Acinar Cell Plasticity Research Articles

Abstract C079: Exploring acinar to ductal metaplasia in pancreatitis and precancerous contexts using FixNCut

Abstract Acinar to ductal metaplasia (ADM) is the process by which acinar cells transition to a duct-like state acquiring ductal markers and morphology. This process can be driven by pancreas inflammation or injury, such as pancreatitis, where it is thought to be an adaptive response to help mitigate damage and facilitate repair. ADM is also observed following acinar-specific oncogenic KRAS mutations and known to precede PDAC precancerous lesions termed pancreatic intraepithelial neoplasias (PanINs). While ADM is well characterized histologically, cellular and molecular details of ADM and PanIN formation remain less well understood, in part due to high levels of digestive enzyme production in acinar cells that hamper characterization. Leveraging our recently described DSP-fixation method, FixNCut, to improve capture and transcriptome quality of acinar and ADM populations in the pancreas, we characterize ADM arising in three contexts: acute pancreatitis, recurrent acute pancreatitis, and acute pancreatitis with an oncogenic KRAS mutation known to drive the formation of PanINs. FixNCut and single-cell RNA-sequencing enabled us to elucidate the transcriptomic landscape of ADM and define changes to the microenvironment in unprecedented detail. Our findings indicate that the cellular and transcriptional landscape of recurrent acute pancreatitis closely mirrors that of acute pancreatitis for up to 6 weeks (or 6 bouts of 2-day acute pancreatitis), which was the longest timepoint tested. In contrast, acute pancreatitis in the presence of an acinar-specific oncogenic KRAS mutation results in a distinct transcriptional profile for both ADM and the surrounding microenvironment compared to pancreatitis alone. Additionally, we use FixNCut to preserve the epigenomes of acinar cells, enabling us to sort acinar cells and profile their histone modifications in various contexts of ADM. We benchmark and validate our results against existing mouse and human datasets of precancer and cancer. Through this work, we aim to enhance our understanding of ADM in various contexts, providing insights into acinar cell plasticity and early PDAC precursor formation that may inform strategies for early detection and interception. Citation Format: Katherine Aney, Woo-Jeong Jeong, Pal Koak, Isabel Kim, Sahar Nissim. Exploring acinar to ductal metaplasia in pancreatitis and precancerous contexts using FixNCut [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C079.

FRA1 controls acinar cell plasticity during murine KrasG12D-induced pancreatic acinar to ductal metaplasia.

Acinar cells have been proposed as a cell-of-origin for pancreatic ductal adenocarcinoma (PDAC) after undergoing acinar-to-ductal metaplasia (ADM). ADM can be triggered by pancreatitis, causing acinar cells to de-differentiate to a ductal-like state. We identify FRA1 (gene name Fosl1) as the most active transcription factor during KrasG12D acute pancreatitis-mediated injury, and we have elucidated a functional role of FRA1 by generating an acinar-specific Fosl1 knockout mouse expressing KrasG12D. Using a gene regulatory network and pseudotime trajectory inferred from single-nuclei ATAC-seq and bulk RNA sequencing (RNA-seq), we hypothesized a regulatory model of the acinar-ADM-pancreatic intraepithelial neoplasia (PanIN) continuum and experimentally validated that Fosl1 knockout mice are delayed in the onset of ADM and neoplastic transformation. Our study also identifies that pro-inflammatory cytokines, such as granulocyte colony stimulating factor (G-CSF), can regulate FRA1 activity to modulate ADM. Our findings identify that FRA1 is a mediator of acinar cell plasticity and is critical for acinar cell de-differentiation and transformation.

The E3 ubiquitin ligase TRIP12 is required for pancreatic acinar cell plasticity and pancreatic carcinogenesis.

