Acinar-ductal Metaplasia Research Articles

Knockout of Acinar Enriched microRNAs in Mice Promote Duct Formation But Not Pancreatic Cancer

The pancreatic acinar-enriched miR-216a, miR-216b and miR-217 are encoded within the miR217HG. These miRNAs have been purported to play a tumor suppressive role as their expression is reduced in both human and mouse pancreatic ductal adenocarcinoma (PDAC). To examine this possibility, we generated individual, germline knockout (KO) mice of miR-216a, miR-216b or miR-217. Unlike our previous study showing germline deletion of the miR217HG was embryonic lethal, CRISPR-Cas9 deleted portions of the 5’ seed region of the miRNAs produced live births. To investigate possible phenotypes during pancreatic acinar ductal metaplasia (ADM), pancreatic acini from wild type and KO mice were plated on collagen and allowed to transdifferentiate over 4 days. Acini from each of the three miRNA KO mice produced greater numbers of ducts compared to controls. Evaluation of the gene expression during in vitro ADM demonstrated an increase in Krt19 and a reduction in acinar genes (Carboxypeptidase A1, Amylase2a) on day 4 of the transdifferentiation. Recovery was delayed for the miR-216a and miR-216b KOs following caerulein-induced acute pancreatitis. Also predominate in the caerulein treated miR-216a and miR-216b KO mice was the presence of pancreatic duct glands (PDGs). To further establish a phenotype, miRNA KO mice were crossed with EL-KRASG12D (EK) mice and followed up to 13 months of age. While all mice developed severe dysplasia and cystic papillary neoplasms, there existed no apparent phenotypic difference in the miRNA KO/EK mice compared to EK mice. Our data does not support a tumor suppressor role for miR-216a, miR-216b or miR-217 in PDAC and emphasizes the need for phenotypic evaluation of miRNAs in complex in vivo models beyond that performed using cell culture.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 1960: Diffusion MR imaging detects differential progression of pathology in two mutant KRas mouse models of pancreatic neoplasia

Abstract A significant clinical need exists for understanding the early changes in pancreatic cancer (PC) progression and employing imaging of cellular and tissue phenotypes to detect neoplasia. We have investigated the development of early pathology in EL-Kras and p48-Cre/LSL-Kras (EK and KC, respectively) mice using diffusion MR imaging. These models express mutant Kras via different mechanisms yet recapitulate pancreatic precursor lesions seen in human PC. Our ex vivo investigation shows that diffusion MR imaging based only on endogenous contrast not only detects pre-neoplastic pathology in EK and KC mice, but also differentiates the progression of pathology in these mouse models. Fixed pancreata from EK and KC mice at 4M, 8M and 12M (n=4/group) were examined by diffusion imaging on a 14.1T MR microimager. Mean diffusivity was calculated for acinar lobules and lesions. Pancreata evaluated by MRI were processed for histology (H&E and trichrome) to determine percent area of tissue containing normal, acinar-ductal metaplasia (ADM), fibrosis and neoplasia. Diffusivity was lower in the acinar compartment of EK mice at 4M (0.83x10-3mm2/s) relative to KC mice (1.21x10-3mm2/s) and normal controls (1.17x10-3mm2/s), suggesting that Kras-related pathology targets the acinar cells of EK mice at a younger age than KC mice. Acinar diffusivity, however, did not change further in EK mice at 8 and 12 months, indicating little or no progression of pathology. In contrast, acinar diffusivity progressively decreased with age in KC mice (8M: 0.68x10-3mm2/s; 12M: 0.59x10-3mm2/s) indicating a later onset, but more advancing pathology. Changes in tissue diffusivity arise from a complex combination of cytological changes induced by pathology. Lower diffusivity in EK mice at 4M probably arises from higher levels of ADM (31.7%) compared to 4M KC mice (ADM 11.7%). Reduced diffusivity in KC mice at 8 and 12M most likely is related to elevated fibrosis in those mice (60.0-62.5%) which is relatively low in EK mice (13.3-18.3%). While diffusion MR images revealed the presence of fluid-filled cysts in both models, only KC mice showed lesions with much lower diffusivity (0.55-0.39x10-3mm2/s). Lower diffusivity indicates high cellularity in these neoplastic lesions which are likely mPanINs. Histology revealed that mPanINs which occupied 11.7-18.8% of the tissue in the KC mouse pancreas, are mostly absent in EK mice. Based on our imaging and histology results, MR-measured diffusivity might be a useful marker to assess the onset and progression of PanIN and cystic disease in PC. Citation Format: Palamadai N. Venkatasubramanian, Matthew Smith, Emman Mascrinas, Andrew Diaz, Karla Castellanos, Brian DeCant, Ron McKinney, Paul J. Grippo, Alice M. Wyrwicz. Diffusion MR imaging detects differential progression of pathology in two mutant KRas mouse models of pancreatic neoplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1960.

