Author Affiliations: Department of Pancreatic Surgery, Union Hospital, Huazhong University of Science and Technology, Wuhan 430022, China (Zhu Z, Liu T, Han F, Zhan SD and Wang CY); Department of General Surgery, First Affiliated Hospital, University of South China, Hengyang 421001, China (Zhu Z)

Corresponding Author: Chun-You Wang, MD, Department of Pancreatic Surgery, Union Hospital, Huazhong University of Science and Technology, Wuhan 430022, China (Tel: +86-27-85351621; Fax: +86-27-85351669; Email: chunyouwang52@126.com)

 

© 2015, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(15)60331-9

Published online January 2, 2015.

 

 

Contributors: WCY proposed the study. ZZ and HF performed research. ZZ and LT wrote the first draft. ZSD collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. WCY is the guarantor.

Funding: This study was supported by a grant from the National Natural Science Foundation of China (30972903).

Ethical approval: The study was approved by the Animal Ethics Committee of Huazhong University of Science and Technology.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: 7, 12-dimethylbenzanthracene (DMBA)-induced pancreatic intraepithelial neoplasia (PanIN) and pancreatic cancer in rats provide a classic model for uncovering the molecular mechanisms underlying pancreatic cancer. However, this model has not been characterized genetically, and in particular, the major genetic alterations in the p16 gene are unknown.

 

METHODS: Lesions of PanIN and pancreatic cancer were induced with DMBA implantation in 40 rats, and control pancreatic tissue was obtained from 10 age-matched rats without exposure to DMBA. Pancreatic tissue was harvested three months after DMBA implantation and DNA was extracted. Homozygous deletions and point mutations of the p16 (exons 1 and 2) gene were detected by PCR amplification and direct sequencing.

 

RESULTS: DMBA implantation in the 40 rats induced 26 PanINs and 9 carcinomas. The overall frequency of p16 alterations in the pancreatic tissue of these rats was 42.86% (15/35), and the changes were point mutations, not homozygous deletions. p16 mutations were present in 30.77% (8/26) of the rats with PanIN and 77.78% (7/9) of the rats with carcinoma (P<0.05). The increasing incidence of p16 alterations was detected in 20.00% (1/5) of PanIN-1, 28.57% (2/7) of PanIN-2 and 35.71% (5/14) of PanIN-3 lesions.

 

CONCLUSION: Our findings indicated that p16 alteration is a common event in the carcinogenesis of this model and that the mutation pattern is analogous to that of human lesions.

 

(Hepatobiliary Pancreat Dis Int 2015;14:208-214)

 

KEY WORDS: homozygous deletion; point mutation; p16; pancreatic intraepithelial neoplasia; pancreatic carcinoma

Pancreatic cancer is the fourth leading cause of cancer-related death in the USA. Approximately 45 220 new patients with pancreatic cancer were diagnosed and 38 460 patients died in 2013.^[1] Because of the silent feature, the patients with pancreatic cancer have no symptoms until progression to an advanced stage and therefore, the mortality rate is high. The overall 5-year survival rate is only 6% after diagnosis.^[1] At the time of diagnosis, 10%-20% of the patients are eligible for potentially curative surgery. However, the 5-year survival rate for those who underwent the surgery is approximately 20%.^[2] To improve the dismal prognosis, attempts have made to explore the molecular mechanisms underlying the development of pancreatic cancer, but many of the factors underlying this disease remain elusive.

 

The recognition of precursor lesions, pancreatic intraepithelial neoplasias (PanINs), is important for early diagnosis of pancreatic cancer. Depending on the degree of architectural and cytological atypia, PanINs are classified into 3 grades: PanIN-1 (which is further subdivided into PanIN-1A and PanIN-1B), PanIN-2 and PanIN-3.^{[3, 4]} Studies^[5-7] showed that PanIN lesions represent the first stage in a multistep model leading to pancreatic carcinogenesis. Other studies^{[6, 8]} showed that pancreatic cancer is fundamentally a genetic disease caused by an inherited germline and acquired somatic mutations in cancer-associated genes in which the p16 locus of tumor tissues is nearly always altered. The p16 tumor suppressor genes are mutated in more than 90% of primary carcinomas and are considered to be an intermediate event in the evolution of pancreatic cancer.^[9-11] Moreover, loss of p16 expression has been observed in 30% of PanIN-1A, 27% of PanIN-1B, 55% of PanIN-2, and 71% of PanIN-3 lesions.^[11] Homozygous deletions and point mutations, accounting for over 80% of p16 mutations, act as major mechanisms for inactivation.^{[9, 10]} Previous studies^[12-14] using tissue specimens of primary pancreatic carcinomas showed that the most frequent mutation sites are located on exons 1 and 2. Therefore, in the current study, we focused on the exons 1 and 2 for assessment of p16 point mutations and also assessed homozygous deletions in this gene.