The E3 ubiquitin ligase thyroid hormone receptor interacting protein 12 (TRIP12) has been implicated in pancreatic adenocarcinoma (PDAC) through its role in mediating the degradation of pancreas transcription factor 1a (PTF1a). PTF1a is a transcription factor essential for the acinar differentiation state that is notably diminished during the early steps of pancreatic carcinogenesis. Despite these findings, the direct involvement of TRIP12 in the onset of pancreatic cancer has yet to be established. In this study, we demonstrated that TRIP12 protein was significantly upregulated in human pancreatic preneoplastic lesions. Furthermore, we observed that TRIP12 overexpression varied within PDAC samples and PDAC-derived cell lines. We further demonstrated that TRIP12 was required for PDAC-derived cell growth and for the expression of E2F-targeted genes. Acinar-to-ductal cell metaplasia (ADM) is a reversible process that reflects the high plasticity of acinar cells. ADM becomes irreversible in the presence of oncogenic Kras mutations and leads to the formation of preneoplastic lesions. Using two genetically modified mouse models, we showed that a loss of TRIP12 prevented acini from developing ADM in response to pancreatic injury. With two additional mouse models, we further discovered that a depletion of TRIP12 prevented the formation of KrasG12D-induced preneoplastic lesions and impaired metastasis formation in the presence of mutated KrasG12D and Trp53R172H genes. In summary our study identified an overexpression of TRIP12 from the early stages of pancreatic carcinogenesis and proposed this E3 ubiquitin ligase as a novel regulator of acinar plasticity with an important dual role in initiation and metastatic steps of PDAC. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract LB235: Prrx1 regulates acinar cell plasticity via TGFβsignaling in pancreatic acinar-to-ductal metaplasia

Abstract Introduction: Acinar cells undergo de-differentiation after injury to a progenitor-like cell type with ductal characteristics termed acinar-to-ductal metaplasia (ADM). In the absence of oncogenic mutation, the ADM lesions resolve and reform the acinar compartment (we terms this as adaptive ADM). However, in the presence of oncogenic Kras mutations (we terms this as oncogenic ADM), acinar cells undergo neoplastic transformation after ADM and evolve to pancreatic intraepithelial neoplasia (PanIN), a well-known precursor of pancreatic ductal adenocarcinoma (PDAC). Herein, we explore the role of the Paired-Related Homeobox1 transcriptional factor (Prrx1) in adaptive ADM and the relationship between Prrx1 and mutant Kras in oncogenic ADM. Methods: We generated tamoxifen-inducible Ptf1aCreERT;Rosa26YFP/YFP (abbreviated as Prrx1cWT) mice and bred in the Prrx1fl/fl allele to make (abbreviated Prrx1cKO ) mice and subjected them to caerulin (CCK analog) induced acute pancreatitis, a well-estabished model. We created a time course for both mouse genotypes subjected to acute pancreatitis for histological analysis. Quantification of ADM was performed through automated cell-counting of immunofluorescence (IF) staining. Then, we performed 3D ex vivo acinar cultures of Prrx1cWT and Prrx1cKO mice. We used an exogenous adenoviral vector to induce Cre expression and knock out Prrx1, using an empty vector as a control. Dissociated acinar cell cultures in collagen were utilized for the evaluation of TGFβ signaling by western blotting (WB). Additionally, we generated novel Pdx1-Cre;LSLKrasG12D/+;Prrx1fl/fl;Rosa26YFP/YFP (KCY Prrx1 KO) mice, in which mutant Kras is efficiently expressed and Prrx1 is deleted in a pancreas-specific manner. KCY Prrx1 WT and KO mice were sacrificed at 3 months and 5 months for histological analysis. Results: IF staining revealed that Prrx1cKO mice had fewer ADM lesions compared to Prrx1cWT mice at Day 3 post-caerulein. Ex vivo cultures demonstrated a substantial increase of cystic structures, which are representative of ADM in Prrx1cWT cultures compared to Prrx1cKO cultures. Additionally, using a novel nucleofection protocol, we demonstrated that overexpression of the Prrx1b isoform (associated with a progenitor cell state) leads to a notable increase in ADM. WB of acinar protein lysates from either Prrx1cWT or Prrx1cKO mice showed significant differences in TGFβ levels, and a >2-fold higher expression of phosphorylated SMAD2 in Prrx1cWT mice. IF staining also showed that KCY Prrx1 KO mice had fewer ADM lesions compared to Prrx1 WT mice at at the 5 months time point. Conclusions: Prrx1 can influence adaptive ADM formation. Moreover, Prrx1 can influence ADM formation in the presence of oncogenic KRAS. Our data suggest that Prrx1 facilitates PDAC progression through ADM formation. Citation Format: Noriyuki Nishiwaki, Kensuke Sugiura, Alina Li, Kensuke Suzuki, Jason R. Pitarresi, Rohit Chandwani, Anil K. Rustgi. Prrx1 regulates acinar cell plasticity via TGFβsignaling in pancreatic acinar-to-ductal metaplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB235.

