Acinar cells contribute to the molecular heterogeneity of pancreatic intraepithelial neoplasia

A number of studies have shown that pancreatic ductal adenocarcinoma develops through precursor lesions termed pancreatic intraepithelial neoplasia (PanIN). PanINs are thought to initiate in the small ducts of the pancreas through activating mutations in the KRAS proto-oncogene. What remains unanswered is the identification of the individual cell type(s) that contributes to pancreatic ductal adenocarcinoma formation. To follow the cellular and molecular changes that occur in acinar and duct cell properties on Kras(G12D) expression, we took advantage of LSL-Kras(G12D/+)/p48(Cre/+) mice, which faithfully mimic the human disease. In young animals (4 weeks), the predominant cellular alteration in the exocrine pancreas was acinar metaplasia in which individual acini consisted of acinar cells and duct-like cells. Metaplastic acinar structures were highly proliferative, expressed Notch target genes, and exhibited mosaic expression patterns for epidermal growth factor receptor, ErbB2, and pErk. This expression pattern paralleled the expression pattern detected in mouse PanINs, suggesting that mouse PanINs and acinar-ductal metaplasia follow similar molecular pathways. Indeed, immunofluorescence studies confirmed the presence of acinar cells within mPanIN lesions, raising the possibility that Kras(G12D)-induced mPanINs develop from acinar cells that undergo acinar-ductal metaplasia. Identification of an acinar contribution to PanIN formation offers new directions for successful targeted therapeutic approaches to combat this disease.     

Glycogen synthase kinase‐3β ablation limits pancreatitis‐induced acinar‐to‐ductal metaplasia

Acinar‐to‐ductal metaplasia (ADM) is a reversible epithelial transdifferentiation process that occurs in the pancreas in response to acute inflammation. ADM can rapidly progress towards pre‐malignant pancreatic intraepithelial neoplasia (PanIN) lesions in the presence of mutant KRas and ultimately pancreatic adenocarcinoma (PDAC). In the present work, we elucidate the role and related mechanism of glycogen synthase kinase‐3beta (GSK‐3β) in ADM development using in vitro 3D cultures and genetically engineered mouse models. We show that GSK‐3β promotes TGF‐α‐induced ADM in 3D cultured primary acinar cells, whereas deletion of GSK‐3β attenuates caerulein‐induced ADM formation and PanIN progression in Kras^G12D transgenic mice. Furthermore, we demonstrate that GSK‐3β ablation influences ADM formation and PanIN progression by suppressing oncogenic KRas‐driven cell proliferation. Mechanistically, we show that GSK‐3β regulates proliferation by increasing the activation of S6 kinase. Taken together, these results indicate that GSK‐3β participates in early pancreatitis‐induced ADM and thus could be a target for the treatment of chronic pancreatitis and the prevention of PDAC progression. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Activated Kras and Ink4a/Arf deficiency cooperate to produce metastatic pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma ranks among the most lethal of human malignancies. Here, we assess the cooperative interactions of two signature mutations in mice engineered to sustain pancreas-specific Cre-mediated activation of a mutant Kras allele (KrasG12D) and deletion of a conditional Ink4a/Arf tumor suppressor allele. The phenotypic impact of KrasG12D alone was limited primarily to the development of focal premalignant ductal lesions, termed pancreatic intraepithelial neoplasias (PanINs), whereas the sole inactivation of Ink4a/Arf failed to produce any neoplastic lesions in the pancreas. In combination, KrasG12D expression and Ink4a/Arf deficiency resulted in an earlier appearance of PanIN lesions and these neoplasms progressed rapidly to highly invasive and metastatic cancers, resulting in death in all cases by 11 weeks. The evolution of these tumors bears striking resemblance to the human disease, possessing a proliferative stromal component and ductal lesions with a propensity to advance to a poorly differentiated state. These findings in the mouse provide experimental support for the widely accepted model of human pancreatic adenocarcinoma in which activated KRAS serves to initiate PanIN lesions, and the INK4A/ARF tumor suppressors function to constrain the malignant conversion of these PanIN lesions into lethal ductal adenocarcinoma. This faithful mouse model may permit the systematic analysis of genetic lesions implicated in the human disease and serve as a platform for the identification of early disease markers and for the efficient testing of novel therapies.     

Pancreatic intraepithelial neoplasia – can we detect early pancreatic cancer?

