pancreatic cancer. Ctnnd1 encodes the adherens junction protein p120 catenin, which is integral in stabilization of cadherin molecules at cell membranes. p120 catenin is misexpressed in an estimated 60% primary pancreatic tumors and low/absent expression levels and predominant cytoplasmic localization of p120 catenin in primary resected pancreatic tumors correlates with worse survival in pancreatic cancer patients. Yet, the mechanisms by which p120 catenin contributes to the pathogenesis of pancreatic cancer are not clear. We have comprehensively examined p120 catenin staining in human Pancreatic Intraepithelial Neoplasia (PanIN) and identified mislocalization of p120 catenin to the cytoplasm as early as PanIN2. 3/5 PanIN3 examined had predominant cytoplasmic staining, which led us to hypothesize that p120 catenin might play a critical role in early pancreatic neoplasia, before the onset of Pancreatic Ductal Adenocarcinoma. To examine the role of p120 catenin during early Kras-driven pancreatic neoplasia, we ablated p120 catenin in a mouse model of preinvasive pancreatic cancer, KCiMist1. KCiMist1p120f/f pancreases display significant acceleration of acinar to ductal metaplasia (ADM) and PanIN formation when compared to KCiMist1p120wt/wt pancreases one month post tamoxifen injection. KCiMist1p120f/f pancreases are significantly larger than KCiMist1p120wt/wt pancreases with 92.89% pancreatic area occupied by Fibrostroma one month post tamoxifen injection. As a result, KCiMist1p120f/f animals have severe exocrine pancreatic insufficiency and die on average 8 weeks earlier than their KCiMist1p120wt/wt controls. Lineage tracing revealed a prominent epithelial cell delamination phenotype in KCiMist1p120f/f pancreases. Quantification of epithelial cells in the extensive stroma revealed a striking 832/7000 CK19+ cells in KCiMist1p120f/f pancreases vs 15/7000 CK19+ cells in KCiMist1p120wt/wt pancreases. Microarray analysis showed >1263 differentially expressed genes! IPA pathway analysis revealed significant gene expression changes in PI3K/AKT and Cdc42 signaling. Taken together, our results suggest a critical role for p120 catenin in regulating epithelial cell delamination in early pancreatic cancer. Citation Format: Audrey M. Hendley, Yue J. Wang, Janivette Alsina, Ishrat Ahmed, Hao Zhang, Samuel Savidge, Hao Ho, Albert Reynolds, Anirban Maitra, Michael Goggins, Christine Iacobuzio-Donahue, Steven D. Leach, Jennifer M. Bailey. p120 catenin: A novel regulator of epithelial cell delamination in early Kras-driven pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4186. doi:10.1158/1538-7445.AM2015-4186

266 Active Kras Leads to Rapid Transdifferentiation of Gastric Chief Cells Into Metaplasia With Progression to Dysplasia and Invasive Cancer

Adenocarcinoma in the human stomach evolves in the setting of Helicobacter pylori-induced oxyntic atrophy and chronic mucous cell metaplasia. Two types of metaplasia associated with development of intestinal-type cancers are observed in the human stomach: intestinal metaplasia (IM; intestinal type goblet cells in the stomach) and Spasmolytic Polypeptide (TFF2) Expressing Metaplasia (SPEM; with the presence of antral type TFF2-expressing mucous cells in the oxyntic region). Our previous study showed that SPEM developed from transdifferentiation of mature chief cells, rather than originating from aberrant differentiation of resident progenitor cells. Recent investigations have noted evidence for increased Ras activity in up to a third of gastric cancers. Thus, we have hypothesized that activated Ras, specifically induced in mature chief cells, elicits transdifferentiation of chief cells into SPEM and progression of SPEM to more intestinalized metaplasia. To test this hypothesis, we have examined a novel mouse model of metaplasia in the stomach in Mist1-CreERT2Tg/+;LSLK-Ras(G12D)Tg/+;mT/mGTg/+ mice. Tamoxifen (TAM) treatment induces the expression of activated K-Ras specifically in mature chief cells and marks the K-Ras-induced chief cells with GFP as a lineage tracer. This mouse model rapidly develops SPEM and IM within 3 months, following TAM induction of activated K-Ras expression in mature chief cells. At 1 week post TAM treatment, we found several groups of metaplastic glands between normal oxyntic glands that were positive for SPEM markers. At 3 months post TAM treatment, oxyntic glands were replaced withmetaplastic glands that showed highly proliferative mucous cell populations at the bases and the cells in the metaplastic glands were strongly positive for clusterin and TFF3 with distinct goblet cell morphologies, and also positive for Pdx1. Some glands were positive for IM markers such as villin and Cdx1. The mice at 4 months post TAM treatment developed severe inflammation, metaplasia, dysplasia and the invasive adenocarcinoma. Consistent with active K-Ras expression, cells with nuclear expression of phospho-ERK1/2 were also observed at the bases of metaplastic glands. Interestingly, we observed that the metaplastic glands were positive for GFP, indicating that they were derived from transdifferentiation of Cre-induced chief cells. Therefore, the induction of activated Ras specifically in mature chief cells initially leads to transdifferentiation of chief cells into SPEM followed by evolution of goblet cell IM and eventual neoplastic lesions. These studies demonstrate that expression of activated Ras in Chief cells can lead to the development of the entire spectrum of metaplastic and neoplastic lineages leading to gastric cancer.

