A new mouse model of pancreatic cancer: PTEN gets its Akt together

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a negative regulator of the oncogenic PI3-K/Akt signaling pathway. Loss-of-function mutations of PTEN are seen in several human solid cancers. A murine model of conditional Pten inactivation in the pancreas is described that leads to acquisition of a profound metaplastic ductal phenotype accompanied by loss of differentiated acinar units. Evidence is presented for a centroacinar cell origin of the metaplastic "neoductules." These mice also develop invasive pancreatic adenocarcinomas at a low frequency, and provide a unique in vivo platform for exploring the role of PI3-K/Akt signaling in pancreatic neoplasia.     

Cytokeratin antigen in BOP-induced pancreatic tumors--implications for histogenesis

Pancreatic carcinoma induced in the Syrian hamster by the carcinogenN-nitrosobis(2-oxopropyl)amine (BOP) is of interest becauseof the ductal/ductular morphology of the tumors, which resembleshuman pancreatic cancer. However, whether hamster tumors arisefrom pre-existing ductal epithelium or from acinar cells hasnot yet been determined. The present study shows that a monoclonalantiserum to cytokeratin (an epithelial marker), when appliedto normal hamster pancreas sections, stained centroacinar, ductularand ductal epithelium but did not stain acinar cells. We thereforeexamined pancreatic tissue from hamsters with benign and malignantneoplasms induced by BOP. The antiserum strongly stained thecells of all BOP-induced lesions (cysts, pseudoductules, hyperplasia,dysplasia and carcinomas). No acinar cell staining was observedin BOP-treated pancreas. These findings support the hypothesisthat BOP-induced neoplasms arise from ductal epithelium andnot from acinar cells.     

A Case of Mistaken Identity? Nonductal Origins of Pancreatic “Ductal” Cancers

In this issue of Cancer Cell, Guerra and colleagues provide important new insights regarding the ability of specific pancreatic cell types to generate invasive pancreatic cancer. First, they demonstrate that classical pancreatic "ductal" neoplasia can be induced by activation of oncogenic Kras in nonductal exocrine cells. Second, they show that, while Kras activation in immature acinar and centroacinar cells is readily able to induce ductal neoplasia, Kras-mediated tumorigenesis in mature exocrine pancreas requires the induction of chronic epithelial injury. The results shed new light on the "cell of origin" of pancreatic ductal cancer and demonstrate that chronic pancreatitis provides a permissive environment for Kras-induced pancreatic neoplasia.     

Experimental pancreatic cancer: role of species, sex and diet

Pancreatic cancer has been experimentally induced in rodents by chemical carcinogens that have been used to establish "animal models" for pancreatic carcinogenesis. Recent work with transgenic mice provided a new model in which a dominantly expressed oncogene is transmitted in the germ cell line of homozygous strains. Carcinogens are not equally effective in all species and the histologic type of carcinoma that develops is strongly influenced by the species. Carcinomas that develop in rats and mice are predominantly acinar cell type. In contrast, hamsters characteristically develop duct-like carcinomas. The histologic type of carcinoma in hamsters resembles more closely the majority of carcinomas in the human pancreas than is the case in the rat or mouse. Studies in rats and guinea pigs have demonstrated that duct-like and undifferentiated carcinomas, as well as acinar cell carcinomas, can arise from acinar cells. Thus, the relative importance of ductal cells, centroacinar cells, acinar cells and putative stem cells in the origin of pancreatic carcinomas remains to be determined. In most rat models, males have developed a higher incidence rate of pancreatic cancers than females. Experimental evidence shows that testosterone promotes and estrogen inhibits the growth of preneoplastic lesions and cancers in the rat pancreas. Dietary composition and additives influence carcinogenesis in the pancreas. High fat diets promote carcinogenesis in rats and hamsters, and dietary trypsin inhibitors promote in rats. Other dietary additives such as retinoids and antioxidants have inhibited carcinogenesis in the animal models.     

Ductal origin of pancreatic adenocarcinomas induced by conditional activation of a human Ha-ras oncogene in rat pancreas

Pancreatic ductal adenocarcinoma is one of the most debilitating malignancies in humans. Currently, radiation and chemotherapy are ineffective, with median survival times after treatment of <12 months. Animal models that reflect the human condition and can be used to explore screening and therapeutic approaches are clearly desirable. One feature of human pancreatic adenocarcinoma is an exceedingly high frequency of K-ras mutation. The present study was conducted to determine if targeted activation of a human oncogenic-ras transgene in rat pancreas would induce carcinomas correspondent to human pancreatic ductal adenocarcinomas. We established transgenic (Hras250) rats in which expression of a human Ha-rasG12V oncogene is regulated by the Cre/lox system. Targeted pancreatic activation of the transgene was accomplished by injection of Cre-carrying adenovirus into the pancreatic ducts and acini through the common bile duct. Adenoviral infection of injected animals was exclusive to the pancreas; infected cells could be identified in duct, intercalated duct, centroacinar and, less frequently, acinar cells, but not in endocrine islet cells. Four weeks after injection, proliferative lesions in the duct epithelium, intercalated ducts and centroacinar cells, but not acinar cells, were widespread. Tumorigenesis in other tissues was not observed. Most lesions, including atypical duct proliferative lesions, PanIN-like lesions and carcinomas, were positive for cytokeratins 19 and 7, cyclooxygenase 2 and MMP-7 but negative for amylase and chymotrypsin. Many adenocarcinoma lesions were positive for EGF and EGFR. Duct epithelial and atypical duct proliferative lesions and carcinoma lesions were all positive for transduced Ha-rasG12V oncogene expression. The cytogenesis of pancreatic ductal type carcinoma was depicted. This model exhibits important similarities to the human disease and promises to advance our understanding of the behavior of pancreas adenocarcinomas and expedite screening and therapy.     

Chronic pancreatitis is essential for induction of pancreatic ductal adenocarcinoma by K-Ras oncogenes in adult mice

Pancreatic ductal adenocarcinoma (PDA), one of the deadliest human cancers, often involves somatic activation of K-Ras oncogenes. We report that selective expression of an endogenous K-Ras(G12V) oncogene in embryonic cells of acinar/centroacinar lineage results in pancreatic intraepithelial neoplasias (PanINs) and invasive PDA, suggesting that PDA originates by differentiation of acinar/centroacinar cells or their precursors into ductal-like cells. Surprisingly, adult mice become refractory to K-Ras(G12V)-induced PanINs and PDA. However, if these mice are challenged with a mild form of chronic pancreatitis, they develop the full spectrum of PanINs and invasive PDA. These observations suggest that, during adulthood, PDA stems from a combination of genetic (e.g., somatic K-Ras mutations) and nongenetic (e.g., tissue damage) events.     

Stage-specific expression of cancer-associated type 1 and type 2 chain polylactosamine antigens in the developing pancreas of human embryos.

Expression of type 1 and type 2 chain carbohydrate antigens during the course of morphogenesis of human embryonic pancreas was investigated using specific monoclonal antibodies and compared with the carbohydrate antigen profiles of human pancreatic cancers. The type 2 chain antigens, such as stage-specific embryonic antigen 1 (Le(x)) and I-antigens, appeared much earlier than the type 1 chain antigens; the epithelial cells of primitive foregut were Le(x)+I-antigen- in the embryos at Carnegie stages 16-23, while the pancreatic primordial cells, which had differentiated from the Le(x)+ gut epithelial cells, were Le(x)-I-antigen+ at Carnegie stages 22-23. The type 1 chain antigens, such as Le(a), Le(b), Le(c), and their sialylated derivatives, were not expressed in any cells at these stages and appeared much later in the pancreas of the 10-12-week embryos, when the primitive pancreatic ductal cells in the primordia exhibited an extensive budding of the daughter cells. At this stage, Le(a) appeared and was expressed strongly in the epithelial cells of primitive pancreatic ducts as well as in the daughter cells that were destined to differentiate into future centroacinar cells; Le(b) was localized in the daughter cells which were to become future acinar cells; and Le(c) was specifically expressed in the daughter cells which were to form future Langerhans islets. With regard to the sialylated derivatives of Le(a), expression of the 2-3 sialyl Le(a) antigen was limited to the epithelial cells of the primitive pancreatic ducts, while the 2-6 sialyl Le(a) antigen was strongly expressed in the future centroacinar cells, which had differentiated from the corresponding daughter cells. Among these antigens, the Le(a) and 2-3 sialyl Le(a) antigens showed the highest incidence in human pancreatic cancer tissues. These results indicate that the expression of these carbohydrate antigens in embryonic pancreas is differentiation dependent and cell lineage specific and that most human pancreatic cancer cells mimic the carbohydrate antigen profile of the epithelial cells of the primitive pancreatic ducts in human embryos.     

Cell proliferation and apoptosis in the exocrine pancreas of azaserine-treated rats and N-nitrosobis(2-oxopropyl)amine-treated hamsters

Altered cell proliferation is an important characteristic of neoplastic development, hence the quantitation of cell growth in normal and (pre)neoplastic pancreatic tissue will provide useful information on the process (and modulation) of pancreatic tumour promotion in experimental animals. Cell proliferation was estimated by means of BrdU-incorporation in normal pancreatic tissue and in putative preneoplastic pancreatic tissue of azaserine-treated rats and of N-nitrosobis(2-oxopropyl)amine (BOP)-treated hamsters at 2, 6, 12, 26 and 52 weeks post-treatment. Furthermore, the effects of diets high in vegetable oil or fish oil on the BrdU labeling index, the Ki-67 labeling index as well as the apoptotic labeling index were determined in azaserine-induced atypical acinar cell foci (AACF) in the pancreas of rats. In saline- and azaserine-treated rats, the BrdU labeling index (LI) in normal pancreatic acinar cells showed a similar time-related decrease. In azaserine-treated rats, the BrdU-LI in AACF was significantly higher than in normal acinar tissue. The BrdU, Ki-67 and apoptotic labeling indices in pancreatic AACF of rats fed diets high in vegetable oil versus fish oil were 24.8+/-1.7 vs 15.5+/-1.2 (BrdU; P<0.05), 9.9+/-0.8 vs 6.5+/-0.5 (Ki-67; P<0.001) and 1.31+/-0.12 vs 0.97+/-0.11 (apoptosis; P<0.05), respectively. In hamsters, up to 26 weeks after BOP-treatment the BrdU-LI in acinar cells was significantly higher than in saline-treated animals. In BOP-treated hamsters, ductal cells showed a significantly increased LI 2 weeks post-treatment, whereas the LI in centroacinar cells had significantly increased 2 and 26 weeks post-treatment. In hamster pancreas the LI was significantly higher in tubular ductal complexes than in cystic ductal complexes and was highest in borderline lesions. It is concluded that (i) determination of BrdU-LI provides a reliable parameter to discriminate between putative preneoplastic lesions with a high or low growth potential, hence with a high or low potential to develop into ultimate carcinomas, (ii) BOP causes increased DNA synthesis in normal acinar cells, leading to increased risk of additional DNA damage, implicating the acinar cell to be putatively involved in the development of ductular adenocarcinomas in the hamster pancreas and (iii) quantitation of cell proliferation and apoptosis may be useful to determine the enhancing or inhibitory effects of life-style factors on pancreatic carcinogenesis.     

Ultrastructural analysis of pancreatic carcinogenesis. V. Changes in differentiation of acinar cells during chronic treatment with N-nitrosobis(2-hydroxypropyl)amine

An electron microscope study was carried out on changes in thepancreatic acinar tissues of male Syrian hamsters during thecourse of induction of pancreatic neoplasia by lifetime weekyinjections of N nitroso bis(2 hydroxypropyl)amine (BHP). Thedominant process observed was a loss of differentiation of theacinar cells, leading to the development of a new populationof cells that closely resemble centro acinar or ductular cells.During this process, the original centroacinar and ductularcells appear to be relatively unaffected by exposure to BHP.Thus, acinar cell de differentiation appears, in this experimentalmodel, to be an important preliminary stage in neoplastic development.It is argued that these findings may have implications for thehistogenesis of human pancreatic adenocarcinoma, consideredhitherto to be ductal in origin.     

Evolution of the expression of fetal acinar antigens during carcinogenesis of the pancreas in hamsters: individual follow-up by open biopsy

Individual lesions in the pancreas and the presence of fetal acinar antigens along with carcinogenesis induced by N-nitrosobis(2-oxopropyl)amine (CAS: 60599-38-4) were studied by open biopsy in 16 Syrian golden hamsters 13, 22, and 40 weeks after initiation of treatment. At 13 weeks, cystadenoma and regular ductal hyperplasia were noted in 3 animals and 1 animal, respectively. Staining for fetal acinar antigens in the pancreas was found in 69% of the hamsters. At 22 weeks, cystadenoma and hyperplastic ducts were common (60 and 53%), and 3 hamsters developed pancreatic adenocarcinoma. Fetal acinar antigens persisted in the acini and extended to irregular hyperplastic ducts and tumor cells. At 40 weeks, ductal proliferation was the main lesion in all pancreatic tissue, and 9 animals had adenocarcinoma. Acinar antigens were found in the remaining acini, in irregular hyperplastic ducts, and in tumor cells. Thus, once reexpressed, fetal acinar antigens persist in pancreatic lesions and pancreatic carcinomas in the hamster.     

Animal models of exocrine pancreatic carcinogenesis

In order to understand the evolution, histogenesis, and biological behaviour of exocrine pancreatic carcinoma, some reproducible experimental models have been developed in certain rodent species. To date, more than 16 chemicals, many of them structurally unrelated, have been shown to induce pancreatic tumors. Although some of these chemicals appear species specific in their effect on the pancreas, others have been shown to be capable of inducing pancreatic tumors in more than one species. In hamsters, the administration of diisopropylnitrosamine or its oxidized metabolites leads to the development of ductal adenocarcinomas that histologically resemble human pancreatic carcinomas. The histogenesis of the ductal type of adenocarcinoma in hamsters is complex, and appears to involve both the duct cells and dedifferentiated acinar cells. All pancreatic tumors in rats develop from acinar cells showing variable degrees of differentiation, regardless of the type of carcinogen used. The type of pancreatic lesions that develop in mice are also of acinar cell origin. In guinea pigs the tumors are adenocarcinomas of the ductal type and are shown to be derived from dedifferentiated acinar cells that have undergone duct-like transformation. Irrespective of the type of tumor that develops in these experimental animals, all of these models can be successfully used to evaluate the various modifying (risk) factors and biological behaviour of these neoplasms.     

Experimental induction of pancreatic carcinomas in the hamster with N delta-(N-methyl-N-nitrosocarbamoyl)-L-ornithine

Carcinomas of the pancreas, stomach, and breast, as well as mesotheliomas and ovarian stromal tumors, were induced in Syrian golden hamsters treated with N delta-(N-methyl-N-nitrosocarbamoyl)-L-ornithine (MNCO), which has previously been shown to cause pancreatic acinar cell carcinomas in rats. The pancreatic carcinomas in hamsters appeared ductlike. The nonneoplastic and preneoplastic lesions induced in the hamster pancreas included cystic ductal complexes, tubular complexes, intraductal hyperplasia and atypical hyperplasia, focal eosinophilic metaplasia, and foci of atypical acinar cells. High doses of 654 mg MNCO/kg body weight were cytotoxic for acinar cells and caused atrophy of the pancreas. Alkaline elution analysis of DNA from acinar cells treated in culture with MNCO showed an increased rate of elution characteristic of single-strand breaks. A group of hamsters treated with a low dose of N-nitrosobis(2-oxopropyl)amine (BOP) developed pancreatic lesions similar to those seen when a subcarcinogenic dose of MNCO was given. The results suggest that MNCO affects both acinar and ductal cells in the hamster and that the response of the hamster pancreas to MNCO and BOP is similar in many respects.     

Protease‐activated receptor 1 drives and maintains ductal cell fates in the premalignant pancreas and ductal adenocarcinoma

Pancreatic acinar cells have high plasticity and can transdifferentiate into ductal‐like cells. This acinar‐to‐ductal metaplasia (ADM) contributes to tissue maintenance but may also contribute to the premalignant transformation that can eventually progress to pancreatic ductal adenocarcinoma (PDAC). Macrophages are key players in ADM, and macrophage‐secreted matrix metalloproteinase (MMP)‐9 induces ADM through yet unknown mechanisms. As we previously identified MMP9 as a novel agonist of protease‐activated receptor 1 (PAR1), a receptor that is known to orchestrate the cross‐talk between macrophages and tumor cells in PDAC, we here assessed the contribution of PAR1 to pancreatic cell fates. We found that genetic deficiency for PAR1 increases acinar gene expression programs in the healthy pancreas and that PAR1 deficiency limits ductal transdifferentiation in experimental systems for ADM. Moreover, PAR1 silencing in PDAC cells increases acinar marker expression. Changes in PDAC cell lines were associated with a downregulation of known Myc‐target genes, and Myc inhibition mimics PAR1 deficiency in enhancing acinar programs in healthy organoids and PDAC cells. Overall, we identify the PAR1‐Myc axis as a driver of ductal cell fates in premalignant pancreas and PDAC. Moreover, we show that cellular plasticity is not unique to acinar cells and that ductal regeneration into acinar‐like cells is possible even in the context of oncogenic KRAS activation.     

Preinvasive pancreatic neoplasia of ductal phenotype induced by acinar cell targeting of mutant Kras in transgenic mice

Activating mutation of the Kras oncogene is the most frequent and perhaps the earliest genetic alteration associated with pancreatic cancer. To examine the link between mutant Kras and exocrine pancreatic cancer, we generated transgenic mice carrying an elastase-mutant Kras transgene, which targets expression to pancreatic acinar cells. Most elastase-Kras founder mice displayed perinatal pancreatic acinar cell hyperplasia and dysplasia. However, adult mice in two surviving lineages displayed preinvasive pancreatic neoplastic lesions with ductal morphology, thereby providing a unique mouse model in which lesion histotype and initiating genetic alteration overlap with the human disease. Our findings suggest that Kras mutation is associated with development of early stage duct-like lesions in pancreas, but that additional alterations must accompany progression to malignancy.     

Cell lineage markers in human pancreatic cancer

The normal pancreas consists of three major cell types or lineages that share a common embryologic origin from pluripotent endodermal precursors. The type of cell that undergoes neoplastic transformation to form a pancreatic carcinoma is controversial and may influence the phenotype and biologic behavior of the tumor. In this study, immunohistologic techniques were used to determine the cell lineage differentiation expressed in 29 primary exocrine pancreatic adenocarcinomas, five metastatic exocrine pancreatic adenocarcinomas, and five islet cell neoplasma. Specimens of normal pancreas and chronic pancreatitis were used for comparison. The cell lineage markers consisted of monoclonal and polyclonal antibodies against trypsin and lipase (acinar cells); secretory component, carbonic anhydrase II, and pancreatic cancer mucin SPan-1 (ductal cells); and chromogranin-A and somatostatin (islet cells). The expression of carcinoembryonic antigen (CEA) and lysozyme were also determined. This collection of markers allowed the differentiation between acinar, ductal, and islet cells of normal pancreas and chronic pancreatitis specimens. The expression of cell lineage markers in islet cell tumors was homogeneous and restricted to chromogranin-A. In contrast, the expression of these markers in primary and metastatic exocrine pancreatic adenocarcinomas was variable. Reactivity with monoclonal anti-CEA was absent in normal pancreas, and was present in 83% of chronic pancreatitis specimens as well as 90% of exocrine pancreatic adenocarcinomas. In addition, lysozyme reactivity was absent in normal pancreas; however, lysozyme was expressed in one case of chronic pancreatitis, 17 cases of primary carcinoma, and three cases of metastatic carcinoma. These findings support the concept that the original transformed cell type in many pancreatic exocrine carcinomas resemble endodermal "stem cells" that retain the capability of differentiation along more than one cell lineage pathway.     

Cripto,a member of the epidermal growth factor family,is over-expressed in human pancreatic cancer and chronic pancreatitis

Cripto is a 188 amino-acid protein containing a central segment that shares amino-acid sequence homology with epidermal growth factor (EGF) and transforming growth factor alpha (TGF-α). The EGF receptor, EGF and TGF-α are expressed in the normal human pancreas, and are over-expressed in human pancreatic cancer. Therefore, in the present study we sought to determine whether cripto is found in the normal human pancreas and whether its expression is altered in pancreatic cancer. Because chronic pancreatitis (CP) is associated with interstitial fibrosis similar to that observed in pancreatic cancer, we also examined cripto expression in pancreatic tissues from patients with CP. In the normal pancreas, cripto immunoreactivity was found at moderate levels in most ductal cells and was present very faintly in a rare acinar cell. In 26 of 58 pancreatic cancers, cripto immunoreactivity was present in many cancer cells. Its presence was associated with advanced tumor stage, but not with shorter post-operative survival. Cripto was also present in acinar and ductal cells adjacent to the cancer cells, and in many ductal and atrophic acinar cells in the CP samples. Northern blot analysis revealed a marked increase in cripto mRNA levels in the cancer and CP samples. By densitometry, there was a 11 - and 4-fold increase in cripto mRNA levels in pancreatic cancer and CP respectively. Southern blot analysis did not reveal an increase in gene copies encoding cripto either in cancer or in CP. These findings indicate that cripto expression may contribute to disease progression in pancreatic cancer, and implicate cripto in the histopathological alterations that occur in the pancreas both in cancer and in CP.     

Neurogenin 3–Directed Cre Deletion of Tsc1 Gene Causes Pancreatic Acinar Carcinoma

The role of tuberous sclerosis complex (TSC) in the pathogenesis of pancreatic cancers remains largely unknown. The present study shows that neurogenin 3 directed Cre deletion of Tsc1 gene induces the development of pancreatic acinar carcinoma. By cross-breeding the Neurog3-cre mice with Tsc1^loxp/loxp mice, we generated the Neurog3-Tsc1−/− transgenic mice in which Tsc1 gene is deleted and mTOR signaling activated in the pancreatic progenitor cells. All Neurog3-Tsc1−/− mice developed notable adenocarcinoma-like lesions in pancreas starting from the age of 100 days old. The tumor lesions are composed of cells with morphological and molecular resemblance to acinar cells. Metastasis of neoplasm to liver and lung was detected in 5% of animals. Inhibition of mTOR signaling by rapamycin significantly attenuated the growth of the neoplasm. Relapse of the neoplasm occurred within 14 days upon cessation of rapamycin treatment. Our studies indicate that activation of mTOR signaling in the pancreatic progenitor cells may trigger the development of acinar carcinoma. Thus, mTOR may serve as a potential target for treatment of pancreatic acinar carcinoma.Abbreviations: ACC, acinar cell carcinoma; 4EBP-1, 4E binding protein 1; mTOR, mammlian target of rapamycin; Neurog3, neurogenin 3; PDA, pancreatic ductal adenocarcinoma; S6, ribosomal protein S6; TSC, tuberous sclerosis complex     

ADAM9 expression in pancreatic cancer is associated with tumour type and is a prognostic factor in ductal adenocarcinoma

Gene expression profiling revealed ADAM9 to be distinctly overexpressed in pancreatic ductal adenocarcinoma (PDAC). We examined the relevance of ADAM9 expression in PDAC diagnosis and prognosis. A total of 59 infiltrating PDACs, 32 specimens from patients with chronic pancreatitis, 11 endocrine tumours and 24 acinar cell carcinomas were immunohistochemically analysed for ADAM9 expression. Staining for ADAM9 was detected in 58 out of 59 (98.3%) PDACs and in two out of 24 (8.3%) acinar cell carcinomas, but not in endocrine tumours. In the non-neoplastic pancreas, whether normal or chronically inflamed, ADAM9 was expressed in centroacinar and intralobular duct cells, but not in interlobular duct cells and their hyperplastic lesions. Pancreatic ductal adenocarcinomas showing cytoplasmic ADAM9 expression correlated with poor tumour differentiation and also with shorter overall survival than in cases showing only an apical membranous staining pattern (P=0.001). Multivariate analysis identified cytoplasmic ADAM9 expression as an independent marker of shortened survival in a set of 42 curatively (R0) resected PDAC (P<0.05, hazard ratio 2.85, 95% confidence interval: 1.21-6.71). The results show that ADAM9 expression distinguishes PDACs from other solid pancreatic tumours. In addition, cytoplasmic ADAM9 overexpression is associated with poor differentiation and shortened survival. Therefore, ADAM9 overexpression might contribute to the aggressiveness of PDACs.