Janivette Alsina

DCLK1 Marks a Morphologically Distinct Subpopulation of Cells With Stem Cell Properties in Preinvasive Pancreatic Cancer

BACKGROUND & AIMS As in other tumor types, progression of pancreatic cancer may require a functionally unique population of cancer stem cells. Although such cells have been identified in many invasive cancers, it is not clear whether they emerge during early or late stages of tumorigenesis. Using mouse models and human pancreatic cancer cell lines, we investigated whether preinvasive pancreatic neoplasia contains a subpopulation of cells with distinct morphologies and cancer stem cell-like properties. METHODS Pancreatic tissue samples were collected from the KC(Pdx1), KPC(Pdx1), and KC(iMist1) mouse models of pancreatic intraepithelial neoplasia (PanIN) and analyzed by confocal and electron microscopy, lineage tracing, and fluorescence-activated cell sorting. Subpopulations of human pancreatic ductal adenocarcinoma (PDAC) cells were similarly analyzed and also used in complementary DNA microarray analyses. RESULTS The microtubule regulator DCLK1 marked a morphologically distinct and functionally unique population of pancreatic cancer-initiating cells. These cells displayed morphological and molecular features of gastrointestinal tuft cells. Cells that expressed DCLK1 also expressed high levels of ATAT1, HES1, HEY1, IGF1R, and ABL1, and manipulation of these pathways in PDAC cell lines inhibited their clonogenic potential. Pharmacological inhibition of γ-secretase activity reduced the abundance of these cells in murine PanIN in a manner that correlated with inhibition of PanIN progression. CONCLUSIONS Human PDAC cells and pancreatic neoplasms in mice contain morphologically and functionally distinct subpopulations that have cancer stem cell-like properties. These populations can be identified at the earliest stages of pancreatic tumorigenesis and provide new cellular and molecular targets for pancreatic cancer treatment and/or chemoprevention.

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 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.

Liver-Directed Therapies in Colorectal Cancer

Colorectal cancer is the third leading cause of cancer-related deaths in the United States. Historically, the majority of patients that presented with metastatic disease to the liver were treated with systemic chemotherapy only but advances in imaging, surgical techniques, and non-resectional approaches have expanded the indications for liver-directed interventions. Current approaches used in patients with liver-only or liver-dominant metastatic disease include surgical resection, direct tumor ablation strategies, the use of intra-arterial infusions, and radiation therapies. The use of these liver-directed therapies in selected patients with colorectal liver metastases has led to significant improvements in overall survival. We review the clinical data and progress using liver-directed therapies in the treatment of colorectal liver metastases.

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

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