ATDC is required for the initiation of KRAS-induced pancreatic tumorigenesis.

Pancreatic adenocarcinoma (PDA) is an aggressive disease driven by oncogenic KRAS and characterized by late diagnosis and therapeutic resistance. Here we show that deletion of the ataxia-telangiectasia group D-complementing (Atdc) gene, whose human homolog is up-regulated in the majority of pancreatic adenocarcinoma, completely prevents PDA development in the context of oncogenic KRAS. ATDC is required for KRAS-driven acinar-ductal metaplasia (ADM) and its progression to pancreatic intraepithelial neoplasia (PanIN). As a result, mice lacking ATDC are protected from developing PDA. Mechanistically, we show ATDC promotes ADM progression to PanIN through activation of β-catenin signaling and subsequent SOX9 up-regulation. These results provide new insight into PDA initiation and reveal ATDC as a potential target for preventing early tumor-initiating events.

MST1R kinase accelerates pancreatic cancer progression via effects on both epithelial cells and macrophages.

The MST1R (RON) kinase is overexpressed in >80% of human pancreatic cancers, but its role in pancreatic carcinogenesis is unknown. In this study, we examined the relevance of Mst1r kinase to Kras driven pancreatic carcinogenesis using genetically engineered mouse models. In the setting of mutant Kras, Mst1r overexpression increased acinar-ductal metaplasia (ADM), accelerated progression of pancreatic intraepithelial neoplasia (PanIN), and resulted in the accumulation of (mannose receptor C type 1) MRC1+, (arginase 1) Arg+ macrophages in the tumor microenvironment. Conversely, absence of a functional Mst1r kinase slowed PanIN initiation, resulted in smaller tumors, prolonged survival and a reduced tumor associated macrophage content. Mst1r expression was associated with increased production of its ligand Mst1, and in orthotopic models, suppression of Mst1 expression resulted in reduced tumor size, changes in macrophage polarization and enhanced T cell infiltration. This study demonstrates the functional significance of Mst1r during pancreatic cancer initiation and progression. Further, it provides proof of concept that targeting Mst1r can modulate pancreatic cancer growth and the microenvironment. This study provides further rationale for targeting Mst1r as a therapeutic strategy.

