Early Stages Of Pancreatic Carcinogenesis Research Articles

Metabolic Reprogramming Is an Initial Step in Pancreatic Carcinogenesis That Can Be Targeted to Inhibit Acinar-to-Ductal Metaplasia.

Metabolic reprogramming is a hallmark of cancer and is crucial for cancer progression, making it an attractive therapeutic target. Understanding the role of metabolic reprogramming in cancer initiation could help identify prevention strategies. To address this, we investigated metabolism during acinar-to-ductal metaplasia (ADM), the first step of pancreatic carcinogenesis. Glycolytic markers were elevated in ADM lesions compared with normal tissue from human samples. Comprehensive metabolic assessment in three mouse models with pancreas-specific activation of KRAS, PI3K, or MEK1 using Seahorse measurements, nuclear magnetic resonance metabolome analysis, mass spectrometry, isotope tracing, and RNA sequencing analysis revealed a switch from oxidative phosphorylation to glycolysis in ADM. Blocking the metabolic switch attenuated ADM formation. Furthermore, mitochondrial metabolism was required for de novo synthesis of serine and glutathione (GSH) but not for ATP production. MYC mediated the increase in GSH intermediates in ADM, and inhibition of GSH synthesis suppressed ADM development. This study thus identifies metabolic changes and vulnerabilities in the early stages of pancreatic carcinogenesis. Significance: Metabolic reprogramming from oxidative phosphorylation to glycolysis mediated by MYC plays a crucial role in the development of pancreatic cancer, revealing a mechanism driving tumorigenesis and potential therapeutic targets. See related commentary by Storz, p. 2225.

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 2184: A high-fat diet accelerates early-stage murine pancreatic carcinogenesis by influencing, in part, mesenteric adipose tissue metabolism

Abstract Increased adiposity represents a modifiable risk factor of pancreatic cancer. Multiple preclinical studies have shown that a high-fat and high-sugar diets on the acceleration of pancreatic carcinogenesis, in the context of obesity. However, the impact of diets resembling the omega-6 to omega-3 fatty acid (FA) ratio in a Western-style diet, remains unclear. Furthermore, the role of the mesenteric adipose tissue (MAT) in pancreatic carcinogenesis is poorly characterized. Thus, we aimed to determine the impact of a high-fat diet with a ratio of 9 parts omega-6 FA to each omega-3 FA, on early stages of pancreatic carcinogenesis in a genetically engineered LSL- KrasLSL-G12D; Ptf1aCre/+ (KC) model of pancreatic cancer, with an emphasis on evaluating the contribution of the MAT within the pancreatic microenvironment. For this purpose, cohorts of male and female KC mice (6-8 males and females per diet group) were randomly assigned to either a control diet (CD) group (9:1 of omega-6 FAs to omega-3 FAs) or a high-fat diet (HFD) group (9:1 of omega-6 FAs to omega-3 FAs) and fed their respective diets until three months of age. After eight weeks on their diets, HFD-fed mice had significantly increased body weight, fat mass, and elevated serum leptin levels. Furthermore, HFD-fed mice had increased acinar-to-ductal metaplasia, associated with increased PCNA expression, ERK and STAT3 phosphorylation and elevated pro-tumorigenic immune cell expression compared to CD-fed mice. Although no significant differences in MAT weight or adipocyte size were observed after 8 weeks between the groups, metabolomics analysis of the MAT revealed that HFD-fed mice had significant upregulation of many key metabolites linked to proliferative and inflammatory pathways. For instance, HFD-fed female KC mice had significant differences in α-linolenic acid metabolism and the biosynthesis of unsaturated fatty acids, while male mice had significant differences in arginine biosynthesis, amino acid metabolism, linoleic acid metabolism, and α-linolenic acid metabolism. HFD-fed female mice had a significant increase in α-linolenic acid and linoleic acid in MAT, concurrent with a reduction in inosine 5-monophosphate and α-tocopherol compared to female CD-fed mice. HFD-fed male mice displayed a more pronounced difference in metabolites, including an increase in α-linolenic acid, linoleic acid, malic acid, glutamine, isoleucine, and several other amino acids, with a concurrent reduction in lauric acid and palmitoleic acid compared to male CD-fed mice. In summary, a HFD accelerates pancreatic carcinogenesis through multifaceted mechanisms, including effects on the tumor and surrounding adipose depots. A HFD increases affects MAT metabolism in a sex-dependent manner, that might ultimately promote early-stage progression of pancreatic carcinogenesis. Citation Format: Aya Ead, Joanna Wirkus, Gerardo G. Mackenzie. A high-fat diet accelerates early-stage murine pancreatic carcinogenesis by influencing, in part, mesenteric adipose tissue metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2184.