Haugk B
(2010) Histopathology 57, 503–514
Pancreatic intraepithelial neoplasia – can we detect early pancreatic cancer? Pancreatic cancer is one of the most lethal cancers, with an incidence equalling mortality. Pancreatic cancer is a heterogeneous group in which pancreatic ductal adenocarcinoma (PDAC) is the most common. It is now established that PDAC develops through stepwise progression from precursor lesions. Detection and treatment of these precursor lesions would allow curative treatment. Three precursor lesions for PDAC have been identified. Two of these – mucinous cystic neoplasms (MCNs) and intraductal papillary mucinous neoplasms (IPMNs) – are rare, radiologically detectable, cystic precursor lesions which can be cured if treated at the preinvasive stage. The third and most common precursor lesion has recently been defined as pancreatic intraepithelial neoplasia (PanIN). PanINs are microscopic lesions with no clinical correlate. They display a spectrum of cyto‐architectural changes (PanIN‐1, PanIN‐2 and PanIN‐3) mirrored in an increasing accumulation of molecular genetic changes, with PanIN‐3 sharing many of the alterations with PDAC. Great advances in the understanding of pancreatic carcinogenesis have opened avenues for diagnosis and chemoprevention. However, access to the pancreas is limited, molecular tests are at the early stages and too little is known about the natural history of early PanINs to justify resection. Currently, screening focuses upon high‐risk individuals only.     

Smad4 is dispensable for normal pancreas development yet critical in progression and tumor biology of pancreas cancer

SMAD4 is inactivated in the majority of pancreatic ductal adenocarcinomas (PDAC) with concurrent mutational inactivation of the INK4A/ARF tumor suppressor locus and activation of the KRAS oncogene. Here, using genetically engineered mice, we determined the impact of SMAD4 deficiency on the development of the pancreas and on the initiation and/or progression of PDAC-alone or in combination with PDAC--relevant mutations. Selective SMAD4 deletion in the pancreatic epithelium had no discernable impact on pancreatic development or physiology. However, when combined with the activated KRAS(G12D) allele, SMAD4 deficiency enabled rapid progression of KRAS(G12D)-initiated neoplasms. While KRAS(G12D) alone elicited premalignant pancreatic intraepithelial neoplasia (PanIN) that progressed slowly to carcinoma, the combination of KRAS(G12D) and SMAD4 deficiency resulted in the rapid development of tumors resembling intraductal papillary mucinous neoplasia (IPMN), a precursor to PDAC in humans. SMAD4 deficiency also accelerated PDAC development of KRAS(G12D) INK4A/ARF heterozygous mice and altered the tumor phenotype; while tumors with intact SMAD4 frequently exhibited epithelial-to-mesenchymal transition (EMT), PDAC null for SMAD4 retained a differentiated histopathology with increased expression of epithelial markers. SMAD4 status in PDAC cell lines was associated with differential responses to transforming growth factor-beta (TGF-beta) in vitro with a subset of SMAD4 wild-type lines showing prominent TGF-beta-induced proliferation and migration. These results provide genetic confirmation that SMAD4 is a PDAC tumor suppressor, functioning to block the progression of KRAS(G12D)-initiated neoplasms, whereas in a subset of advanced tumors, intact SMAD4 facilitates EMT and TGF-beta-dependent growth.     

The foamy variant of pancreatic intraepithelial neoplasia

Foamy gland adenocarcinoma is a variant of pancreatic ductal carcinoma, whose precursor has not been described. We describe here the morphologic and immunohistochemical features of the pancreatic intraepithelial neoplasia (PanIN) lesions associated with invasive foamy pancreatic adenocarcinoma. The staining properties and morphologic and immunohistochemical features of 3 foamy PanIN lesions were compared with those of 7 pancreatic foamy gland adenocarcinomas. Hematoxylin and eosin, Mayer mucicarmine, periodic acid-Schiff, and Alcian blue stains were available for review in all cases. Immunohistochemical labeling for cytokeratin (CK)7, CK20, carcinoembryonic antigen polyclonal, MUC1, MUC2, CDX2, p53, and cyclin D1 was performed. The PanIN-1 lesions were found in the nonneoplastic pancreas and were similar to the PanIN-1 lesions of ordinary pancreatic ductal carcinoma. The PanIN-2 and -3 lesions were recognized immediately adjacent to or within the invasive foamy gland carcinoma. In these lesions, small or markedly dilated ducts were lined by cuboidal and columnar dysplastic nonfoamy cells and foamy cells. Hobnail cells were present in 2 cases. The PanIN-1, 2, and 3 lesions and the invasive foamy gland adenocarcinomas stained with mucicarmine, periodic acid-Schiff, and Alcian blue. The 3 PanIN-2 and -3 lesions and all 7 invasive foamy adenocarcinomas labeled with CK7, carcinoembryonic antigen polyclonal, and MUC1, whereas only 2 PanIN-2 and -3 lesions and 5 invasive adenocarcinomas showed immunoreactivity for cyclin D1 and p53. Three distinctive foamy PanIN lesions were identified within 7 invasive foamy gland pancreatic adenocarcinomas. The gradual progression of cytological and architectural abnormalities of the PanIN lesions from PanIN-1 to PanIN-3 excludes neoplastic ductal spread. These foamy PanIN lesions probably represent cancer precursors.     