Pancreatic ductal adenocarcinoma with acinar-to-ductal metaplasia-like cancer cells shows increased cellular proliferation

Background/objectivesAcinar-to-ductal metaplasia (ADM) has been shown to contribute to the development of pancreatic ductal adenocarcinoma (PDAC) in genetically engineered mouse models, but little is known about whether acinar cell plasticity contributes to carcinogenesis in human PDAC. We aimed to assess whether cancer cells that stain positive for amylase and CK19 (ADM-like cancer cells) are present in human resected PDAC and to investigate their role in tumor progression. MethodsWe immunohistochemically investigated the presence of ADM-like cancer cells, and compared the clinical and histological parameters of PDAC patients with and without ADM-like cancer cells. ResultsADM-like cancer cells were detected in 16 of 60 (26.7%) PDAC specimens. Positive staining for anterior gradient protein 2 (AGR2) was observed in 14 of 16 (87.5%) PDAC specimens with ADM-like cancer cells. On the other hand, the intensity of AGR2 expression (negative, low/moderate or high) was lower in PDAC with ADM-like cancer cells (9/7) than in PDAC without these cells (11/33) (P = 0.032). The presence of ADM-like cancer cells was significantly correlated with increased cell proliferation (P = 0.012) and tended to be associated with MUC1 expression (P = 0.067). ConclusionsThese results indicated that acinar cells may act as the origin of human PDAC, and that their presence may be useful for the stratification of human PDAC to predict prognosis.

Molecular profile of metastasis, cell plasticity and EMT in pancreatic cancer: a pre-clinical connection to aggressiveness and drug resistance.

The metastasis is a multistep process in which a small proportion of cancer cells are detached from the colony to enter into blood cells for obtaining a new place for metastasis and proliferation. The metastasis and cell plasticity are considered major causes of cancer-related deaths since they improve the malignancy of cancer cells and provide poor prognosis for patients. Furthermore, enhancement in the aggressiveness of cancer cells has been related to the development of drug resistance. Metastasis of pancreatic cancer (PC) cells has been considered one of the major causes of death in patients and their undesirable prognosis. PC is among the most malignant tumors of the gastrointestinal tract and in addition to lifestyle, smoking, and other factors, genomic changes play a key role in its progression. The stimulation of EMT in PC cells occurs as a result of changes in molecular interaction, and in addition to increasing metastasis, EMT participates in the development of chemoresistance. The epithelial, mesenchymal, and acinar cell plasticity can occur and determines the progression of PC. The major molecular pathways including STAT3, PTEN, PI3K/Akt, and Wnt participate in regulating the metastasis of PC cells. The communication in tumor microenvironment can provide by exosomes in determining PC metastasis. The components of tumor microenvironment including macrophages, neutrophils, and cancer-associated fibroblasts can modulate PC progression and the response of cancer cells to chemotherapy.

Hematopoietic progenitor kinase 1 inhibits the development and progression of pancreatic intraepithelial neoplasia.

Ras plays an essential role in the development of acinar-to-ductal metaplasia (ADM) and pancreatic ductal adenocarcinoma (PDAC). However, mutant Kras is an inefficient driver for PDAC development. The mechanisms of the switching from low Ras activity to high Ras activity that are required for development and progression of pancreatic intraepithelial neoplasias (PanINs) are unclear. In this study, we found that hematopoietic progenitor kinase 1 (HPK1) was upregulated during pancreatic injury and ADM. HPK1 interacted with the SH3 domain and phosphorylated Ras GTPase-activating protein (RasGAP) and upregulated RasGAP activity. Using transgenic mouse models of HPK1 or M46, a kinase-dead mutant of HPK1, we showed that HPK1 inhibited Ras activity and its downstream signaling and regulated acinar cell plasticity. M46 promoted the development of ADM and PanINs. Expression of M46 in KrasG12D Bac mice promoted the infiltration of myeloid-derived suppressor cells and macrophages, inhibited the infiltration of T cells, and accelerated the progression of PanINs to invasive and metastatic PDAC, while HPK1 attenuated mutant Kras-driven PanIN progression. Our results showed that HPK1 plays an important role in ADM and the progression of PanINs by regulating Ras signaling. Loss of HPK1 kinase activity promotes an immunosuppressive tumor microenvironment and accelerates the progression of PanINs to PDAC.