 

To decipher the molecular mechanisms underlying pancreatic cancer, numerous in vivo systems, including xenografts and chemically induced and genetically engineered models, have been developed.^[15] 7, 12-di-methylbenzanthracene (DMBA)-induced PanIN and pancreatic cancer, a chemically induced model that rapidly and accurately recapitulates the process of pancreatic cancer, has been generally acknowledged as a classic tool for clarifying the pathogenesis of pancreatic cancer.^{[16, 17]} This model has at least two advantages over other models. First, the DMBA model is ideal for studying genetic events induced by carcinogens in which the multistep progression occurs during the development of pancreatic cancer.^[18,19] Second, DMBA promotes an inflammation-induced carcinogenesis, characterized by the induction of cytokines, including IL-1, IL-12, IL-17, and TNF-alpha.^[20-23] Hence, DMBA-induced pancreatic cancer provides a powerful tool for unveiling the mechanism of inflammation-associated carcinogenesis.

 

The rat model shares several similar biological characteristics with humans including a ductal phenotype, the potential for local invasion and peritoneal metastasis with pancreatic cancer.^{[17, 24]} Genetic similarities include frequent and early k-ras mutations.^[24] However, to our knowledge, whether and to what extent p16 alteration is involved in this model has not been characterized.

 

To further clarify the genetic alterations contributing to this poorly understood model, we assessed the alterations of the p16 gene in DMBA-induced PanIN and pancreatic cancer in rats using PCR and DNA sequencing. The purpose of the present study was to evaluate potential homozygous deletions and point mutations in the p16 gene and to determine whether the alteration pattern in this model is consistent with that of human lesions.

 

 

Fifty male Sprague-Dawley rats weighing 150 to 200 g were purchased from the Laboratory Animal Center of Huazhong University of Science and Technology. All rats were free access to food and water and were maintained under standard conditions of temperature and humidity with an alternating 12-hour light/dark cycle. All experiments were conducted with the approval of the Animal Ethics Committee of Huazhong University of Science and Technology.

 

The DMBA induction model was established according to previously published protocol.^[25] Briefly, forty rats were anesthetized with sodium pentobarbital (40 mg/kg, intraperitoneally) and subjected to a midline laparotomy. A pocket was made in the parenchyma of the pancreatic tail, where approximately 5 mg of DMBA crystals were implanted. The pocket was closed with a 6-0 prolene purse-string suture. To obtain normal control pancreas tissue, the remaining 10 animals were left untreated and maintained free from carcinogen exposure throughout the experiment.

 

Animals were sacrificed at 3 months following DMBA implantation. After sacrifice, tissue near the implantation site and normal pancreatic tissue were harvested. Samples were divided into two parts: one was frozen and stored at -80 �� for gene analysis, and the other was fixed in 10% formalin for pathologic examination.

 

Paraffin-embedded tissue sections were stained with hematoxylin and eosin (HE). All slides were evaluated in a blinded fashion by a pathologist and assessed by the PanIN classification system described previously.^[3] Reactive hyperplasia is characterized by the presentation of an increase in the number of epithelial cells along the DMBA implantation site.^[25] Pancreatic carcinoma was diagnosed based on the presence of infiltrating irregular neoplastic glands within desmoplastic stroma.^[26] When numerous lesions were observed on the same sample, the final diagnosis was dependent on the most advanced lesion.