Update on pancreatic intraepithelial neoplasia

Pancreatic intraepithelial neoplasia (PanIN) is a histologically well-defined precursor to invasive ductal adenocarcinoma of the pancreas. PanINs are remarkably common lesions, particularly in the elderly population. Molecular studies have helped establish the progression of PanIN to invasive cancer, and recently genetically engineered mouse models have been generated that recapitulate the entire spectrum of lesions from precursor to invasive pancreatic cancer. Some PanIN lesions produce lobulocentric atrophy of the pancreatic parenchyma, and, when multifocal, this lobulocentric atrophy may be detectable using currently available imaging techniques such as endoscopic ultrasound. The association of acinar-ductal metaplasia with PanIN lesions has led some to hypothesize that PanINs develop from acinar cells that undergo acinar-ductal metaplasia.     

Expression of urocortin in pancreatic ductal adenocarcinoma and pancreatic intraepithelial neoplasia

Urocortin (UCN) is a 40‐aminoacid neuropeptide that regulates angiogenesis and inhibits cell proliferation. Our aim was to examine the relationship of UCN expression to the clinicopathological parameters of pancreatic ductal adenocarcinoma (PDAC) and histological grade of pancreatic intraepithelial neoplasia (PanIN). Tissue microarray was used to analyze UCN protein expression in 89 surgical specimens including 21 PanIN, 3 PDAC arising from PanIN, and 65 PDAC without PanIN. UCN immunoscores ranging from 0 to 12 were obtained by multiplying intensity (scored on a 3‐point scale) by the percentage of stained cells (scored on a 4‐point scale). Strong expression of UCN was detected in 5 specimens of non‐neoplastic pancreatic ductal epithelia. UCN immunoscore was significantly higher in PanIN‐1 than in PanIN‐2 and PanIN‐3 (p = 0.038) and significantly higher in well‐differentiated PDAC or early American Joint Committee on Cancer (AJCC) stage PDAC than in poorly differentiated or advanced stage PDAC (p = 0.025, p = 0.018). Higher expression of UCN correlates with PDAC tumor grade and AJCC pathologic stage as well as PanIN grade. Immunohistochemical assessment of UCN may help clinicians predict tumor recurrence rate and help pathologists make a proper diagnosis.     

The epigenetic regulators Bmi1 and Ring1B are differentially regulated in pancreatitis and pancreatic ductal adenocarcinoma

Chronic pancreatitis and pancreatic ductal adenocarcinoma (PDAC) are associated with major changes in cell differentiation. These changes may be at the basis of the increased risk for PDAC among patients with chronic pancreatitis. Polycomb proteins are epigenetic silencers expressed in adult stem cells; up‐regulation of Polycomb proteins has been reported to occur in a variety of solid tumours such as colon and breast cancer. We hypothesized that Polycomb might play a role in preneoplastic states in the pancreas and in tumour development/progression. To test these ideas, we determined the expression of PRC1 complex proteins (Bmi1 and Ring1b) during pancreatic development and in pancreatic tissue from mouse models of disease: acute and chronic pancreatic injury, duct ligation, and in K‐Ras^G12V conditional knock‐in and caerulein‐treated K‐Ras^G12V mice. The study was extended to human pancreatic tissue samples. To obtain mechanistic insights, Bmi1 expression in cells undergoing in vitro exocrine cell metaplasia and the effects of Bmi1 depletion in an acinar cancer cell line were studied. We found that Bmi1 and Ring1B are expressed in pancreatic exocrine precursor cells during early development and in ductal and islet cells—but not acinar cells—in the adult pancreas. Bmi1 expression was induced in acinar cells during acute injury, in acinar–ductal metaplastic lesions, as well as in pancreatic intraepithelial neoplasia (PanIN) and PDAC. In contrast, Ring1B expression was only significantly and persistently up‐regulated in high‐grade PanINs and in PDAC. Bmi1 knockdown in cultured acinar tumour cells led to changes in the expression of various digestive enzymes. Our results suggest that Bmi1 and Ring1B are modulated in pancreatic diseases and could contribute differently to tumour development. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Genetic analyses of isolated high‐grade pancreatic intraepithelial neoplasia (HG‐PanIN) reveal paucity of alterations in TP53 and SMAD4