Abstract B032: Sox4-dependent acinar cell plasticity in pancreatic regeneration and cancer initiation

Abstract Gene alterations play a prominent role in driving cancer initiation and progression. Yet, mutations on oncogenes (those genes that promote tumorigenesis) only transform cells under certain cellular contexts. The mechanisms controlling neoplastic transformation (oncogenic competence) are poorly understood in pancreatic ductal adenocarcinoma (PDAC). Our laboratory investigates the interplay of mutations on the Kirsten Rat Sarcoma oncogene (Kras), developmental transcriptional programs, and the tissue microenvironment PDAC initiation. Our data demonstrate that Sox4 is necessary for the specification of cellular states in preinvasive cancer lesions and regulates the characteristics and cellular compositions of the tumor microenvironment in an autochthonous genetic model of PDAC. The pancreas has a remarkable ability to regenerate and recover from acute pancreatitis. In this process, acinar cells repress the expression of genes associated with acinar function and transiently activate a gene program that resembles pancreas progenitors of development. This mechanism, defined as cellular plasticity, alleviates tissue damage, stimulates acinar proliferation, and is necessary for pancreas regeneration. However, it makes the acinar cells competent to malignant transformation mediated by Kras. To investigate SOX4 function in pancreas regeneration and cancer, we used the KCacinar mouse model (Ptf1a-CreER; Kras G12DLSL). After injury, we observed a transient four-fold increase in the expression of SOX4, which correlates with the morphological and molecular evidence of cellular plasticity. KCacinar mice rapidly develop mucinous pancreatic intraepithelial neoplasias (PanINs) after caerulein-induced pancreatitis. Histological analysis KCAcinar Sox4-depleted pancreas (KCSAcinar) reveals a distinct cystic lesion lined by cuboidal or flattened epithelium with large irregular and hyperchromatic nuclei and absence of mucin-producing cells. Mice lacking Sox4 showed a significant reduction in the number of tuft cells. Furthermore, we observed an extensive reduction of the tumor stroma surrounding epithelial acinar-derived lesions in KCSAcinar compared to KC littermates. Next, we perform gene expression analysis of lesions 21 days after the induction of pancreatitis. Principal component analysis clusters the samples according to the genotype. Differential gene expression validated our histopathological analysis and showed a significant reduction in the expression of mucins and tuft cell markers. Cumulative, our data suggest that Sox4 is necessary for the specification of cellular states in the precursor lesions of PDAC, and that the cellular state of the tumor cell of origin determines the characteristics and cellular composition of the tumor microenvironment. Our work will continue to unravel the function of Sox4 in cancer initiation and the interaction of signaling pathways activated in pancreas regeneration with Kras mutations. Citation Format: Jonathan Baldan, Juan Camacho Roda, Charlotte Vestrup Rift, Jane Preuss Hasselby, Patrick Jacquemin, Ilse Rooman, Véronique Lefebvre, Luis Arnes. Sox4-dependent acinar cell plasticity in pancreatic regeneration and cancer initiation [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B032.

Abstract 788: Prrx1 regulates acinar cell plasticity in Kras-driven pancreatic acinar-to-ductal metaplasia