Abstract 66: p120 catenin: A novel regulator of PanIN epithelial cell delamination in preinvasive pancreatic cancer

Pancreatic cancer is among the deadliest human malignancies ranking 4 th in the United States for cancer-related deaths among both men and women. Due to the invasive nature of pancreatic cancer, metastasis to the lymphatic system and distant organs is a major contributor to pancreatic cancer-related death. Genetic alterations in cell adhesion molecules contribute to human disease including developmentally related syndromes and cancer. In a sequencing study of 24 primary pancreatic tumor exomes published by the Hopkins pancreatic cancer team in 2008, 79% of tumors had at least one mutation in a homophilic cell adhesion molecule, and this class of molecules was named as one of the twelve core signaling pathways in pancreatic cancer. Among this class of cell adhesion molecules are adherens junctions. Mislocalization of the adherens protein p120 catenin has been identified in almost all of the major types of human carcinomas including pancreatic cancer. An accumulating body of evidence has identified p120 catenin as a prognostic marker in pancreatic cancer. Based on relevant literature, we hypothesize that misexpression and mislocalization of p120 catenin in pancreatic cancer is pathologic in the progression of this deadly disease. To test this hypothesis, we have ablated p120 catenin in the KC iMist1 mouse model of pancreatic cancer. The KC iMist1 mouse model activates oncogenic Kras in adult pancreatic acinar cells and displays the preinvasive PanIN 1 - PanIN 3 lesions in a manner that faithfully recapitulates the human disease. Homozygous deletion of p120 catenin in this model resulted in an almost complete replacement of acinar cells by acinar to ductal metaplastic lesions, accelerated PanIN formation, and stromal infiltration at 1 month. At 2 months, the stromal infiltrate persists, and we see considerable evidence of fibrosis and fatty deposition in the pancreas, which are pathologic features of pancreatitis. We used the KC iMist1 Gp120 f/f model to trace the lineage of acinar cells undergoing Kras activation and p120 catenin excision, and observed a marked increase in isolated GFP+ Ecadherin+ cells located in the extensive stroma at 1 and 2 months suggesting that p120 catenin normally restrains PanIN epithelial cell delamination. We have begun to study potential mechanisms for this delamination phenotype including epithelial-mesenchymal transition (EMT) and non-EMT based mechanisms. Research suggests that EMT is a contributing factor to the development of drug resistance, which makes EMT a promising target for the development of future therapies that reduce invasion and drug resistance leading to better prognosis for patients with pancreatic cancer. Our preliminary findings suggest that the KC iMist1 Gp120 f/f mouse model represents a valuable tool to study both EMT and non-EMT based mechanisms for PanIN epithelial cell delamination. Citation Format: Audrey M. Hendley, Jennifer M. Bailey, Janivette Alsina, Christine Iacobuzio-Donahue, Anirban Maitra, Albert Reynolds, Steven D. Leach. p120 catenin: A novel regulator of PanIN epithelial cell delamination in preinvasive pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 66. doi:10.1158/1538-7445.AM2014-66