Obesity-induced pancreatopathy in rats is reversible after bariatric surgery

Obesity is a risk factor for pancreatic diseases. Bariatric surgery is one of the most efficient treatments of morbid obesity. The aims were to assess pancreatic endocrine and exocrine lesions in obese rats, to analyze effects of bariatric surgery. Sixty-three male Wistar rats were included in five groups: 2 fed with high fat diet (HFD) or normal diet for 3 months, 2 fed with HFD or normal diet for 6 months; 1 group fed with HFD and undergoing bariatric surgery (n = 30). Quantitative MR imaging was performed in HFD6, ND6 and HFD3-BS. Pancreas specimens were analyzed after sacrifice for adipocyte infiltration, fibrosis, acinar-ductal metaplasia, abnormality of Langerhans islets (HHF: hypertrophy, hypervascularisation, fibrosis), and hemosiderin deposits in acinar or endocrine locations. We found that HFD6 rats had more fibro-inflammatory islets (P = 0.0139) and acinar-ducal metaplasia (P = 0.0843) than HFD3 rats. Rats with HFD3+6 had more fibro-inflammatory islets (P < 0.0001), hemosiderin deposits (p < 0.0001), fat infiltration (P = 0.0008) and acinar-ductal metaplasia lesions (P = 0.0424). Weight increase was associated with glycoregulation abnormalities (r = 0.44, P = 0.08) and adipocyte infiltrations (P = 0.009). After surgery, less fibro-inflammatory islets (P = 0.0004), fat and iron infiltrates (P = 0.005 and P = 0.06), and acino-ductal metaplasia (P = 0.05) were observed compared to HFD6 rats. MR image quantifications revealed increased elasticity, fat fraction, and R2 and a decreased elasticity wave dispersion coefficient in the high fat groups that reversed after surgery. MRI parameters were in strong correlation with respective histological counterparts. In conclusion, obese rats develop pancreatic inflammatory lesions with acinar-ductal metaplasia in acinar location and the endocrine-exocrine interface. These changes can be prevented by bariatric surgery. Quantitative MR imaging is accurate in identifying early pancreatic lesions.

Human CPA1 mutation causes digestive enzyme misfolding and chronic pancreatitis in mice

ObjectiveChronic pancreatitis is a progressive, relapsing inflammatory disorder of the pancreas, which often develops in the background of genetic susceptibility. Recently, loss-of-function mutations in CPA1, which encodes the digestive enzyme...

Abstract 4499: Determining the role of discoidin domain receptors 1 and 2 in the pathogenesis of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) is an aggressive and devastating cancer often characterized by an intense collagen-rich, fibrotic response. In the presence of an oncogenic Kras mutation, the pancreas undergoes an initial morphological event in which normal acinar cells transdifferentiate into a ductal-like phenotype in a process called acinar-ductal metaplasia (ADM). ADM advances into pre-cancerous lesions, followed by a subsequent replacement of epithelium with a collagen-dense stromal reaction as PDA progresses. Discoidin domain receptors, DDR1 and DDR2, are a unique family of tyrosine receptor kinases that bind to collagen and activate downstream cellular responses that affect cell proliferation, migration, and adhesion. Our preliminary data shows that during Kras-induced ADM in vitro DDR1 is downregulated and is accompanied by an upregulation of DDR2 expression. Immunohistochemistry on human tissue microarrays reveals DDR1 is expressed in normal epithelia and pre-neoplastic lesions, but downregulated during advanced PDA. Conversely, DDR2 is upregulated in metaplastic lesions and advanced carcinoma as well as in the stroma at all stages of progression, suggesting DDRs may play a differential role throughout the PDA. DDR1 is expressed in epithelial cells and associated with disease states such as fibrosis and various cancers, including PDA. To study the role of DDR1 in pancreatitis, a known risk factor of PDA, we used DDR1-null mice (DDR1-/-) with our cerulein-induced pancreatitis protocol. DDR1 ablation induces tissue damage and impairs recovery from extended cerulein treatment. To understand the role of DDR1 in tumorigenesis, we mated DDR1-/- mice in the KrasG12D/+; Ptf1aCre/+ (KC model) of pancreatic neoplasia. The absence of DDR1 in the KC model does not inhibit nor delay tumorigenesis, however, pancreata are smaller with less differentiated morphology than wild-type KC animals. In contrast, DDR2 is expressed in mesenchymal cells and has been implicated in EMT, cell proliferation, tumor invasion, and required for metastasis in breast cancer, however, its significance in PDA remains unknown. Our preliminary data shows DDR2 co-stains with neuroendocrine positive cells within neoplastic lesions that are associated with poor patient survival. To further investigate the role of DDR2 in tumor progression we have successfully mated a conditional, global DDR2 knockout mouse (DDR2fl/fl; β-actinCreERT2) with our novel KrasFSF-G12D/+;p53FRT/+ ;Ptf1a-FlpO/+ (KPF) aggressive PDA mouse model. Collectively, the results from these studies will help determine the role of DDRs throughout the initiation and progression of PDA and help define a potential point of regulation at the interface between the epithelial-stromal interactions. Citation Format: Jeanine Ruggeri, Christopher Halbrook, Anjum Sohail, Rafael Fridman, Howard Crawford. Determining the role of discoidin domain receptors 1 and 2 in the pathogenesis of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4499.