Abstract Introduction: Acinar cells in the adult pancreas demonstrate cellular plasticity and undergo de-differentiation to a progenitor-like cell type with ductal characteristics after injury. This process, termed acinar-to-ductal metaplasia (ADM), is an important feature facilitating pancreas regeneration after injury. In the absence of oncogenic mutations, the ADM lesions resolve and reform the acinar compartment (adaptive ADM). However, in the presence of oncogenic Kras mutations (oncogenic ADM), acinar cells undergo neoplastic transformation after ADM and evolve to pancreatic intraepithelial neoplasia (PanIN), a well-known precursor of pancreatic ductal adenocarcinoma (PDAC). We have characterized the role of Paired-Related Homeobox1 (PRRX1) in adaptive ADM. We demonstrated through our novel conditional Prrx1 knock-out mouse model that loss of Prrx1 abrogated ADM formation. Here we explore the relationship between Prrx1 and mutant Kras on promoting ADM and a pro-ADM microenvironment. Methods: We generated novel Pdx1-Cre;LSLKrasG12D/+;Prrx1fl/fl;Rosa26YFP/YFP (KCY Prrx1 KO) mice, in which mutant Kras is efficiently expressed and Prrx1 is deleted in a pancreas-specific manner. KCY Prrx1 WT and KO mice were sacrificed at 3 months and 5 months for histological analysis. Immunofluorescence (IF) staining for CK19, characterized for the rate of ADM formation and evaluated for F4/80 and SMA. Quantification of ADM regions, F4/80 and SMA was performed through automated cell-counting of immunofluorescence staining (IF). Dissociated acinar cell culture in collagen was utilized for the evaluation of ADM under ex vivo conditions. Results: IF staining revealed that KCY Prrx1 KO mice had fewer ADM lesions compared to Prrx1 WT mice at 3 months. This difference became dramatically apparent at the 5 month timepoint. Additionally, lower areas of fibrosis were identified via H&E staining in KCY Prrx1 KO mice, which was accompanied with lower F4/80 and SMA positivity at both 3 months and 5 months. Ex vivo cultures also demonstrated significant reduction in ADM formation in the context of oncogenic Kras and loss of Prrx1. Conclusions: PRRX1 can influence ADM formation in both a cell-intrinsic and cell-extrinsic manner in the presence of oncogenic KRAS. Our preliminary data suggest Prrx1 facilitates PDAC progression through PanIN formation. We will continue to investigate the mechanisms driving Prrx1-dependent ADM formation. Citation Format: Alina L. Li, Kensuke Sugiura, Kensuke Suzuki, Jason R. Pitarresi, Anna M. Chiarella, Gizem Efe, Rohit Chandwani, Anil K. Rustgi. Prrx1 regulates acinar cell plasticity in Kras-driven pancreatic acinar-to-ductal metaplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 788.

Interleukin 22 Signaling Regulates Acinar Cell Plasticity to Promote Pancreatic Tumor Development in Mice

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy that invades surrounding structures and metastasizes rapidly. Although inflammation is associated with tumor formation and progression, little is known about the mechanisms of this connection. We investigate the effects of interleukin (IL) 22 in the development of pancreatic tumors in mice. We performed studies with Pdx1-Cre;LSL-KrasG12D;Trp53+/-;Rosa26EYFP/+ (PKCY) mice, which develop pancreatic tumors, and PKCY mice with disruption of IL22 (PKCY Il22-/-mice). Pancreata were collectedat different stages of tumor development and analyzed by immunohistochemistry, immunoblotting, real-time polymerase chain reaction, and flow cytometry. Some mice were given cerulean to induce pancreatitis. Pancreatic cancer cell lines (PD2560) were orthotopically injected into C57BL/6 mice or Il22-/-mice, and tumor development was monitored. Pancreatic cells were injected into the tail veins of mice, and lung metastases were quantified. Acini were collected from C57BL/6 mice and resected human pancreata and were cultured. Cell lines and acini cultures were incubated with IL22 and pharmacologic inhibitors, and protein levels were knocked down with small hairpin RNAs. We performed immunohistochemical analyses of 26 PDACs and 5 nonneoplastic pancreas specimens. We observed increased expression of IL22 and the IL22 receptor (IL22R) in the pancreas compared with other tissues in mice; IL22 increased with pancreatitis and tumorigenesis. Flow cytometry indicated that the IL22 was produced primarily by T-helper 22 cells. PKCY Il22-/-mice did not develop precancerous lesions or pancreatic tumors. The addition of IL22 to cultured acinar cells increased their expression of markers of ductal metaplasia; these effects of IL22 were prevented with inhibitors of Janus kinase signaling to signal transducer and activator of transcription (STAT) (ruxolitinib) or mitogen-activated protein kinase kinase (MEK) (trametinib) and with STAT3 knockdown. Pancreatic cells injected into Il22-/- mice formed smaller tumors than those injected into C57BL/6. Incubation of IL22R-expressing PDAC cells with IL22 promoted spheroid formation and invasive activity, resulting in increased expression of stem-associated transcription factors (GATA4, SOX2, SOX17, and NANOG), and increased markers of the epithelial-mesenchymal transition (CDH1, SNAI2, TWIST1, and beta catenin); ruxolitinib blocked these effects. Human PDAC tissues had higher levels of IL22, phosphorylated STAT3, and markers of the epithelial-mesenchymal transition than nonneoplastic tissues. An increased level of STAT3 in IL22R-positive cells was associated with shorter survival times of patients. We found levels of IL22 to be increased during pancreatitis and pancreatic tumor development and to be required for tumor development and progression in mice. IL22 promotes acinar to ductal metaplasia, stem cell features, and increased expression of markers of the epithelial-mesenchymal transition; inhibitors of STAT3 block these effects. Increased expression of IL22 by PDACs is associated with reduced survival times.

