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.     

The cellular origins of cancer with particular reference to the gastrointestinal tract

Stem cells or their closely related committed progenitor cells are the likely founder cells of most neoplasms. In the continually renewing and hierarchically organized epithelia of the oesophagus, stomach and intestine, homeostatic stem cells are located at the beginning of the cell flux, in the basal layer of the oesophagus, the isthmic region of gastric oxyntic glands and at the bottom of gastric pyloric‐antral glands and colonic crypts. The introduction of mutant oncogenes such as Kras^G12D or loss of Tp53 or Apc to specific cell types expressing the likes of Lgr5 and Mist1 can be readily accomplished in genetically engineered mouse models to initiate tumorigenesis. Other origins of cancer are discussed including ‘reserve’ stem cells that may be activated by damage or through disruption of morphogen gradients along the crypt axis. In the liver and pancreas, with little cell turnover and no obvious stem cell markers, the importance of regenerative hyperplasia associated with chronic inflammation to tumour initiation is vividly apparent, though inflammatory conditions in the renewing populations are also permissive for tumour induction. In the liver, hepatocytes, biliary epithelial cells and hepatic progenitor cells are embryologically related, and all can give rise to hepatocellular carcinoma and cholangiocarcinoma. In the exocrine pancreas, both acinar and ductal cells can give rise to pancreatic ductal adenocarcinoma (PDAC), although the preceding preneoplastic states are quite different: acinar‐ductal metaplasia gives rise to pancreatic intraepithelial neoplasia culminating in PDAC, while ducts give rise to PDAC via. mucinous cell metaplasia that may have a polyclonal origin.     

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.     

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.     

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     

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.     

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.     

Origin of pancreatic ductal adenocarcinoma from atypical flat lesions: a comparative study in transgenic mice and human tissues

Pancreatic ductal adenocarcinoma (PDAC) and its precursor lesions, pancreatic intraepithelial neoplasia (PanIN), display a ductal phenotype. However, there is evidence in genetically defined mouse models for PDAC harbouring a mutated kras under the control of a pancreas-specific promoter that ductal cancer might arise in the centroacinar-acinar region, possibly through a process of acinar-ductal metaplasia (ADM). In order to further elucidate this model of PDAC development, an extensive expression analysis and molecular characterization of the putative and already established (PanIN) precursor lesions were performed in the Kras(G12D/+) ; Ptf1a-Cre(ex1/+) mouse model and in human tissues, focusing on lineage markers, developmental pathways, cell cycle regulators, apomucins, and stromal activation markers. The results of this study show that areas of ADM are very frequent in the murine and human pancreas and represent regions of increased proliferation of cells with precursor potential. Moreover, atypical flat lesions originating in areas of ADM are the most probable precursors of PDAC in the Kras(G12D/+); Ptf1a-Cre(ex1/+) mice and similar lesions were also found in the pancreas of three patients with a strong family history of PDAC. In conclusion, PDAC development in Kras(G12D/+); Ptf1a-Cre(ex1/+) mice starts from ADM and a similar process might also take place in patients with a strong family history of PDAC.     

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.     

Anti‐stromal treatment together with chemotherapy targets multiple signalling pathways in pancreatic adenocarcinoma