PO-196 Vav1 and Mutant K-Ras synergize in pancreatic ductal adenocarcinoma development: lessons from mouse models

Introduction The overall 5 year survival rate of Pancreatic Ductal Adenocarcinoma (PDAC) is less than 5% and has remained stubbornly unchanged long time ago, despite the efforts in preclinical and clinical science. PDAC, the main form of pancreatic cancer, develops via acinar-ductal metaplasia (ADM) and neoplastic precursor lesions, such as pancreatic intraepithelial neoplasia (PanIN). Mutant K-Ras is present in >90% of PDAC and is the most frequent and the earliest genetic alteration, being found in low-grade lesions. Identification of other molecular lesions that affect PDAC is of cardinal importance. One such potential protein is Vav1, a hematopietic specific signal transducer. Overexpression of wild-type Vav1 is implicated in human cancers, such as neuroblastoma, lung and PDAC. The expression of Vav1 in PDAC is indicative of a worse survival rate. Our goal was to determine whether Vav1 plays a causative or facilitatory role in-vivo in PDAC development. Material and methods We generated several transgenic mouse models that express Vav1, K-Ras G12D , or Vav1 and K-Ras G12D in the pancreas. K-Ras was induced by tamoxifen and Vav1 by Dox. Pancreata were analysed at different times post transgenes induction. Results and discussions Vav1 Expression together with K-Ras G12D in the pancreas has a dramatic synergistic effect enhancing ADM formation already at 3 months post transgene induction, resulting in at least 3 times the number of lesions in the pancreata of Vav1;K-Ras G12D mice compared to K-Ras G12 mice. No lesions were observed in the pancreas of Vav1 mice. The number of Ki-67 positive cells in Vav1;K-Ras G12D mice was significantly higher than in Vav1 or K-Ras G12D transgenic mice. The increase of pancreatic lesions was also accompanied with an increase in staining of Sox9 and Keratin and various pathways such as pERK, pEGFR and Rac1-GTP., highlighting the synergistic effect of Vav1 and K-Ras G12D in the development of PDAC. Notably, removal of Dox, thus ablating the expression of Vav1 in the pancreas of Vav1;K-Ras G12D led to a significant reduction in malignant lesions, thus further highlighting the necessity of expression of both oncogenes to cancer development in the pancreas. Conclusion Vav1 contributes to the development of malignant lesions in the pancreas when expressed with mutant K-Ras. Identification of Vav1 as a protein that synergizes with mutant K-Ras in PDAC development might pave the way to choosing good candidates for therapeutic drug design.

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

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.

Luteolin inhibits pancreatitis‑induced acinar‑ductal metaplasia, proliferation and epithelial‑mesenchymal transition of acinar cells.

Luteolin, a flavone, has been demonstrated to have anti-cancer properties. In the current study, the effects of luteolin on certain carcinogenesis-associated changes induced by pancreatitis, which are significant risk factors for pancreatic cancer, were investigated. Male six-week-old C57BL6 mice used in the current study were divided into three groups; the control group, acute pancreatitis group and luteolin group. Intra-peritoneal injection of cearulein was performed in the acute pancreatitis group and luteolin group to induce acute pancreatitis whereas the luteolin group received intra-peritoneal injection of luteolin. The control group received intra-peritoneal injection of normal saline. Then, the expression of SOX9, phosphorylated (p-) STAT3, p-EGFR, cytokeratin-19, Ki67 and N-cadherin were determined by immunohistochemistry. Morphological changes of acinar cells were determined by hematoxylin and eosin staining. The mRNA expression of the epithelial-mesenchymal transition markers CDH1, CDH2, Slug, Zeb1, EpCAM, ZO1, Vimentin, Snail and Twist was determined by reverse transcription-quantitative polymerase chain reaction. It was identified that luteolin inhibits the formation of tubular complexes and ectopic expression of cytokeratin-19 and luteolin also decreased proteins of SOX9, p-STAT3 and p-EGFR. In addition, luteolin inhibits proliferation and epithelial-mesenchymal transition of acinar cells induced by acute pancreatitis. As tubular complex formation and ectopic expression of cytokeratin-19 were two prominent characters of acinar-ductal metaplasia, it was concluded that luteolin inhibits acinar-ductal metaplasia induced by pancreatitis and also inhibits pancreatitis-induced proliferation and epithelial-mesenchymal transition of acinar cells. Acinar-ductal metaplasia and proliferation have close associations with pancreatic carcinogenesis. It is suggested that luteolin has potential anti-pancreatic carcinogenesis effects and merits further investigation.