 

DNA was extracted from 35 samples diagnosed as PanINs or carcinomas and 10 normal controls with the TAINamp Genomic DNA Kit (Tiangen, Beijing, China). Primer sequences of p16 were as follows: p16 exon 1 forward, 5'-GTG TGG AAC CAG GTC AGG AG-3' and reverse, 5'-ATG GGA CAT TCC TTG CCT ACC-3'. p16 exon 2 forward, 5'-GGT CAC ACC ACT GGC GAC T-3' and reverse, 5'-GTA TCG GGG TAC GAC CGA AA-3'. Furthermore, we amplified beta-actin as an internal control using the following specific intron-derived primers: forward, 5'-GGC TTT AGG AGC TTG ACA ATA CTG-3' and reverse, 5'-GCA TTG GTC ACC TTT AGA TGG A-3'.^[27]

 

PCR amplification was performed in a GeneAmp PCR System 2400 thermal cycler (Perkin-Elmer, Foster City, CA, USA) with a 26 µL mixture containing template DNA, 100 nmol/L of each PCR primer (Sangon, Shanghai, China), buffer consisting of 10 mmol/L Tris-HCl (pH 8.4) and 1 mmol/L MgCl₂, 200 µmol/L of each deoxyribonucleoside triphosphate and 1.25 U of Taq polymerase (Promega, Madison, WI, USA). The PCR program consisted of preheating at 94 �� for 10 minutes; 30 cycles of 94 �� for 30 seconds, 58.5 �� for 30 seconds, and 72 �� for 1 minute; and incubation at 72 �� for 7 minutes.

 

The PCR products of p16 (exons 1 and 2) and beta-actin were electrophorased on a 1.5% agarose gel and visualized under a UV illuminator for the detection of homozygous deletions. Band intensities were quantified by ImageJ software and used to calculate the copy number of p16 (exons 1 and 2) and beta-actin. The p16 gene dosage was determined using the p16/beta-actin ratio.^[28] Then, the PCR products were purified using a TIANgel Midi Purification Kit (Tiangen) and sequenced with an ABI-PRISM 3730 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA).

 

Student's t test was used to evaluate significant differences between p16 gene dosage in normal controls and experimental lesions. Mutation frequencies were evaluated using Fisher's exact test. P<0.05 was considered statistically significant.

 

 

Among the 40 rats implanted with DMBA crystals, one who died in the early post-operative period was excluded from the study. After 3 months, histological evaluation was performed for the remaining 39 rats. Four (10.26%) of the specimens taken from the DMBA-treated group showed reactive hyperplasia, 26 (66.67%) showed PanIN lesions (classified as PanIN-1, PanIN-2 or PanIN-3 according to the criteria of Hruban et al^[3]), and 9 (23.08%) showed carcinomas (Table 1). Photomicrographs of the lesions of each type are shown in Fig. 1. PanIN-1A lesions were flat and were composed of tall columnar cells and abundant supranuclear mucin (Fig. 1B). PanIN-1B lesions had papillary projections but were otherwise identical to PanIN-1A (Fig. 1C). PanIN-2 lesions were flat or papillary with nuclear abnormalities, including nuclear crowding and pseudostratification (Fig. 1D). PanIN-3 lesions had a cribriform architecture and a loss of nuclear polarity (Fig. 1E). A classification of pancreatic carcinoma required the presence of infiltrating irregular neoplastic glands into the adjacent desmoplastic stroma (Fig. 1F).

 

To determine whether the induction of pancreatic abnormalities in the DMBA model correlates with the alterations in the p16 gene, we isolated DNA from the rats that presented with PanIN or pancreatic cancer and PCR-amplified and sequenced the p16 exons 1 and 2, which were shown to carry the most mutations in human pancreatic cancer.^[12-14] The amplification was successful for all samples, and no homozygous deletions were detected (Fig. 2). The relative amount of the p16 gene in the normal control, PanIN-1, PanIN-2, PanIN-3, and pancreatic carcinoma was not significantly different (P>0.05) (Table 2). The overall frequency of p16 alteration was 42.86% (15/35) and all of the changes were point mutations. Histological classifications showed that 30.77% (8/26) of PanINs and 77.78% (7/9) of carcinomas harbored p16 mutations (P<0.05). Similar to the results reported for human PanIN,^[11] increasing p16 mutations were observed in 20.00% (1/5) of PanIN-1, 28.57% (2/7) of PanIN-2, and 35.71% (5/14) of PanIN-3; however, these differences were not statistically significant (Table 3). Direct sequencing showed that most samples contained mutations in two or more codons, with mutations occurring at codons 2, 5, 7, 8, 18, 20, 28, 31, 33 and 35 in exon 1; and at codon 44 in exon 2 (Table 4). Alterations at codons 5, 7 and 20 were silent; however, the other mutations were predicted to cause amino acid changes or the introduction of a stop codon, suggesting a possible association of p16 mutation with the incidence and severity of PanIN and pancreatic cancer.