Stromal targeting for pancreatic ductal adenocarcinoma (PDAC) is rapidly becoming an attractive option, due to the lack of efficacy of standard chemotherapy and increased knowledge about PDAC stroma. We postulated that the addition of stromal therapy may enhance the anti‐tumour efficacy of chemotherapy. Gemcitabine and all‐trans retinoic acid (ATRA) were combined in a clinically applicable regimen, to target cancer cells and pancreatic stellate cells (PSCs) respectively, in 3D organotypic culture models and genetically engineered mice (LSL‐Kras^G12D/+;LSL‐Trp53^R172H/+;Pdx‐1‐Cre: KPC mice) representing the spectrum of PDAC. In two distinct sets of organotypic models as well as KPC mice, we demonstrate a reduction in cancer cell proliferation and invasion together with enhanced cancer cell apoptosis when ATRA is combined with gemcitabine, compared to vehicle or either agent alone. Simultaneously, PSC activity (as measured by deposition of extracellular matrix proteins such as collagen and fibronectin) and PSC invasive ability were both diminished in response to combination therapy. These effects were mediated through a range of signalling cascades (Wnt, hedgehog, retinoid, and FGF) in cancer as well as stellate cells, affecting epithelial cellular functions such as epithelial–mesenchymal transition, cellular polarity, and lumen formation. At the tissue level, this resulted in enhanced tumour necrosis, increased vascularity, and diminished hypoxia. Consequently, there was an overall reduction in tumour size. The enhanced effect of stromal co‐targeting (ATRA) alongside chemotherapy (gemcitabine) appears to be mediated by dampening multiple signalling cascades in the tumour–stroma cross‐talk, rather than ablating stroma or targeting a single pathway. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Dichotomous role of the serine/threonine kinase MAP4K4 in pancreatic ductal adenocarcinoma onset and metastasis through control of AKT and ERK pathways

MAP4K4 is a serine/threonine kinase of the STE20 family involved in the regulation of actin cytoskeleton dynamics and cell motility. It has been proposed as a target of angiogenesis and inhibitors show potential in cardioprotection. MAP4K4 also mediates cell invasion in vitro, is overexpressed in various types of cancer, and is associated with poor patient prognosis. Recently, MAP4K4 has been shown to be overexpressed in pancreatic cancer, but its role in tumour initiation, progression, and metastasis is unknown. Here, using the Kras^G12D Trp53^R172H Pdx1-Cre (KPC) mouse model of pancreatic ductal adenocarcinoma (PDAC), we show that deletion of Map4k4 drives tumour initiation and progression. Moreover, we report that the acceleration of tumour onset is also associated with an overactivation of ERK and AKT, two major downstream effectors of KRAS, in vitro and in vivo. In contrast to the accelerated tumour onset caused by loss of MAP4K4, we observed a reduction in metastatic burden with both the KPC model and in an intraperitoneal transplant assay indicating a major role of MAP4K4 in metastatic seeding. In summary, our study sheds light on the dichotomous role of MAP4K4 in the initiation of PDAC onset, progression, and metastatic dissemination. It also identifies MAP4K4 as a possible druggable target against pancreatic cancer spread, but with the caveat that targeting MAP4K4 might accelerate early tumorigenesis. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.     

Decreased expression and promoter methylation of the menin tumor suppressor in pancreatic ductal adenocarcinoma

Loss of menin, a tumor suppressor coded by the MEN1 gene, is a key factor in the pathogenesis of multiple endocrine neoplasia type I and in a percentage of sporadic endocrine tumors of the pancreas and parathyroid glands. This study investigated expression of the menin protein in the normal exocrine pancreas and in pancreatic ductal adenocarcinoma (PDAC), the most common pancreatic tumor. Immunofluorescence (IF) analyses showed that menin is expressed at high levels in normal acinar and duct cells. Examination of 24 clinical samples of PDAC revealed a pronounced decrease in menin expression in all tumors examined. To identify alterations underlying this defect, we searched for disruption and epigenetic silencing of the MEN1 gene. Analysis of nine laser‐microdissected tumors revealed loss of heterozygosity of intragenic (one tumor) or adjacent (three tumors) MEN1 microsatellite markers. Methylation of CpG sites in the MEN1 promoter was documented in five of 24 tumors. IF analyses also revealed low to undetectable menin expression in the PDAC cell lines MiaPaCa‐2 and Panc‐1. Ectopic expression of menin in these cells resulted in a marked alteration of the cell cycle, with an increase in the G1/S+G2 ratio. These findings represent the first evidence that the MEN1 gene is a target of mutation and methylation in PDAC and that menin influences the cell cycle profile of duct cells. © 2009 Wiley‐Liss,Inc.     

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.     