Metaplasia: tissue injury adaptation and a precursor to the dysplasia-cancer sequence.

Metaplasia is the replacement of one differentiated somatic cell type with another differentiated somatic cell type in the same tissue. Typically, metaplasia is triggered by environmental stimuli, which may act in concert with the deleterious effects of microorganisms and inflammation. The cell of origin for intestinal metaplasia in the oesophagus and stomach and for pancreatic acinar-ductal metaplasia has been posited through genetic mouse models and lineage tracing but has not been identified in other types of metaplasia, such as squamous metaplasia. A hallmark of metaplasia is a change in cellular identity, and this process can be regulated by transcription factors that initiate and/or maintain cellular identity, perhaps in concert with epigenetic reprogramming. Universally, metaplasia is a precursor to low-grade dysplasia, which can culminate in high-grade dysplasia and carcinoma. Improved clinical screening for and surveillance of metaplasia might lead to better prevention or early detection of dysplasia and cancer.

Ductular and proliferative response of esophageal submucosal glands in a porcine model of esophageal injury and repair.

Esophageal injury is a risk factor for diseases such as Barrett's esophagus (BE) and esophageal adenocarcinoma. To improve understanding of signaling pathways associated with both normal and abnormal repair, animal models are needed. Traditional rodent models of esophageal repair are limited by the absence of esophageal submucosal glands (ESMGs), which are present in the human esophagus. Previously, we identified acinar ductal metaplasia in human ESMGs in association with both esophageal injury and cancer. In addition, the SOX9 transcription factor has been associated with generation of columnar epithelium and the pathogenesis of BE and is present in ESMGs. To test our hypothesis that ESMGs activate after esophageal injury with an increase in proliferation, generation of a ductal phenotype, and expression of SOX9, we developed a porcine model of esophageal injury and repair using radiofrequency ablation (RFA). The porcine esophagus contains ESMGs, and RFA produces a consistent and reproducible mucosal injury in the esophagus. Here we present a temporal assessment of this model of esophageal repair. Porcine esophagus was evaluated at 0, 6, 18, 24, 48, and 72 h and 5 and 7 days following RFA and compared with control uninjured esophagus. Following RFA, ESMGs demonstrated an increase in ductal phenotype, echoing our prior studies in humans. Proliferation increased in both squamous epithelium and ESMGs postinjury with a prominent population of SOX9-positive cells in ESMGs postinjury. This model promises to be useful in future experiments evaluating mechanisms of esophageal repair.NEW & NOTEWORTHY A novel porcine model of injury and repair using radiofrequency ablation has been developed, allowing for reproducible injury to the esophagus to study repair in an animal model with esophageal submucosal glands, a key anatomical feature and missing in rodent models but possibly harboring progenitor cells. There is a strong translational component to this porcine model given the anatomical and physiological similarities between pigs and humans.

Smad2/3 Linker Phosphorylation Is a Possible Marker of Pancreatic Stem/Progenitor Cells in the Regenerative Phase of Acute Pancreatitis.