 

 

The experimental model of DMBA-induced PanIN and pancreatic cancer is useful for the study of the molecular mechanisms underlying the carcinogenesis of pancreatic cancer. Osvaldt et al^[25] reported that the frequency of PanIN lesions was 35% after 3 months DMBA treatment. To our knowledge, the present study was the first report of the prevalence of PanINs in the rat DMBA model. We found that the frequency of PanINs in rats was higher than Osvaldt's study in mice. The difference is perhaps due to the different species. Rat is better than mouse in the induction of PanINs. In 1997, Rivera and colleagues^[29] found that 39% of rats which exposed to DMBA for 10 months developed pancreatic cancer. Z'graggen et al^[24] reported that the frequency of carcinoma induced by DMBA in rats was 29% at 1 month, but dropped to 17% at 9 months following DMBA implantation. The inconsistence of the prevalence of carcinoma in DMBA-implanted rats was associated with various experimental conditions and the duration of induction. Interestingly, studies^{[24, 29]} showed that the incidence of carcinoma was within the range of 17%-39%.

 

Our study demonstrated that point mutations are the major mechanism for p16 alterations in the DMBA model of pancreatic cancer in rats. The prevalence is higher than that of pancreatic cancer in humans, which varies from 15% to 67.5%.^{[9, 10, 13, 30, 31]} In our study, the point mutations in PanINs were statistically lower than those in carcinomas. Interestingly, the frequency of point mutation tended to be higher in higher grade PanINs lesions. Similar to the results of carcinomas, the frequency of point mutation in PanIN-1 and PanIN-2 was somewhat higher than that of human lesions (9.6%, 20.0%).^[32] Differences in the genetic background between rats and humans and the unique chemically-induced carcinogenesis of the DMBA model may contribute to these differences in mutation incidence. Additionally, the present study is limited to an analysis of the mutations in exons 1 and 2, which are the most frequent mutation sites;^[12-14] however, it is possible that mutation may occur elsewhere in the p16 gene. Furthermore, despite the preponderance of carcinomas with mutation identified in our study, laser microdissection may provide an improved alternative for future studies of p16 mutations.^{[33, 34]} Nevertheless, the similar trends of p16 mutation in pancreatic carcinomas and PanINs suggest a fundamental similarity of the DMBA rat model to human pancreatic cancer in terms of the incidence of p16 point mutations as a mechanism of carcinogenesis.

 

Interestingly, our results were consistent with the loss of p16 expression in the background of chronic pancreatitis.^[35] PanIN, which usually occurs in chronic pancreatitis, is observed in the DMBA model as early as 4 days following implantation and is sustained throughout the process of carcinogenesis, with prominent chronic inflammation 1 month after the implantation.^[16] Pancreatitis has been shown to be a risk factor for pancreatic cancer.^{[36, 37]} Studies^{[38, 39]} showed that the combination of both genetic events and extrinsic factors that produce tissue injury, such as oxidative stress and associated inflammatory damage observed during pancreatitis, result in pancreatic cancer. Our data suggested that p16 alterations in DMBA-induced PanINs probably resulted from chronic pancreatitis in the microenvironment. Thus, the DMBA-induced rat model may be useful in elucidating the correlation between chronic pancreatitis and pancreatic cancer.

 

We did not detect any homozygous deletions in p16 in the DMBA rat model. Though p16 alterations are universally recognized in human pancreatic cancer, the prevalence of homozygous deletions is controversial. Some studies^{[9, 10]} reported that approximately 40% of pancreatic cancer cases had homozygous deletion, and another study^[13] reported no homozygous deletion. Methylation of the p16 gene promoter provides an additional mechanism of suppressing this gene by inactivating its expression. Methylation is often in conjunction with other genetic p16 alterations and in up to 50% of p16 genetic alterations.^{[10, 13, 30]} Thus, future study is necessary to identify the role of methylation in point mutations or homozygous deletions in pancreatic carcinomas.

 

In conclusion, p16 mutation is a common event in the carcinogenesis of pancreatic cancer. The mutation pattern in this model is analogous to that of human lesions. This model may provide a basis for the further study of genetic mechanisms of PanINs and pancreatic cancer.

 

 

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