Acetyl-CoA Metabolism Supports Multistep Pancreatic Tumorigenesis.

Pancreatic ductal adenocarcinoma (PDA) has a poor prognosis, and new strategies for prevention and treatment are urgently needed. We previously reported that histone H4 acetylation is elevated in pancreatic acinar cells harboring Kras mutations prior to the appearance of premalignant lesions. Because acetyl-CoA abundance regulates global histone acetylation, we hypothesized that altered acetyl-CoA metabolism might contribute to metabolic or epigenetic alterations that promote tumorigenesis. We found that acetyl-CoA abundance is elevated in KRAS-mutant acinar cells and that its use in the mevalonate pathway supports acinar-to-ductal metaplasia (ADM). Pancreas-specific loss of the acetyl-CoA-producing enzyme ATP-citrate lyase (ACLY) accordingly suppresses ADM and tumor formation. In PDA cells, growth factors promote AKT-ACLY signaling and histone acetylation, and both cell proliferation and tumor growth can be suppressed by concurrent BET inhibition and statin treatment. Thus, KRAS-driven metabolic alterations promote acinar cell plasticity and tumor development, and targeting acetyl-CoA-dependent processes exerts anticancer effects. SIGNIFICANCE: Pancreatic cancer is among the deadliest of human malignancies. We identify a key role for the metabolic enzyme ACLY, which produces acetyl-CoA, in pancreatic carcinogenesis. The data suggest that acetyl-CoA use for histone acetylation and in the mevalonate pathway facilitates cell plasticity and proliferation, suggesting potential to target these pathways.See related commentary by Halbrook et al., p. 326.This article is highlighted in the In This Issue feature, p. 305.

Epithelial-Myeloid cell crosstalk regulates acinar cell plasticity and pancreatic remodeling in mice.

Dedifferentiation of acini to duct-like cells occurs during the physiologic damage response in the pancreas, but this process can be co-opted by oncogenic Kras to drive carcinogenesis. Myeloid cells infiltrate the pancreas during the onset of pancreatic cancer, and promote carcinogenesis. Here, we show that the function of infiltrating myeloid cells is regulated by oncogenic Kras expressed in epithelial cells. In the presence of oncogenic Kras, myeloid cells promote acinar dedifferentiation and carcinogenesis. Upon inactivation of oncogenic Kras, myeloid cells promote re-differentiation of acinar cells, remodeling of the fibrotic stroma and tissue repair. Intriguingly, both aspects of myeloid cell activity depend, at least in part, on activation of EGFR/MAPK signaling, with different subsets of ligands and receptors in different target cells promoting carcinogenesis or repair, respectively. Thus, the cross-talk between epithelial cells and infiltrating myeloid cells determines the balance between tissue repair and carcinogenesis in the pancreas.

Acinar cell plasticity and development of pancreatic ductal adenocarcinoma.

Acinar cells in the adult pancreas show high plasticity and can undergo transdifferentiation to a progenitor-like cell type with ductal characteristics. This process, termed acinar-to-ductal metaplasia (ADM), is an important feature facilitating pancreas regeneration after injury. Data from animal models show that cells that undergo ADM in response to oncogenic signalling are precursors for pancreatic intraepithelial neoplasia lesions, which can further progress to pancreatic ductal adenocarcinoma (PDAC). As human pancreatic adenocarcinoma is often diagnosed at a stage of metastatic disease, understanding the processes that lead to its initiation is important for the discovery of markers for early detection, as well as options that enable an early intervention. Here, the critical determinants of acinar cell plasticity are discussed, in addition to the intracellular and extracellular signalling events that drive acinar cell metaplasia and their contribution to development of PDAC.