The aims of this study are to characterize cell proliferation and differentiation during regeneration after pancreatitis and pancreatic buds during development to evaluate the role of Smad2/3, phosphorylated at the specific linker threonine residues (pSmad2/3L-Thr) in positive cells. Male C57BL/6 mice received hourly intraperitoneal injections of cerulein and were analyzed after induced pancreatitis. Pancreatitis-affected tissue sections and pancreatic buds were immunostained for pSmad2/3L-Thr, with other markers thought to be stem/progenitor markers of the pancreas. pSmad2/3L-Thr immunostaining-positive cells increased as the pancreatitis progressed. The expression of pSmad2/3L-Thr was seen in acinar cells and ductlike tubular complexes. These results suggest that pSmad2/3L-Thr is expressed during acinar-ductal metaplasia. Immunohistochemical colocalization of pSmad2/3L-Thr with Ki67 was never observed. pSmad2/3L-Thr-positive cells may remain in an undifferentiated state. During the pancreatic development process, pSmad2/3L-Thr was expressed as other markers. pSmad2/3L-Thr develops in duct structure of the undifferentiated cell population in the last part of viviparity that acinar structure is formed clearly. pSmad2/3L-Thr expression occurs during acinar-ductal metaplasia after pancreatitis and may represent the contribution of stem cells and/or progenitor cells to the differentiation of the pancreas.

Origin of Barrett's Epithelium: Esophageal Submucosal Glands.

The origin of the progenitor cell for Barrett’s esophagus remains a major unsolved mystery. Understanding the source of this progenitor may improve strategies to prevent the development of esophageal adenocarcinoma. Esophageal submucosal glands (ESMGs) and ducts may serve as a potential source of progenitor cells that respond to esophageal injury. Through the use of human histologic and molecular analysis, ESMGs and ducts have been described in physical continuity with areas of columnar esophagus, and shared mutations have been described between ESMG ducts and Barrett’s esophagus. Acinar ductal metaplasia, associated with carcinogenesis in other organs, occurs within ESMGs with human esophageal injury and esophageal adenocarcinoma. By using atypical animal models, a squamous epithelial defect well above the gastroesophageal junction healed to columnar epithelium and continuity of ESMG ducts was noted in the new epithelium. Increased proliferation in ESMGs and ducts in response to injury also has been noted in human beings and animals.

Abstract A62: ATDC is required for KRAS-induced pancreatic tumorigenesis

Abstract We have recently demonstrated that ATDC, a novel oncogenic protein, serves as an invasive switch in pancreatic cancer (PDA) by activation of beta–catenin signaling and upregulation of CD44, resulting in EMT and an invasive phenotype during PanIN progression. To further explore the tumorigenic function of ATDC, we generated a floxed ATDC mouse (A F/F) to evaluate the impact of conditional knockout of ATDC on oncogenic Kras-induced PDA initiation and progression. Pancreas-specific ATDC knockout did not cause any histologic abnormalities in pancreas, up to 1 year of age (n=8). Through a series of crosses of LSL-KrasG12D (K), p53F/+ (P), RosaYFP (Y), Pdx1-Cre (C) and AF/F mice, KrasG12D; CY (KCY); KrasG12D; p53+/-; CY (KPCY), KCYA-/- KPCYA-/- mice were generated. Knockout of ATDC in KPCY mice completely prevented the development of ADM and PanIN lesions in 3 month old mice (n= 8), and resulted in the formation of very rare ADM and PanIN1 lesions (2 out of 8) in KPCYA-/- mice at 12 months of age (n=8). In contrast, all KPCY mice developed extensive PanIN (low and high grade) at 3 months of age (n= 8), with the subsequent development of invasive and metastatic cancer at frequencies similar to that reported in the literature. To determine the possible mechanisms by which ATDC inhibited KrasG12D-induced acinar-ductal metaplasia (ADM), we isolated acini from 1.5 month old KCY and KCYA-/- pancreata and performed in vitro 3D cultures and ADM assays. ADM lesions readily formed in 3-D cultures of acini from KCY mice at 5 days, and this was significantly inhibited in acini isolated from KCYA-/- mice (duct-like structures: 95.1±3.5% to 28.0±2.2%*, KCY vs KCYA-/-, n=3, *p&amp;lt;0.05). Expression of ATDC specific shRNA in acini from KCY mice also effectively decreased ADM formation in 3D culture, an effect that was completely reversed by ATDC overexpression using an ATDC-shRNA-resistant expression vector. To further evaluate the role of ATDC in ADM and PanIN formation, we induced caerulein-mediated acute pancreatitis in 1.5 month old WT, CYA-/-, KCY, KCYA-/- mice and analyzed pancreatic tissue 1 and 7 days following cerulein treatment. 1 day post-caerulein treatment, KCY, KCYA-/-, CYA-/- and WT mice exhibited widespread ADM, which was replaced by normal acini by 7 days in WT, CYA-/- and KCYA-/- mice. However, in KCY mice 7 days post-cerulein treatment, extensive ADM and PanIN lesions were present, suggesting that ATDC is required for oncogenic KRAS to promote ADM and PanIN formation. Conclusions: Knockout of ATDC markedly reduces KrasG12D-induced ADM and PanIN formation, highlighting a key biologic function for ATDC in this process and its role in driving progression of KRAS-induced tumorigenesis in the pancreas. Citation Format: Lidong Wang, Huibin Yang, Ethan V. Abel, Phillip L. Palmbos, Christophe Halbrook, Kenneth Takeuchi, Jiaqi Shi, Yaqing Zhang, Sumithra Urs, Meghna Waghray, Marina Pasca di Magliano, Andrew D. Rhim, Howard C. Crawford, Diane M. Simeone.{Authors}. ATDC is required for KRAS-induced pancreatic tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A62.

Abstract A47: The acinar differentiation factor PTF1A negatively regulates EGFR-mediated acinar cell reprogramming

Abstract The primary goal of this study is to characterize interactions between the master acinar transcription factor, Ptf1a, and signal transduction pathways implicated in acinar reprogramming. Animal models suggest that acinar-ductal metaplasia (ADM) is one of the first steps in the initiation of pancreatic cancer. It is now established that acinar-specific activation of the Kras oncogene is sufficient at the whole-organ level to cause ADM and PanIN formation in mice, although it does so only inefficiently at the level of individual cells. Recently, our lab demonstrated that deletion of the master acinar transcriptional regulator, Ptf1a, facilitates ADM. Additionally, loss of Ptf1a dramatically accelerates PanIN formation when compounded with an oncogenic Kras mutation. Mechanistically, loss of Ptf1a activates transcriptional signatures of several signaling pathways that have been implicated in promoting acinar to ductal metaplasia (ADM) as well as PDAC progression. Prime among these candidate pathways is epidermal growth factor receptor (EGFR) signaling, which promotes acinar to ductal metaplasia (ADM) via the Ras-Raf-MEK-ERK pathway. In this study we test whether Ptf1a negatively regulates EGFR signaling to restrain acinar reprogramming and acinar-to-ductal metaplasia. To test this hypothesis, we performed in vitro culture assays on acinar clusters isolated from mice containing activated oncogenic KRAS alone (KrasG12D) or KrasG12D-Ptf1a cKO double mutant mice. We find that acinar clusters from KrasG12D-Ptf1a cKO double mutant mice undergo reprogramming, giving rise to duct-like cysts, both more rapidly and at a greater frequency than KrasG12D mice. In congruence with previous work, stimulation with the EGFR ligand, TGFα, increases the size and frequency of cyst formation in both KrasG12D and KrasG12D-Ptf1a cKO mice, suggesting that EGFR activation increases the transforming ability of oncogenic KRAS. Consistent with previous studies, blocking EGFR signaling using the pharmacological inhibitor Erlotinib, prevented ADM in acinar clusters from mice expressing oncogenic KRAS alone. Surprisingly, however, Erlotinib does not prevent ADM and cyst formation in KrasG12D-Ptf1a cKO mice, suggesting that acinar differentiation programs are required for the inhibitory effects of this drug. These results additionally indicate that EGFR is dispensable for ADM when Ptf1a is deleted. On the other hand, restriction of downstream signaling using the MEK inhibitor PD-0325901 inhibits reprogramming in acinar clusters, despite deletion of Ptf1a in KrasG12D mice. Since Ptf1a is downregulated in both mouse and human PDAC, these results highlight the potential of MEK inhibitors as feasible therapeutics, and the possibility that varying differentiation competence of tumor cells underlies the variable clinical effectiveness of Erlotinib and other EGFR inhibitors. Going forward, we will investigate whether reintroducing Ptf1a into dedifferentiated cells is sufficient to reprogram them back to an acinar-like phenotype. Using in vitro culture, we will determine if cells re-differentiate and regain sensitivity to Erlotinib with ectopic expression of Ptf1a. We will also characterize target genes of Ptf1a whose dysregulation leads to EGFR independence. Ultimately, these studies will lead us to investigate if reactivation of Ptf1a expression could represent a novel therapeutic avenue for PDAC. Citation Format: Shuba M. Narayanan, Nathan M. Krah, L. Charles Murtaugh.{Authors}. The acinar differentiation factor PTF1A negatively regulates EGFR-mediated acinar cell reprogramming. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A47.