Abstract A065: A Western-style mimicking high-fat diet increases early stage pancreatic carcinogenesis in a KC murine model

Abstract Background: Pancreatic cancer is the 3rd leading cause of cancer-related deaths in the USA. Given the limited clinical benefit of current treatments, pancreatic cancer prevention appears as a key approach to lowering the mortality and suffering caused by this disease. A key modifiable risk factor for pancreatic cancer is the diet. Previous studies have indicated that high fat diets, but also high sugar diets, accelerate pancreatic carcinogenesis. Although the link between increased body fatness and pancreatic cancer risk is apparent, the specific role played by the different dietary fats remains unclear. Moreover, the impact of only diets rich in fats with an omega-6 to omega-3 fatty acid (FA) ratio resembling that of a Western-style diet, remains unclear. Objective: To determine the impact of a high fat diet, that mimics the Western-style diet with a ratio of about 10 parts of omega-6 FA to each omega-3 FA, on early stages of pancreatic carcinogenesis in a clinically relevant, genetically engineered LSL- KrasLSL-G12D; Ptf1aCre/+ (KC) model of pancreatic cancer. Methods: Cohorts of male and female KC mice (N= 44; 10-12 males and females per diet group) were weaned at three weeks of age, single housed, and placed on the AIN93G diet for two weeks. At five weeks of age, the KC mice were randomly assigned to either a control diet (CD) group (12% calories from fat with an omega-6:omega-3 FA ratio of 10:1) or a diet induced obesity (DIO) group (60% calories from fat with an omega-6:omega-3 FA ratio of 10:1) and fed their respective diets until three months of age. Mice were euthanized and organs and serum were collected for further analysis. Results: After 7 weeks on their respective diets, DIO-fed mice had higher body weight compared to CD-fed mice; however, it only reached statistically significance in female mice. Male and female DIO-fed mice had significantly higher fat mass, and significantly lower lean, compared to CD-fed mice. Additionally, male, but not female, DIO-fed mice had a significantly larger pancreas’ weight compared to CD-fed mice. Moreover, upon histological analysis, DIO-fed mice had a significantly increased rate of acinar-to-ductal metaplasia, compared to CD-fed mice. In addition, there were high serum concentrations of leptin, insulin as well as the pro-inflammatory cytokines IL-6, and TNF-α in KC mice fed a DIO compared to the CD-fed mice. In contrast, no differences were noted in MCP-1 and IL-1β between groups. Conclusion: Our results indicate that feeding a high-fat diet (mimicking a Western-style diet) for 7 weeks leads to increased body fatness, acinar-to-ductal metaplasia, and increased levels of leptin, insulin and pro-inflammatory cytokines. Current work is underway to characterize the cellular mechanisms on how DIO induces pancreatic carcinogenesis, as well as the specific roles played by select FA in pancreatic carcinogenesis. Citation Format: Aya Ead, Joanna Wirkus, Gerardo G. Mackenzie. A Western-style mimicking high-fat diet increases early stage pancreatic carcinogenesis in a KC murine model [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 A065.

Highlights of This Issue

Satellite RNA transcribed from tandem repeat arrays near centromeric regions in chromosomes are aberrantly and highly expressed in the early stages of pancreatic carcinogenesis. Kishikawa and colleagues generated a mouse major satellite (MajSAT) RNA-expressing mouse model and demonstrated that MajSAT RNA increases DNA damage by inhibiting stress-responded nuclear translocation of YBX1 and accelerates tumor formation in the pancreas of Kras-mutant mice, which is consistent with previous in vitro findings. This study indicates that MajSAT RNA play a role as an intrinsic mutagen in the process of pancreatic cancer development.Unrestricted replicative growth conferred by stable telomere maintenance is one of the hallmarks of cancer, and most of human cancers achieve this through the aberrant expression of telomerase. Therapeutic use of telomerase targeting agents was limited by a substantial lag phase preceding telomere shortening and the onset of cytotoxicity. This study, by Thompson and colleagues, demonstrates that telomerase facilitates the kinetics of telomere cap formation and passage through G2 phase. Transient telomerase inhibition results in a prolonged ATM-dependent DNA-damage signal that alters the cell cycle profile. This data provides the biological rationale for exploring clinical telomerase inhibition strategies that leverage telomere-length-independent mechanisms of activity.Cancer-associated adipocytes play a vital role in ovarian cancer (OC) progression. Previous studies demonstrate effective treatment of platinum-resistant OC with epigenetic therapy. However, effect of epigenetic therapy on the tumor microenvironment (TME) is not well understood. Tang and colleagues examined the effects of a DNMT inhibitor, guadecitabine, on adipocytes in the context of the tumor microenvironment. Guadecitabine treatment of adipocytes decreased OC cell migration and invasion partly through upregulation of SUSD2, a secreted tumor suppressor. These findings suggest that in addition to direct effects on cancer cells, epigenetic therapy impcats the tumor microenvironment through altered cytokine secretion to prevent OC metastasis.The nuclear receptor CAR, activated by phenobarbital, preferentially promotes hepatocellular carcinoma (HCC) development in male mice. Now growth arrest and DNA-damage-inducible 45 beta (GADD45B) is characterized as a key target of CAR in hepatocyte proliferation. CAR directly interacts and prevents GADD45B from forming a complex with MKK6 to repress the activity of the p38 MAPK tumor suppressor. Experiments with GADD45B knockout mice demonstrate that GADD45B mediates a phenobarbital-induced and male-specific hepatocyte proliferative phenotype. Thus, this study provides new mechanistic insight into phenobarbital-promoted HCC development.

Abstract 3403: Telomere shortening in pancreatic cancer is correlated to KRAS mutation

Abstract Background: Pancreatic cancer is characterized by genomic complexity and chromosomal instability, and atypical mitotic figures are morphological features of this phenotype. Previously we have reported that approximately 30% of total mitosis in pancreatic cancer was atypical including multipolar, lag-type, ring and asymmetrical mitosis, and anaphase bridges, and the number of total mitosis and atypical mitosis in pancreatic cancers was correlated with aggressive phenotype and prognosis (Pancreatology, 2016). In the present study, we clarified the relation between atypical mitotic figures, telomere length, and genetic abnormality in the pancreatic cancer. Methods: We surveyed the mitotic figures of the normal epithelium, pancreatic intraepithelial neoplasias (PanINs), and pancreatic cancers using surgically resected pancreatic cancer specimens (n=40). Telomere length was analyzed using quantitative fluorescent in situ hybridization technique. We also analyzed mutations of Kras codon 12 and 13 by PCR and microsatellite instability by immunohistochemical staining of MLH1, MSH2, MSH6 and PMS2. Results: Pancreatic cancer and duodenal epithelium showed significantly higher mitotic indices as compared with the duct, acinar cells, and PanINs. Normal mitosis was also higher in pancreatic cancers and the duodenal epithelium, while atypical mitosis was significantly elevated only in pancreatic cancers. Number of total mitosis and atypical mitosis were negatively correlated with telomere length, suggesting that telomere shortening plays important roles in cancer proliferation and chromosomal instability. In comparison with normal ducts, telomere length was decreased in PanIN-1, -2 and -3 and cancer. Furthermore, telomere length was gradually shorter among PanIN grades. Most of pancreatic cancers harbored mutations in Kras codon 12, and pancreatic cancer cases with Kras mutation showed shorter telomere length as compared with cases without Kras mutation. All pancreatic cancer cases in the present study were microsatellite stable. Conclusion: Our data strongly suggest that telomere shortening occurs in the early stages of pancreatic carcinogenesis and progresses with precancerous development. Telomere shortening and chromosomal instability in the duct epithelium plays key roles on carcinogenesis of the pancreas. Citation Format: Yoko Matsuda, Naotaka Izumiyama, Mutsunori Fujiwara, Naoshi Ishikawa, Junko Aida, Kaiyo Takubo, Toshiyuki Ishiwata, Tomio Arai. Telomere shortening in pancreatic cancer is correlated to KRAS mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3403. doi:10.1158/1538-7445.AM2017-3403

Proteasome activity is required for the initiation of precancerous pancreatic lesions.

Proteasome activity is significantly increased in advanced cancers, but its role in cancer initiation is not clear, due to difficulties in monitoring this process in vivo. We established a line of transgenic mice that carried the ZsGreen-degronODC (Gdeg) proteasome reporter to monitor the proteasome activity. In combination with Pdx-1-Cre;LSL-KrasG12D model, proteasome activity was investigated in the initiation of precancerous pancreatic lesions (PanINs). Normal pancreatic acini in Gdeg mice had low proteasome activity. By contrast, proteasome activity was increased in the PanIN lesions that developed in Gdeg;Pdx-1-Cre;LSL-KrasG12D mice. Caerulein administration to Gdeg;Pdx-1-Cre;LSL-KrasG12D mice induced constitutive elevation of proteasome activity in pancreatic tissues and accelerated PanIN formation. The proteasome inhibitor markedly reduced PanIN formation in Gdeg;Pdx-1-Cre;LSL-KrasG12D mice (P = 0.001), whereas it had no effect on PanIN lesions that had already formed. These observations indicated the significance of proteasome activity in the initiation of PanIN but not the maintenance per se. In addition, the expressions of pERK and its downstream factors including cyclin D1, NF-κB, and Cox2 were decreased after proteasome inhibition in PanINs. Our studies showed activation of proteasome is required specifically for the initiation of PanIN. The roles of proteasome in the early stages of pancreatic carcinogenesis warrant further investigation.

KLF4 Initiates Acinar Cell Reprogramming and Is Essential for the Early Stages of Pancreatic Carcinogenesis

Pancreatic ductal adenocarcinoma (PDA) has a dismal prognosis and is minimally responsive to current chemotherapies. In this issue of Cancer Cell, Xie et al. (2016) identify the transcription factor KLF4 as essential for the early stages of pancreatic carcinogenesis, expanding the repertoire of targets for early intervention strategies.

Pancreatic alteration induced by incretins is consistent with the changes at the early stages of pancreatic carcinogenesis in the hamster model

Pancreatic alteration induced by incretins is consistent with the changes at the early stages of pancreatic carcinogenesis in the hamster model

Abstract 1415: Telomere shortening in centroacinar-acinar region of the pancreas: relationships with aging, cancers and tissue stem cells

Abstract Background: Telomeres play a key role in preventing chromosomal instability. There is a striking link between advanced age and increased incidence of pancreatic cancer, suggesting a combined effect of mutation load, epigenetic regulation, telomere dysfunction, and an altered stromal milieu. Pancreatic intraepithelial neoplasia (PanIN) is a major precursor lesion of human pancreatic cancers. However, in genetically engineered mice, there is evidence that pancreatic cancer might arise in the centroacinar-acinar region, possibly through a process of acinar-to-ductal metaplasia (ADM). Determining whether ADM is a precursor lesion of human pancreatic cancers is not clear yet. In this study, we analyzed telomere length in the centroacinar-acinar region of the human pancreas with and without cancer. Methods: We used surgically resected and autopsied pancreatic specimens without cancer (n = 27) and with invasive ductal carcinoma (n = 11). We selected areas without inflammation. Immunohistochemical staining was performed for acinar, ductal, and stem cell markers. Using our quantitative fluorescence in situ hybridization (Q-FISH) technique (Takubo, et al. J Pathol, 2010), we estimated the telomere lengths of acinar and centroacinar cells. Results: Immunohistochemical staining revealed centroacinar cells with cytokeratin (CK) 19 (+)/CK7 (+)/trypsin (-)/CD133 (focal +) and acinar cells with CK19 (-)/CK7 (-)/ trypsin (+)/ CD133 (-). Telomeres in the centroacinar cells was significantly longer than in the acinar cells. In addition, telomere length in centroacinar and acinar cells decreased with age. The rate of decline in telomere length due to age was greater in centroacinar cells than in acinar cells. However, in elderly people at least 60 years of age, telomere length in the centroacinar cells did not decrease, while those in acinar cells continued to decrease after 60 years. The length of telomeres in the centroacinar and acinar cells of pancreatic cancer cases (minimum age, 60 years) was shorter than that in the cells of the cases without cancers. Discussion: Centroacinar cells expressed the stem cell marker, CD133, and had longer telomeres, suggesting that the centroacinar compartments are frequently included among cell types proposed as candidate pancreatic stem. The fact that the maximum regression of telomeres was found in centroacinar cells suggests that the number of stem cells and cells with the longest telomeres decreases with age, as previously described (Aida, et al. Exp Gerontol, 2008). Furthermore, in pancreatic cancer cases, telomere shortening in centroacinar and acinar cells occurs in the early stages of pancreatic carcinogenesis without histological changes. In conclusion, critical telomere dysfunction in the centroacinar-acinar region is possibly due to high annual telomere attrition, which leads to chromosomal instability and carcinogenesis in the pancreas. Citation Format: Yoko Matsuda, Naotaka Izumiyama-Shimomura, Toshiyuki Ishiwata, Mutsunori Fujiwara, Ken-ichiro Tomita, Naoki Hiraishi, Hideki Hamayasu, Ken-ichi Nakamura, Naoshi Ishikawa, Steven Poon, Junko Aida, Kaiyo Takubo, Tomio Arai. Telomere shortening in centroacinar-acinar region of the pancreas: relationships with aging, cancers and tissue stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1415. doi:10.1158/1538-7445.AM2015-1415

Orthotopic inflammation-related pancreatic carcinogenesis in a wild-type mouse induced by combined application of caerulein and dimethylbenzanthracene.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human malignancies, with a poor long-term prognosis, and effective therapeutic options are lacking. Observing the dynamics of the pathogenesis of pancreatic intraepithelial neoplasia (PanIN) and PDAC in tumor models can facilitate understanding of the molecular mechanisms involved in early PDAC. Furthermore, it can compensate for the research limitations associated with analyzing clinical specimens of late-stage PDAC. In this study, we orthotopically treated the pancreas with dimethylbenzanthracene (DMBA) combined with caerulein in wild-type C57BL/6 J mice to induce inflammation-related pancreatic carcinogenesis. We observed that DMBA and caerulein treatment induced a chronic consumptive disease, which caused a decrease in the relative body and pancreas weights, diminishing the health status of the mice and enhancing the inflammation-related histological changes. Moreover, mid-dose and high-frequency treatment with caerulein caused prolonged inflammatory damage to the pancreas and contributed to a permissive environment for the development of PDAC. CXCL12/CXCR4, CCL2/CCR2, and several cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were upregulated in the tumor tissue of DMBA and caerulein-induced PDAC mice. This orthotopic mouse pancreatic carcinogenesis model mimic human disease because it reproduces a spectrum of pathological changes observed in human PDAC, ranging from inflammatory lesions to pancreatic intraepithelial neoplasia. Thus, this mouse model may improve the understanding of molecular mechanisms underlying the injury-inflammation-cancer pathway in the early stages of pancreatic carcinogenesis.

Gradual telomere shortening and increasing chromosomal instability among PanIN grades and normal ductal epithelia with and without cancer in the pancreas.

A large body of evidence supports a key role for telomere dysfunction in carcinogenesis due to the induction of chromosomal instability. To study telomere shortening in precancerous pancreatic lesions, we measured telomere lengths using quantitative fluorescence in situ hybridization in the normal pancreatic duct epithelium, pancreatic intraepithelial neoplasias (PanINs), and cancers. The materials employed included surgically resected pancreatic specimens without cancer (n = 33) and with invasive ductal carcinoma (n = 36), as well as control autopsy cases (n = 150). In comparison with normal ducts, telomere length was decreased in PanIN-1, −2 and −3 and cancer. Furthermore, telomeres were shorter in cancer than in PanIN-1 and −2. Telomere length in cancer was not associated with histological type, lesion location, or cancer stage. PanINs with or without cancer showed similar telomere lengths. The incidences of atypical mitosis and anaphase bridges, which are morphological characteristics of chromosomal instability, were negatively correlated with telomere length. The telomeres in normal duct epithelium became shorter with aging, and those in PanINs or cancers were shorter than in age-matched controls, suggesting that telomere shortening occurs even when histological changes are absent. Our data strongly suggest that telomere shortening occurs in the early stages of pancreatic carcinogenesis and progresses with precancerous development. Telomere shortening and chromosomal instability in the duct epithelium might be associated with carcinogenesis of the pancreas. Determination of telomere length in pancreatic ductal lesions may be valuable for accurate detection and risk assessment of pancreatic cancer.

CD271+ Subpopulation of Pancreatic Stellate Cells Correlates with Prognosis of Pancreatic Cancer and Is Regulated by Interaction with Cancer Cells

Pancreatic stellate cells (PSCs) play a crucial role in the aggressive behavior of pancreatic cancer. Although heterogeneity of PSCs has been identified, the functional differences remain unclear. We characterized CD271+ PSCs in human pancreatic cancer. Immunohistochemistry for CD271 was performed for 31 normal pancreatic tissues and 105 pancreatic ductal adenocarcinomas (PDACs). We performed flow cytometry and quantitative RT-PCR, and assessed CD271 expression in PSCs isolated from pancreatic tissues and the changes in CD271 expression in PSCs cocultured with cancer cells. We also investigated the pattern of CD271 expression in a SCID mouse xenograft model. In the immunohistochemical analyses, the CD271-high staining rates in pancreatic stroma in normal pancreatic tissues and PDACs were 2/31 (6.5%) and 29/105 (27.6%), respectively (p = 0.0069). In PDACs, CD271+ stromal cells were frequently observed on the edge rather than the center of the tumors. Stromal CD271 high expression was associated with a good prognosis (p = 0.0040). Flow cytometric analyses demonstrated CD271-positive rates in PSCs were 0–2.1%. Quantitative RT-PCR analyses revealed that CD271 mRNA expression was increased in PSCs after coculture with pancreatic cancer cells. However, the level of CD271 mRNA expression subsequently decreased after the transient increase. Furthermore, CD271 mRNA expression was decreased in PSCs migrating toward pancreatic cancer cells through Matrigel. In the xenograft model, CD271+ PSCs were present at tumor margins/periphery and were absent in the tumor core. In conclusion, CD271 was expressed in PSCs around pancreatic tumors, but not in the center of the tumors, and expression decreased after long coculture with pancreatic cancer cells or after movement toward pancreatic cancer cells. These findings suggest that CD271+ PSCs appear at the early stage of pancreatic carcinogenesis and that CD271 expression is significantly correlated with a better prognosis in patients with PDAC.

Oct4 and Nanog Expression Is Associated With Early Stages of Pancreatic Carcinogenesis

To characterize the role of Oct4 and Nanog, two important homeobox transcription factors of embryonic development, in pancreatic carcinogenesis. Using a tissue microarray of human pancreatic carcinoma and adjacent noncancerous tissues as well as the N-nitrosobis(2-oxopropyl)amine-induced Syrian golden hamster pancreatic cancer model, we characterized the expression of Oct4 and Nanog. The presence of K-ras mutation with the time course of carcinogenesis in hamster model was also evaluated. Oct4 expression in metaplastic ducts was significantly stronger than in normal acini and pancreatic carcinoma (P < 0.05). Of 24 cases, 19 (79.2%) showed a strong Oct4 expression in metaplastic ducts. In contrast, only 6 (19.4%) of 31 cancer tissues and 3 (16.7%) of 18 noncancer tissues showed a strong Oct4 expression. Nanog also showed similar patterns as Oct4. Restriction fragment length polymorphism-polymerase chain reaction showed the overt K-ras mutation after the expression of Oct4 in the hamster model. The strong expression of Oct4 and Nanog in metaplastic ducts and Oct4 expression preceding Ras mutation suggests that these homeobox transcription factors are associated with the early stage of pancreatic cancer carcinogenesis and may play an important role in that process.

S100A11, A Putative Tumor Suppressor Gene, Is Overexpressed in Pancreatic Carcinogenesis

Recent microarray analyses revealed that expression of S100A11 is up-regulated in pancreatic cancer. The aim of the present study was to evaluate the association of S100A11 with pancreatic carcinogenesis. We measured S100A11 mRNA expression in various clinical samples related to pancreatic cancer and its precursor lesions, intraductal papillary mucinous neoplasm (IPMN) and pancreatic intraepithelial neoplasia, by quantitative reverse transcription-PCR. Levels of S100A11 were significantly higher in pancreatic cancer (n=22) and IPMN (n=18) bulk tissues than in nonneoplastic bulk tissues (n=22; P<0.0001 for both). Levels of S100A11 did not differ between pancreatic cancer and IPMN bulk tissues. In microdissection analyses, however, IPMN cells (n=21) expressed significantly higher levels of S100A11 than did cancer cells (n=23; P=0.003). The median level of S100A11 expression was higher in pancreatic intraepithelial neoplasia cells (n=6) than in cancer cells. In pancreatic juice analyses, cancer-related (n=24; P=0.004) and IPMN-related (n=18; P=0.001) juice expressed significantly higher levels of S100A11 than did chronic pancreatitis-related juice (n=23). The present data suggest that expression of S100A11, a putative tumor suppressor gene, is increased in the early stage of pancreatic carcinogenesis and decreased during subsequent progression to cancer. Analysis of the S100A11 level in pancreatic juice may be an effective tool for screening of patients with high-risk lesions that could progress to pancreatic cancer or detecting early-stage pancreatic cancer.

Overexpression of c-met in the early stage of pancreatic carcinogenesis; altered expression is not sufficient for progression from chronic pancreatitis to pancreatic cancer

To investigate c-met expression during early pancreatic carcinogenesis. We used 46 bulk tissues and 36 micro-dissected samples, including normal pancreas, chronic pancreatitis, and pancreatic cancer, for quantitative real-time reverse transcription-polymerase chain reaction. In bulk tissue analyses, pancreatic cancer tissues expressed significantly higher levels of c-met than did chronic pancreatitis and normal pancreas tissues. c-met levels did not differ between chronic pancreatitis and normal pancreas tissues. In microdissection-based analyses, c-met was expressed at higher levels in microdissected pancreatic cancer cells and pancreatitis-affected epithelial cells than in normal ductal epithelial cells (both, P < 0.01). Interestingly, pancreatitis-affected epithelial cells expressed levels of c-met similar to those of pancreatic cancer cells. Overexpression of c-met occurs during the early stage of pancreatic carcinogenesis, and a single alteration of c-met expression is not sufficient for progression of chronic pancreatitis-affected epithelial cells to pancreatic cancer cells.

Chronic exposure of transforming growth factor beta 1 confers a more aggressive tumor phenotype through downregulation of p21WAF1/CIP1 in conditionally immortalized pancreatic epithelial cells

BackgroundRecent studies have demonstrated that transforming growth factor beta 1 (TGF-β1) expression is markedly enhanced in invasive ductal pancreatic adenocarcinomas, although the precise role of TGF-β1 in pancreatic carcinogenesis remains unclear. We analyzed TGF-β1 expression in pancreatic intraepithelial neoplasias (PanINs) and the effects of chronic TGF-β1 exposure on conditionally immortalized pancreatic epithelial (IMPE) cells. MethodsSixty-one PanIN lesions were immunohistochemically stained with a polyclonal rabbit antibody against human TGF-β1. Growth-inhibitory effects of short-term exposure to TGF-β1 were examined in IMPE cells. IMPE cells resistant to TGF-β1 (IMPE-Tr cells) were generated by continuous exposure to 1 ng/mL of TGF-β1 for more than 50 days. Phenotypic alterations of IMPE-Tr cells were examined by soft agar and Matrigel assay and Western blot analysis. IMPE and IMPE-Tr cells were injected subcutaneously into nude mice for an in vivo tumorigenicity assay. ResultsForty-six percent of PanINs (28/61) were positive for TGF-β1 expression, whereas all the epithelia of normal pancreatic ducts were negative. TGF-β1 treatment showed the marked growth-inhibitory effects (>75%) in IMPE cells, whereas its effects were not observed in IMPE-Tr cells. IMPE-Tr cells were more spindle shaped compared with IMPE cells. In soft agar and Matrigel, formations of many colonies were observed in IMPE-Tr cells, but not in IMPE cells. Interestingly, the expression of p21WAF1/CIP1 was induced by short-term exposure to TGF-β1 in IMPE cells, whereas the induction was decreased in IMPE-Tr cells. All of the IMPE-Tr cell-injected mice (5/5) had subcutaneous tumors, although no tumor was found in the IMPE cell-injected mice. ConclusionsTGF-β1 expression in PanINs and neoplastic transformation of IMPE cells by long-term exposure to TGF-β1 suggest that TGF-β1 may act as a tumor promoter in the early stage of pancreatic carcinogenesis.

Characterization of Gene Expression Profiles in Intraductal Papillary-Mucinous Tumors of the Pancreas

The molecular pathology of precursor lesions leading to invasive pancreatic ductal adenocarcinomas remains relatively unknown. We have applied cDNA microarray analysis to characterize gene expression profiles in a series of intraductal papillary-mucinous tumors (IPMTs) of the pancreas, which represents one of the alternative routes of intraepithelial progression to full malignancy in the pancreatic duct system. Using a cDNA microarray containing 4992 human genes, we screened a total of 13 IPMTs including nine noninvasive and four invasive cases. Expression change in more than half of the tumors was observed for 120 genes, ie, 62 up-regulated and 58 down-regulated genes. Some of the up-regulated genes in this study have been previously described in classical pancreatic carcinomas such as lipocalin 2, galectin 3, claudin 4, and cathepsin E. The most highly up-regulated genes in IPMTs corresponded to three members of the trefoil factor family (TFF1, TFF2, and TFF3). Immunohistochemistry performed on five genes found to be differentially expressed at the RNA level (TFF1, TFF2, TFF3, lipocalin 2, and galectin 3) showed a good concordance between transcript level and protein abundance, except for TFF2. Hierarchical clustering organized the cases according to the dysplastic and invasive phenotype of theIPMTs. This analysis has permitted us to implicate several genes (caveolin 1, glypican 1, growth arrest-specific 6 protein, cysteine-rich angiogenic inducer 61) in tumor progression. The observation that several genes are differentially expressed both in IPMTs and pancreatic carcinomas suggests that they may be involved at an early stage of pancreatic carcinogenesis.

K-ras gene mutations and loss of heterozygosity at the p53 gene locus relative to histological characteristics of mucin-producing tumors of the pancreas

Mucin-producing tumors (MPTs) of the pancreas accompanied by carcinomas usually include various grades of dysplasia in the ductal epithelium, and invasive areas are histologically similar to those of common invasive ductal carcinomas, suggesting that MPTs provide a good tool to investigate early stages of pancreatic carcinogenesis. Thus, to clarify genetic alterations in the early stage of pancreatic carcinogenesis, we analyzed K-ras gene mutations and loss of heterozygosity (LOH) at the p53 gene locus using 37 cases of MPTs harboring dysplastic epithelium. Further, we conducted an extended, multifocal microdissection analysis focusing on the histological features of ductal epithelium and the distribution of genetic alterations for 3 cases of MPT positive for LOH of the p53 gene to determine the relation to tumor progression. K-ras gene mutations were detected with high frequency in 50% or more cases of the adenomas (14 of 19), borderline tumors (4 of 7), and carcinomas (8 of 11), whereas LOH of the p53 gene was limited to carcinomas (3 of 5 informative cases, 60%) and always accompanied by K-ras gene mutation. Investigation of a total of 126 microdissection sites from 3 cases showed the presence of K-ras gene mutations in mild dysplasia and all (100%) regions of moderate or more marked dysplasia, whereas LOH of the p53 gene showed more gradual tendency to increase with grade from moderate dysplasia. In addition, the multifocal genetic analysis showed K-ras gene mutations to be widely distributed throughout tumors, whereas LOH of the p53 gene was localized to 1 or a few areas. Further, topographically delimited areas with the same histology in the same tumor did not always show the same genetic alteration. In conclusion, we could confirm that both the K-ras and p53 gene alterations occur in the intraductal stage of MPT, and the latter is superimposed on the former during the course of tumor progression. However, the pattern of association of histological features with genetic alteration differs from tumor to tumor. HUM PATHOL 31:795-803.

A rat model of pancreatic ductal adenocarcinoma: Targeting chemical carcinogens

Background. Current experimental models of pancreatic cancer either fail to reproduce the ductal phenotype or cause simultaneous cancers in other organs also. To develop an animal model of pancreatic cancer that accurately mimics the human condition, we restricted carcinogenic exposure to the pancreas and specifically targeted ductal epithelial cells. Three different carcinogens were either implanted directly into the pancreas or infused into the pancreatic duct, with or without near-total pancreatectomy (as a means of inducing pancreatic ductal cell proliferation). Methods. Groups of male Sprague-Dawley rats were exposed to varying doses of dimethylbenzanthracine (DMBA), methynitronitrosoguanidine, or ethylnitronitrosoguanidine either through direct implantation into the pancreas or infusion into the pancreatic duct. Near-total pancreatectomy was added in all groups except two DMBA implantation groups. Surviving rats were killed at 3, 6, 9, or 12 months, and the pancreata were evaluated histologically. Results. All three carcinogens caused pancreatic inflammation, ductal hyperplasia, atypia, and dysplasia beginning by 3 months and becoming more prominent at later time points. Only DMBA caused frequent invasive pancreatic ductal adenocarcinoma, which was first evident by 6 months. The prevalence, of pancreatic cancer among DMBA-treated rats evaluated after 10 months was 39% (19 of 49). The addition of pancreatic resection did not enhance pancreatic cancer development. Conclusions. Of the strategies tested, only direct implantation of DMBA into the rat pancreas frequently produces pancreatic cancer histologically similar to human ductal adenocarcinoma. The development of hyperplastic, atypical, and dysplastic changes preceding and accompanying carcinomas suggests that these lesions are preneoplastic. This model recapitulates the progression from normal to neoplastic epithelium and is likely to be useful for the study of morphologic and molecular mechanisms underlying the early stages of pancreatic carcinogenesis and for the investigation of novel diagnostic and therapeutic techniques.