High‐grade pancreatic intraepithelial neoplasia (HG‐PanIN) is the major precursor of pancreatic ductal adenocarcinoma (PDAC) and is an ideal target for early detection. To characterize pure HG‐PanIN, we analysed 23 isolated HG‐PanIN lesions occurring in the absence of PDAC. Whole‐exome sequencing of five of these HG‐PanIN lesions revealed a median of 33 somatic mutations per lesion, with a total of 318 mutated genes. Targeted next‐generation sequencing of 17 HG‐PanIN lesions identified KRAS mutations in 94% of the lesions. CDKN2A alterations occurred in six HG‐PanIN lesions, and RNF43 alterations in five. Mutations in TP53, GNAS, ARID1A, PIK3CA, and TGFBR2 were limited to one or two HG‐PanINs. No non‐synonymous mutations in SMAD4 were detected. Immunohistochemistry for p53 and SMAD4 proteins in 18 HG‐PanINs confirmed the paucity of alterations in these genes, with aberrant p53 labelling noted only in three lesions, two of which were found to be wild type in sequencing analyses. Sixteen adjacent LG‐PanIN lesions from ten patients were also sequenced using targeted sequencing. LG‐PanIN harboured KRAS mutations in 94% of the lesions; mutations in CDKN2A, TP53, and SMAD4 were not identified. These results suggest that inactivation of TP53 and SMAD4 are late genetic alterations, predominantly occurring in invasive PDAC. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Pancreatic cell plasticity and cancer initiation induced by oncogenic Kras is completely dependent on wild-type PI 3-kinase p110α

Increased PI 3-kinase (PI3K) signaling in pancreatic ductal adenocarcinoma (PDAC) correlates with poor prognosis, but the role of class I PI3K isoforms during its induction remains unclear. Using genetically engineered mice and pharmacological isoform-selective inhibitors, we found that the p110α PI3K isoform is a major signaling enzyme for PDAC development induced by a combination of genetic and nongenetic factors. Inactivation of this single isoform blocked the irreversible transition of exocrine acinar cells into pancreatic preneoplastic ductal lesions by oncogenic Kras and/or pancreatic injury. Hitting the other ubiquitous isoform, p110β, did not prevent preneoplastic lesion initiation. p110α signaling through small GTPase Rho and actin cytoskeleton controls the reprogramming of acinar cells and regulates cell morphology in vivo and in vitro. Finally, p110α was necessary for pancreatic ductal cancers to arise from Kras-induced preneoplastic lesions by increasing epithelial cell proliferation in the context of mutated p53. Here we identify an in vivo context in which p110α cellular output differs depending on the epithelial transformation stage and demonstrate that the PI3K p110α is required for PDAC induced by oncogenic Kras, the key driver mutation of PDAC. These data are critical for a better understanding of the development of this lethal disease that is currently without efficient treatment.     

p21WAF1/Cip1 limits senescence and acinar‐to‐ductal metaplasia formation during pancreatitis

Trans‐differentiation of pancreatic acinar cells into ductal‐like lesions, a process defined as acinar‐to‐ductal metaplasia (ADM), is observed in the course of organ regeneration following pancreatitis. In addition, ADM is found in association with pre‐malignant PanIN lesions and correlates with an increased risk of pancreatic adenocarcinoma (PDAC). Human PDAC samples show down‐regulation of p21^WAF1/Cip1, a key regulator of cell cycle and cell differentiation. Here we investigated whether p21 down‐regulation is implicated in controlling the early events of acinar cell trans‐differentiation and ADM formation. p21‐mediated regulation of ADM formation and regression was analysed in vivo during the course of cerulein‐induced pancreatitis, using wild‐type (WT) and p21‐deficient (p21^?/?) mice. Biochemical and immunohistochemical methods were used to evaluate disease progression over 2 weeks of the disease and during a recovery phase. We found that p21 was strongly up‐regulated in WT acinar cells during pancreatitis, while it was absent in ADM areas, suggesting that p21 down‐regulation is associated with ADM formation. In support of this hypothesis, p21^?/? mice showed a significant increase in number and size of metaplasia. In addition, p21 over‐expression in acinar cells reduced ADM formation in vitro, suggesting that the protein regulates the metaplastic transition in a cell‐autonomous manner. p21^?/? mice displayed increased expression and relocalization of β‐catenin both during pancreatitis and in the subsequent recovery phase. Finally, loss of p21 was accompanied by increased DNA damage and development of senescence. Our findings are consistent with a gate‐keeper role of p21 in acinar cells to limit senescence activation and ADM formation during pancreatic regeneration. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd