Basil Bakir

Isolation, culture and genetic manipulation of mouse pancreatic ductal cells

The most common subtype of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). PDAC resembles duct cells morphologically and, to some extent, at a molecular level. Recently, genetic-lineage labeling has become popular in the field of tumor biology in order to study cell-fate decisions or to trace cancer cells in the mouse. However, certain biological questions require a nongenetic labeling approach to purify a distinct cell population in the pancreas. Here we describe a protocol for isolating mouse pancreatic ductal epithelial cells and ductlike cells directly in vivo using ductal-specific Dolichos biflorus agglutinin (DBA) lectin labeling followed by magnetic bead separation. Isolated cells can be cultured (in two or three dimensions), manipulated by lentiviral transduction to modulate gene expression and directly used for molecular studies. This approach is fast (∼4 h), affordable, results in cells with high viability, can be performed on the bench and is applicable to virtually all genetic and nongenetic disease models of the pancreas.

Prrx1 isoform switching regulates pancreatic cancer invasion and metastatic colonization

The two major isoforms of the paired-related homeodomain transcription factor 1 (Prrx1), Prrx1a and Prrx1b, are involved in pancreatic development, pancreatitis, and carcinogenesis, although the biological role that these isoforms serve in the systemic dissemination of pancreatic ductal adenocarcinoma (PDAC) has not been investigated. An epithelial-mesenchymal transition (EMT) is believed to be important for primary tumor progression and dissemination, whereas a mesenchymal-epithelial transition (MET) appears crucial for metastatic colonization. Here, we describe novel roles for both isoforms in the metastatic cascade using complementary in vitro and in vivo models. Prrx1b promotes invasion, tumor dedifferentiation, and EMT. In contrast, Prrx1a stimulates metastatic outgrowth in the liver, tumor differentiation, and MET. We further demonstrate that the switch from Prrx1b to Prrx1a governs EMT plasticity in both mouse models of PDAC and human PDAC. Last, we identify hepatocyte growth factor ( HGF) as a novel transcriptional target of Prrx1b. Targeted therapy of HGF in combination with gemcitabine in a preclinical model of PDAC reduces primary tumor volume and eliminates metastatic disease. Overall, we provide new insights into the isoform-specific roles of Prrx1a and Prrx1b in primary PDAC formation, dissemination, and metastatic colonization, allowing for novel therapeutic strategies targeting EMT plasticity.

Spontaneous Pancreatitis Caused by Tissue-Specific Gene Ablation of Hhex in Mice.

Background & Aims Perturbations in pancreatic ductal bicarbonate secretion cause chronic pancreatitis. The physiologic mechanism of ductal secretion is known, but its transcriptional control is not. We determine the role of the transcription factor hematopoietically expressed homeobox protein (Hhex) in ductal secretion and pancreatitis. Methods We derived mice with pancreas-specific, Cre-mediated Hhex gene ablation to determine the requirement of Hhex in the pancreatic duct in early life and in adult stages. Histologic and immunostaining analyses were used to detect the presence of pathology. Pancreatic primary ductal cells were isolated to discover differentially expressed transcripts upon acute Hhex ablation on a cell autonomous level. Results Hhex protein was detected throughout the embryonic and adult ductal trees. Ablation of Hhex in pancreatic progenitors resulted in postnatal ductal ectasia associated with acinar-to-ductal metaplasia, a progressive phenotype that ultimately resulted in chronic pancreatitis. Hhex ablation in adult mice, however, did not cause any detectable pathology. Ductal ectasia in young mice did not result from perturbation of expression of Hnf6, Hnf1β, or the primary cilia genes. RNA-seq analysis of Hhex-ablated pancreatic primary ductal cells showed mRNA levels of the G-protein coupled receptor natriuretic peptide receptor 3 (Npr3), implicated in paracrine signaling, up-regulated by 4.70-fold. Conclusions Although Hhex is dispensable for ductal cell function in the adult, ablation of Hhex in pancreatic progenitors results in pancreatitis. Our data highlight the critical role of Hhex in maintaining ductal homeostasis in early life and support ductal hypersecretion as a novel etiology of pediatric chronic pancreatitis.