Hes1 Controls Exocrine Cell Plasticity and Restricts Development of Pancreatic Ductal Adenocarcinoma in a Mouse Model

Perturbation of pancreatic acinar cell state can lead to acinar-to-ductal metaplasia (ADM), a precursor lesion to the development of pancreatic ductal adenocarcinoma (PDAC). In the pancreas, Notch signaling is active both during development and in adult cellular differentiation processes. Hes1, a key downstream target of the Notch signaling pathway, is expressed in the centroacinar compartment of the adult pancreas as well as in both preneoplastic and malignant lesions. In this study, we used a murine genetic invivo approach to ablate Hes1 in pancreatic progenitor cells (Ptf1a+/Cre; Hes1fl/fl). Using this model, we studied the role of Hes1 in both acinar cell plasticity and pancreatic regeneration after caerulein-induced pancreatitis and in KrasG12D-driven PDAC development. We show that, although pancreatic development is not perturbed on the deletion of Hes1, terminal acinar differentiation in the adult pancreas is compromised. Moreover, the loss of Hes1 leads to the impaired regeneration of the exocrine compartment, accelerated fatty metaplasia, and persistent ADM after acute caerulein-induced pancreatitis. In KrasG12D-driven carcinogenesis, Hes1 ablation resulted in increased ADM, decreased formation of high-grade pancreatic intraepithelial neoplasias, and accelerated development of PDAC with shortened survival time. In conclusion, Hes1 plays a key role in acinar cell integrity and plasticity on cellular insults. Furthermore, Hes1 is an essential component of the pancreatic intraepithelial neoplasias-to-PDAC route in KrasG12D-driven mouse pancreatic carcinogenesis.

NFATc4 Regulates Sox9 Gene Expression in Acinar Cell Plasticity and Pancreatic Cancer Initiation.

Acinar transdifferentiation toward a duct-like phenotype constitutes the defining response of acinar cells to external stress signals and is considered to be the initial step in pancreatic carcinogenesis. Despite the requirement for oncogenic Kras in pancreatic cancer (PDAC) development, oncogenic Kras is not sufficient to drive pancreatic carcinogenesis beyond the level of premalignancy. Instead, secondary events, such as inflammation-induced signaling activation of the epidermal growth factor (EGFR) or induction of Sox9 expression, are required for tumor formation. Herein, we aimed to dissect the mechanism that links EGFR signaling to Sox9 gene expression during acinar-to-ductal metaplasia in pancreatic tissue adaptation and PDAC initiation. We show that the inflammatory transcription factor NFATc4 is highly induced and localizes in the nucleus in response to inflammation-induced EGFR signaling. Moreover, we demonstrate that NFATc4 drives acinar-to-ductal conversion and PDAC initiation through direct transcriptional induction of Sox9. Therefore, strategies designed to disrupt NFATc4 induction might be beneficial in the prevention or therapy of PDAC.

Activation of protein kinase Cδ leads to increased pancreatic acinar cell dedifferentiation in the absence of MIST1