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

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.

Pancreatic fibroblasts smoothen their activities via AKT-GLI2-TGFα.

Pancreatic stromal fibroblasts provide structural support. Activated fibroblasts are critical in the tumor microenvironment. In this issue of Genes & Development, Liu and colleagues (pp. 1943-1955) unravel the finding that depletion of Smoothened (Smo) in pancreatic stromal fibroblasts results in AKT activation and noncanonical GLI2 activation with subsequent TGFα secretion, activation of EGFR in pancreatic epithelial cells, and augmentation of acinar-ductal metaplasia. Additionally, Smo-mediated signaling has proproliferative effects on pancreatic tumor cells.

Abstract A26: The RON tyrosine kinase accelerates pancreatic duct neoplasia and activates a positive autocrine/paracrine signaling loop that promotes alternative macrophage activation

Abstract Introduction: The RON (receptor d'origine nantais) tyrosine kinase is overexpressed in &amp;gt;80% of human pancreatic cancers, but its effect on pancreatic carcinogenesis is largely unknown. Methods: We developed a transgenic mouse that overexpresses RON specifically in the pancreas (Pdx-1-RON). Additionally, we utilized an existing transgenic mouse with a functionally inactive RON tyrosine kinase domain (Pdx-1-RTK-/-). These strains were crossed with the established CK murine model of pancreatic cancer yielding compound strains, RCK and CK/TK-/-. We performed immunofluorescent staining, flow cytometry, and western blot analysis on the tissues from these animals to study the effects of RON overexpression and the loss of RON signaling in the setting of oncogenic Kras. Results: RON overexpression results in increased acinar-ductal metaplasia, accelerates PanIN progression to invasive cancer, and results in an accumulation of alternatively-activated (MRC1+) macrophages in the tumor microenvironment. We found that RON overexpression results in upregulation of its own ligand, macrophage-stimulating protein (MSP), activating a positive autocrine/paracrine signaling loop that effects tumor growth and the differentiation of tumor-associated macrophages. Knockdown of RON expression and/or MSP expression results in decreased tumor growth in vivo. Additionally, loss of RON signaling in CK/TK-/- mice leads to a profound decrease in alternatively-activated (MRC1+) macrophages in the tumor microenvironment. In contrast, these animals demonstrate an accumulation of classically activated (CD11c+) macrophages. RON signaling is also associated with downregulation of the chemokine, monocyte chemoattractant protein-1 (MCP-1), a known driver of CD11c+ macrophage activation. The correlation of RON and MSP and the inverse correlation with MCP-1 expression were validated in a database analysis of human pancreatic tumor specimens. Conclusion: Our studies demonstrate that murine RON overexpression accelerates the development of invasive pancreatic cancer in the setting of oncogenic Kras. Active RON signaling results in upregulation of its own ligand, MSP, initiating a positive autocrine/paracrine feedback loop that promotes tumor growth and alters the differentiation of macrophages in the tumor microenvironment. Citation Format: Michele L. Babicky, Megan M. Harper, Evangeline S. Mose, Dawn J. Jaquish, Randall P. French, Susan E. Waltz. The RON tyrosine kinase accelerates pancreatic duct neoplasia and activates a positive autocrine/paracrine signaling loop that promotes alternative macrophage activation. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A26.