Loss of natural killer T cells promotes pancreatic cancer in LSL‐KrasG12D/+ mice

The role of the unique T‐cell population, natural killer T (NKT) cells, which have similar functions to NK cells in pancreatic cancer (PC), is not yet evaluated. To address the regulatory roles of NKT cells on tumour progression through tumour‐associated macrophages (TAM) and their production of microsomal prostaglandin E synthase‐1 (mPGES‐1) and 5‐lipoxygenase (5‐LOX) in (Kras)‐driven pancreatic tumour (KPT) progression, we crossed CD1d^−/− mice deficient in both invariant and variant NKT cells with the Kras^G12D mice. Loss of NKT cells significantly increased pancreatic intraepithelial neoplasia (PanIN) lesions and also increased 5‐LOX and mPGES‐1 expression in M2‐type macrophages and cancer stem‐like cells in pancreatic tumours. Pharmacological inhibition of mPGES‐1 and 5‐LOX in M2 macrophages with specific inhibitor YS‐121 in KPT‐CD1d^−/− mice decreased PanIN lesions and suppressed tumour growth in association with elevated levels of active CD8a cells. Hence, NKT cells regulate PC by modulating TAMs (M2) through mPGES‐1 and 5‐LOX; and the absence of NKT cells leads to aggressive development of PC.     

ATDC induces an invasive switch in KRAS-induced pancreatic tumorigenesis

The initiation of pancreatic ductal adenocarcinoma (PDA) is linked to activating mutations in KRAS. However, in PDA mouse models, expression of oncogenic mutant KRAS during development gives rise to tumors only after a prolonged latency or following induction of pancreatitis. Here we describe a novel mouse model expressing ataxia telangiectasia group D complementing gene (ATDC, also known as TRIM29 [tripartite motif 29]) that, in the presence of oncogenic KRAS, accelerates pancreatic intraepithelial neoplasia (PanIN) formation and the development of invasive and metastatic cancers. We found that ATDC up-regulates CD44 in mouse and human PanIN lesions via activation of β-catenin signaling, leading to the induction of an epithelial-to-mesenchymal transition (EMT) phenotype characterized by expression of Zeb1 and Snail1. We show that ATDC is up-regulated by oncogenic Kras in a subset of PanIN cells that are capable of invading the surrounding stroma. These results delineate a novel molecular pathway for EMT in pancreatic tumorigenesis, showing that ATDC is a proximal regulator of EMT.     

The vacuolar-ATPase modulates matrix metalloproteinase isoforms in human pancreatic cancer

The vacuolar-ATPase (v-ATPase) is a proton transporter found on many intracellular organelles and the plasma membrane (PM). The v-ATPase on PMs of cancer cells may contribute to their invasive properties in vitro. Its relevance to human cancer tissues remains unclear. We investigated whether the expression and cellular localization of v-ATPase corresponded to the stage of human pancreatic cancer, and its effect on matrix metalloproteinase (MMP) activation in vitro. The intensity of v-ATPase staining increased significantly across the range of pancreatic histology from normal ducts to pancreatic intraepithelial neoplasms (PanIN), and finally pancreatic ductal adenocarcinoma (PDAC). Low-grade PanIN lesions displayed polarized staining confined to the basal aspect of the cell in the majority (86%) of fields examined. High-grade PanIN lesions and PDAC showed intense and diffuse v-ATPase localization. In pancreatic cancer cells, PM-associated v-ATPase colocalized with cortactin, a component of the leading edge that helps direct MMP release. Blockade of the v-ATPase with concanamycin or short-hairpin RNA targeting the V?E subunit reduced MMP-9 activity; this effect was greatest in cells with prominent PM-associated v-ATPase. In cells with detectable MMP-2 activities, however, treatment with concanamycin markedly increased MMP-2's most activated forms. V-ATPase blockade inhibited functional migration and invasion in those cells with predominantly MMP-9 activity. These results indicate that human PDAC specimens show loss of v-ATPase polarity and increased expression that correlates with increasing invasive potential. Thus, v-ATPase selectively modulates specific MMPs that may be linked to an invasive cancer phenotype.     

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.