Pancreas 3D Organoids: Current and Future Aspects as a Research Platform for Personalized Medicine in Pancreatic Cancer

Pancreatic ductal adenocarcinoma is one of the most aggressive forms of cancer, and the third leading cause of cancer-related mortality in the United States. Although important advances have been made in the last decade, the mortality rate of pancreatic ductal adenocarcinoma has not changed appreciably. This review summarizes a rapidly emerging model of pancreatic cancer research, focusing on 3-dimensional organoids as a powerful tool for several applications, but above all, representing a step toward personalized medicine.

ETV5 regulates ductal morphogenesis with Sox9 and is critical for regeneration from pancreatitis: ETV5 in Pancreatic Regeneration

Background: The plasticity of pancreatic acinar cells to undergo acinar to ductal metaplasia (ADM) has been demonstrated to contribute to the regeneration of the pancreas in response to injury. Sox9 is critical for ductal cell fate and important in the formation of ADM, most likely in concert with a complex hierarchy of, as yet, not fully elucidated transcription factors. Results: By using a mouse model of acute pancreatitis and three dimensional organoid culture of primary pancreatic ductal cells, we herein characterize the Ets‐transcription factor Etv5 as a pivotal regulator of ductal cell identity and ADM that acts upstream of Sox9 and is essential for Sox9 expression in ADM. Loss of Etv5 is associated with increased severity of acute pancreatitis and impaired ADM formation leading to delayed tissue regeneration and recovery in response to injury. Conclusions: Our data provide new insights in the regulation of ADM with implications in our understanding of pancreatic homeostasis, pancreatitis and epithelial plasticity. Developmental Dynamics 247:854–866, 2018.

Abstract PR04: p120 catenin loss drives pancreatic cancer EMT and metastasis through activation of calcium signaling

Purpose: We have generated a mouse model to delete the gene Ctnnd1, whose gene product p120 catenin (p120ctn) is necessary for E-CADHERIN stability, resulting in enhanced metastasis in the conventional KPC pancreatic tumor mouse model. An unbiased screen of tumor cells isolated from these mice identified misregulated calcium signaling as a previously unappreciated contributor to epithelial-to-mesenchymal transition (EMT) and metastasis. Thus, our overarching hypothesis is that p120ctn loss in pancreatic cancer drives EMT and metastasis through functional upregulation of the calcium signaling component PTHLH. Background: Pancreatic ductal adenocarcinoma (PDAC) is a major health issue, with only 7% of patients surviving beyond 5 years, and increases in PDAC-associated deaths project this disease to be the second leading cause of cancer deaths by 2020. An unbiased approach to discover candidate cancer genes in PDAC identified the p120 catenin gene as one of the top 20 PDAC cancer genes, and further analysis revealed that p120ctn loss was associated with reduced PDAC patient survival. Recent work from our lab further demonstrated that conditional p120ctn/Ctnnd1 deletion in the esophagus was sufficient to drive invasive squamous cell carcinoma, establishing p120ctn/Ctnnd1 as a bona fide tumor-suppressor gene. Results presented herein determined the in vivo role of p120ctn loss in PDAC tumorigenesis and metastasis through genetic mouse models and unbiased RNA-seq analysis. Methods: A PDAC mouse model was established to study the role of p120ctn in pancreatic carcinogenesis and metastasis. Specifically, LSL-KrasG12D/+; p53fl/+; Pdx1cre; Rosa26LSL-YFP; Ctnnd1fl/wt mice (herein KPCY-p120CKO) were generated to determine the effect of conditional p120ctn loss on pancreatic cancer. Furthermore, RNA-seq was performed on p120-intact or p120-null pancreatic tumor cells isolated from these mice to identify novel mechanisms of pancreatic tumorigenesis and metastasis. Results: We demonstrate p120ctn loss as a catastrophic event for tumor epithelial cell identity in vivo, leading to enhanced EMT and metastasis. Specifically, we show that KPCY-p120CKO mice have an enhanced metastatic phenotype relative to KPCY controls. Our data therefore suggest that p120ctn is a critical factor in metastatic cell dissemination, and that p120ctn loss results in tumor cells being “locked” in a mesenchymal phenotype by failing to stabilize E-cadherin at the metastatic site. To determine the mechanism of enhanced EMT and metastasis, RNA-seq analysis of p120ctn-null tumor cells was performed, which surprisingly revealed aberrant activation of calcium signaling. Unexpectedly, two of the top five most upregulated genes in p120ctn-null cells were the secreted factor Pthlh and the kinase Camk2b, both of which are key signaling molecules involved in calcium signaling. We demonstrate that PTHLH binding to its cognate receptor leads to cytosolic calcium ion (Ca2+) release, resulting in phosphorylation and activation of CaMKII. We further establish that genetic deletion or pharmacologic inhibition of Pthlh results in proliferation and migration defects. Moreover, orthotopic implantation of KPC-PthlhNULL tumor cell lines reduced tumor growth and metastasis in vivo. Finally, we show that PDAC patients with high expression of Pthlh have significantly decreased survival, suggesting that calcium signaling may be a potent oncogenic pathway in pancreatic cancer and that blocking this pathway may be of therapeutic benefit. Conclusions: This work has demonstrated the importance of the previously unappreciated role of calcium signaling in pancreatic cancer progression and metastasis, and future studies will look to determine the efficacy of calcium-modulating therapeutics in preclinical models of pancreatic cancer. This abstract is also being presented as Poster A42. Citation Format: Jason R. Pitarresi, Maximilian Reichert, Basil Bakir, Leticia Moreira, Lauren Simon, Anil K. Rustgi. p120 catenin loss drives pancreatic cancer EMT and metastasis through activation of calcium signaling [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr PR04.

756 ETV5, an ETS-Transcription Factor, Regulates Ductal Morphogenesis in Association With SOX9 and Is Critical for Regeneration From Acute Pancreatitis

NA not assessed, INT intensive therapy, NPDR non proliferative diabetic retinopathy, a Values are numbers (%) or mean (SEM) unless stated otherwise. b CAN prevalence is defined as any of: R-R variation 300 mg/day, renal transplant or dialysis. Table 2. Multiple Variable Predictor Models for Delayed Gastric Emptying in Type 1 Diabetes Mellitus

Abstract PR05: p120 catenin mediated epithelial-to-mesenchymal plasticity determines the metastatic potential of pancreatic ductal adenocarcinoma

Introduction: The majority of pancreatic ductal adenocarcinoma (PDAC) patients present with metastases and nearly all will succumb to disease within 6-12 months of clinical presentation (Hidalgo, 2010). Recently, p120catenin (p120) was identified as a “cancer candidate gene” in sleeping beauty-induced PDAC and p120 loss is associated with poor patient survival (Mann et al, 2012). P120 is critical in stabilizing E-cadherin (E-cad) at the adherens junctions. In this study, we wished to unravel new roles for p120 in the context of PDAC initiation and progression (epithelial-mesenchymal transition or EMT), as well as metastasis. Results: We have introduced a floxed p120 allele into the Pdx1cre;LSL-KrasG12D;R26YFP (KCY) PDAC mouse model. Mice with homozygous p120 deletion in the context of a LSL-KrasG12D allele (KCYp120fl/fl) are not viable. However, mice with heterozygous p120 loss (KCYp120fl/wt) are born according to Mendelian ratios. At 20 weeks of age, KCYp120fl/wt mice show a remarkable acceleration of the Kras-driven phenotype. KCYp120fl/wt mice (n=21) harbor the entire spectrum of precursor lesions, including PanINs (1-3), mucinous cystic neoplasms (MCN) and intraductal papillary mucinous neoplasms (IPMN) and metastatic PDAC. The phenotype in control KCYp120wt/wt mice (n=12) was restricted to early PanINs. We next asked whether the second allele of p120 is lost during metastatic dissemination. We demonstrated that the remaining p120 allele is expressed in liver metastases, indicating that p120 loss-of-heterozygosity did not occur. Of note, in liver metastases, p120 co-localizes with E-cad, thereby indicating that a single p120 allele is sufficient to stabilize E-cad. This raised the question of whether one p120 allele is required to establish epithelial integrity at the metastatic site. In order to address this mechanistically, and since KCYp120fl/fl are not viable, we performed three-dimensional (3D) culture experiments with pancreatic cells isolated from non-recombined LSL-KrasG12D/+;p120wt/wt;R26YFP (KYp120wt/wt), KYp120fl/wt and KYp120fl/fl mice. Cells were Cre-recombined in vitro. In 3D culture, KYp120wt/wt cells form round multicellular cysts. Monoallelic p120 loss only disrupts the symmetry of cysts while biallelic loss completely prevents cells from forming organized, cyctic structures. We next injected these cell lines orthotopically into the pancreata of immunodeficient mice. KYp120wt/wt cells form PanIN-like structures. Interestingly, KYp120fl/wt establish large cysts reminiscent of IPMN/MCN lesions. In the cysts, p120 is localized at the plasma membrane with E-cad. However, the invasive fronts of tumors show significantly less p120 expression. Finally, KYp120fl/fl cells form poorly differentiated tumors invading into the surrounding stroma. Given the fact that p120 stabilizes E-cad and that E-cad is critical in EMT and MET, we injected the KYp120wt/wt, KYp120fl/wt and KYp120fl/fl cell lines directly into the portal veins of these mice. KYp120fl/wt cells colonize the liver and retain the remaining p120 allele and display membranous E-cad localization. By contrast, although KYp120fl/fl cells are able to generate invasive primary pancreatic tumors when being injected orthotopically, these cells fail to colonize the liver. Conclusions: Our findings demonstrate that monoallelic loss of p120 accelerates Kras-driven PDAC formation. However, one allele of p120 is required to establish epithelial integrity and metastases. Taken together, these results for the first time underscore the importance of p120-mediated EMT plasticity in order to complete the metastatic cascade in vivo. Furthermore, therapeutic strategies to prevent EMT alone may not be sufficient in light of our results but rather require novel approaches to target EMT plasticity so as to enhance survival in metastatic PDAC. This abstract is also presented as Poster A63. Citation Format: Maximilian Reichert, Basil S. Bakir, Christopher M. Hahn, Gregory P. Botta, Andrew D. Rhim, Robert H. Vonderheide, Albert B. Reynolds, Yingtao Bi, Ramana Davuluri, Burcu Saka, N. Volkan Adsay, Anil K. Rustgi. p120 catenin mediated epithelial-to-mesenchymal plasticity determines the metastatic potential of pancreatic ductal adenocarcinoma. [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 PR05.

Abstract A11: Ets transcription factor Etv5 regulates ductal morphogenesis and differentiation in association with Sox9 in vitro and increases susceptibility and delays recovery from pancreatitis in vivo

Introduction: The exocrine pancreas comprises a branched network of ducts that are connected to acini and lined by a monolayered epithelium that derives from the endoderm and is surrounded by mesenchyme. The formation and maturation of pancreatic ductal cells during branching morphogenesis is controlled by a complex hierarchy of transcription factors, which is not fully understood. The Ets-transcription factor Etv5 has been reported to play an important role during the development of organs that undergo branching morphogenesis such as mammary and salivary glands. We therefore aimed to characterize the functional role of Etv5 in pancreatic ductal development and differentiation. Methods: Cells were isolated from wild-type mouse pancreata and lentiviral transduction was used to induce Etv5 overexpression. Cells were grown in 3D organotypic culture and analyzed with time lapsed microscopy for morphology and dynamics in the formation of duct-like cystic structures. Differentiation status of cells grown in 3D was analyzed by immunofluorescence-staining (IF) and qPCR. To asses the role of Etv5 in the normal mouse pancreas and its role in inflammation and regeneration in vivo, Pdx1cre;Etv5-/-; RosaYFP mice were generated and aged up to six months and subjected to a cerulein induced acute pancreatitis protocol. Results: Normal pancreatic ductal cells grown in 3D-organotypic culture form spheroid cysts that resemble pancreatic ductal structures. Etv5 overexpression leads to early and exuberant formation of spheroid cysts within 2 days after seeding. Time-lapse microscopy demonstrated a significantly increased movement of cellular structures along the cyst as well as fusion of cysts into larger tubular structures. mRNA analysis of cysts harvested at day 7 displayed a strong upregulation of Sox9 and Foxa2, important regulators of ductal differentiation. Concurrently, E-cadherin was upregulated significantly whereas N-cadherin was downregulated indicating the terminal differentiation of these cells. IF-staining revealed co-localization of Etv5 and Sox9 in spheroid cysts of Etv5-overexpressing cells. Knockdown of Sox9 in Etv5-overexpressing cells with siRNA partially abrogated the formation of tubular structures and disrupted cyst architecture. Etv5 knockout mice (Pdx1Cre;Etv5-/-) were generated and we performed a cerulein-induced acute pancreatitis model. We found significantly elevated levels of serum amylase in Etv5-/- mice on Days 1 and 3 of the protocol. In a semiquantitative, blinded review of the histology, there was significantly more intense edema, inflammation, vacuolization and necrosis in Etv5-/- mice in all time points, which was also confirmed by quantitative amylase area scoring. Conclusion: Our data suggest a novel role for Etv5 in pancreatic ductal morphogenesis and lumen formation that is at least in part mediated by Sox9. In addition, our data suggests that the loss of Etv5 expression increases susceptibility to pancreatitis and results in persistent pancreatitis with delayed regeneration. Citation Format: Koushik K. Das, Steffen Heeg, Maximilian Reichert, Shigetsugu Takano, Basil S. Bakir, Gregory P. Botta, Christopher Hahn, Andrew D. Rhim, Anil K. Rustgi. Ets transcription factor Etv5 regulates ductal morphogenesis and differentiation in association with Sox9 in vitro and increases susceptibility and delays recovery from pancreatitis in vivo. [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 A11.

Abstract A46: The Ets-transcription factor Etv1 induces epithelial-mesenchymal transition (EMT) and invasion as well as expands the stromal compartment in vivo

Introduction: The Ets-transcription factor Etv1 is involved in epithelial-mesenchymal interactions during pancreatic development and shows enhanced expression in PanIN as well as in pancreatic ductal adenocarcinoma (PDAC). Therefore, we aimed to identify the mechanistic roles of Etv1 in EMT and invasion in PanIN and PDAC. Methods: Cells were isolated from Pdx1Cre;Kras G12D/+ -mice (PanIN) and Pdx1Cre;Kras G12D/+ ;p53 R175H/+ -mice (PDAC) and lentiviral tranduction was used to induce overexpression or knockdown (shRNA) of Etv1, respectively. Cells were grown in 3D organotypic culture to analyze morphology and growth behavior. Invasion and sphere formation (self-renewal) assays were performed. FACS-sorting was used to isolate CD44+/CD24+ cells as a putative cancer stem cell population in PDAC and analyzed for Etv1 expression. To assess the role of Etv1 in PDAC in vivo, orthotopic pancreatic xenograft transplantation of Etv1 overexpressing PDAC cells was performed. Results: Etv1 overexpression in PanIN cells grown in 3D-organotypic culture induces a spindle shaped morphology and highly perturbed cyst architecture in contrast to control PanIN cells that form spheroid cysts that resemble normal pancreatic ductal structures. Concurrently and consistently, EMT-related genes (snail, twist, zeb1 and zeb2, as well as vimentin and N-cadherin) were found upregulated by >2-fold in PanIN-mEtv1 cells. Moreover, the expression of Mmp3 and Mmp9 was significantly increased. Functionally, the invasive capacity of PanIN-mEtv1 cells was more than twice that of control cells; knockdown of Etv1 in PDAC-cells significantly abrogated their invasive capacity. In sphere forming assays PanIN-mEtv1 cells revealed an increased self-renewal capacity, whereas sphere formation is reduced by knockdown of Etv1 in PanIN as well as in PDAC cells. FACS-sorting and subsequent expression analysis revealed increased Etv1 levels in the putative cancer stem cell population of CD44+/CD24+ PDAC-cells. Data from orthotopic xenografts showed significantly larger tumors, significantly increased stromal expansion measured by trichrome staining, and increased local tumor invasion in Etv1-overexpressing cells compared to controls. The increased stromal expansion significantly correlated to the increased tumor volume observed in Etv1 over-expressing xenografts. Conclusion: Our novel data indicate that Etv1 induces EMT as well as invasion both in vitro and in vivo and mediates self-renewal capacity of premalignant cells derived from PanIN- as well as PDAC-cells. Our in vivo data suggest a role for Etv1 in expanding the stromal compartment. These striking data suggest that Etv1 is biologically important in EMT. In vivo experiments with conditional knockout of Etv1 in Pdx1Cre;Kras G12D/+ ;p53 R175H/+ -mice are ongoing to further elucidate the role of Etv1 in PanIN and PDAC initiation and progression. Citation Format: Koushik K. Das, Steffen Heeg, Maximilian Reichert, Shigetsugu Takano, Basil S. Bakir, Gregory P. Botta, Christopher Hahn, Andrew D. Rhim, Anil K. Rustgi. The Ets-transcription factor Etv1 induces epithelial-mesenchymal transition (EMT) and invasion as well as expands the stromal compartment in vivo. [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 A46.