Pancreatic ductal adenocarcinoma (PDAC) has a 5 year survival rate post-diagnosis of < 5%. Individuals with chronic pancreatitis (CP) are 20-fold more likely to develop PDAC, making it a significant risk factor for PDAC. While the relationship for the increased susceptibility to PDAC is unknown, loss of the acinar cell phenotype is common to both pathologies. Pancreatic acinar cells can dedifferentiate or trans-differentiate into a number of cell types including duct cells, β cells, hepatocytes and adipocytes. Knowledge of the molecular pathways that regulate this plasticity should provide insight into PDAC and CP. MIST1 (encoded by Bhlha15 in mice) is a transcription factor required for complete acinar cell maturation. The goal of this study was to examine the plasticity of acinar cells that do not express MIST1 (Mist1(-/-) ). The fate of acinar cells from C57Bl6 or congenic Mist1(-/-) mice expressing an acinar specific, tamoxifen-inducible Cre recombinase mated to Rosa26 reporter LacZ mice (Mist1(CreERT/-) R26r) was determined following culture in a three-dimensional collagen matrix. Mist1(CreERT/-) R26r acini showed increased acinar dedifferentiation, formation of ductal cysts and transient increases in PDX1 expression compared to wild-type acinar cells. Other progenitor cell markers, including Foxa1, Sox9, Sca1 and Hes1, were elevated only in Mist1(-/-) cultures. Analysis of protein kinase C (PKC) isoforms by western blot and immunofluorescence identified increased PKCε accumulation and nuclear localization of PKCδ that correlated with increased duct formation. Treatment with rottlerin, a PKCδ-specific inhibitor, but not the PKCε-specific antagonist εV1-2, reduced acinar dedifferentiation, progenitor gene expression and ductal cyst formation. Immunocytochemistry on CP or PDAC tissue samples showed reduced MIST1 expression combined with increased nuclear PKCδ accumulation. These results suggest that the loss of MIST1 is a common event during PDAC and CP and events that affect MIST1 function and expression may increase susceptibility to these pathologies.

Lineage Tracing Evidence for Transdifferentiation of Acinar to Duct Cells and Plasticity of Human Pancreas

Animal studies have indicated that pancreatic exocrine acinar cells have phenotypic plasticity. In rodents, acinar cells can differentiate into ductal precursors that can be converted to pancreatic ductal adenocarcinoma or insulin-producing endocrine cells. However, little is known about human acinar cell plasticity. We developed nongenetic and genetic lineage tracing methods to study the fate of human acinar cells in culture. Human exocrine tissue was obtained from organ donors, dissociated, and cultured. Cell proliferation and survival were measured, and cell phenotypes were analyzed by immunocytochemistry. Nongenetic tracing methods were developed based on selective binding and uptake by acinar cells of a labeled lectin (Ulex europaeus agglutinin 1). Genetic tracing methods were developed based on adenoviral introduction of a Cre-lox reporter system, controlled by the amylase promoter. Both tracing methods showed that human acinar cells can transdifferentiate into cells that express specific ductal markers, such as cytokeratin 19, hepatocyte nuclear factor 1β, SOX9, CD133, carbonic anhydrase II, and cystic fibrosis transmembrane conductance regulator. Within 1 week of culture, all surviving acinar cells had acquired a ductal phenotype. This transdifferentiation was decreased by inhibiting mitogen-activated protein kinase signaling. Human acinar cells have plasticity similar to that described in rodent cells. These results might be used to develop therapeutic strategies for patients with diabetes or pancreatic cancer.

Improved Retention of Zymogen Granules in Cultured Murine Pancreatic Acinar Cells and Induction of Acinar-Ductal Transdifferentiation In Vitro

To establish a primary culture model for murine pancreatic acinar cells and to investigate the effects of different culture conditions on the phenotype and plasticity of these cells in extended culture. Acinar cells, cultured in Chee (CME) or Waymouth/Ham F-12 (WHME) media, exhibited 2 markedly dissimilar phenotypes. We employed 5'-bromo-2'-deoxyuridine (BrdU) incorporation, immunocytochemistry, and electron microscopy to investigate differences in cell cycle status and phenotype. CME-cultured cells grew as discrete epithelial islands and retained zymogen granules and endoplasmic reticulum stacks, yet expressed cytokeratin 7, suggesting that they comprise an intermediate between the acinar and ductal cell types. Observed by time-lapse videomicroscopy, cells transferred to WHME formed a confluent monolayer, flattened and dedifferentiated to a duct-like phenotype typified by loss of secretory apparatus, variable beta-catenin expression, an elongated teardrop shape, increased cell size, and nuclear pleomorphism. Transition between phenotypes did not involve apoptosis as assessed by morphologic criteria in Feulgen-stained cultures. The flattened cells exhibited increased BrdU incorporation and mitotic index, suggesting that dedifferentiation precedes the capacity for increased cell cycle entry, while the appearance of meganuclei is consistent with amplified DNA content. We also demonstrate greatly improved gene delivery to cultured acinar cells by adenovirus-mediated transduction compared with lipid-mediated transfection. This in vitro model confirms the plasticity of acinar cells and may serve to delineate the changes underlying acinar cell dedifferentiation and acquisition of a duct-like pluripotent phenotype.