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A better molecular characterization of Intraductal Papillary Mucinous Neoplasm (IPMN), the most frequent cystic precursor lesion of pancreatic adenocarcinoma, may have a pivotal role in its early detection and in the development of effective therapeutic strategies. BRG1, a central component of the chromatin remodeling complex SWI/SNF regulating transcription, is inactive in several malignancies. In this study we evaluate the Brg1 expression in IPMN in order to better understand its role in the pancreatic carcinogenesis. Tissue microarrays (TMAs) of 66 surgically resected IPMNs were immunolabeled for the Brg1 protein. Expression patterns were then correlated with clinicopathologic parameters. Normal pancreatic epithelium strongly immunolabeled for Brg1. Reduced Brg1 expression was observed in 32 (53.3%) of the 60 evaluable IPMN lesions and occurred more frequently in high-grade IPMNs (13 of 17 showed loss; 76%) compared to intermediate-grade (15 of 29 showed loss; 52%) and low-grade IPMNs (4 of 14 showed loss; 28%) (p=0.03). A complete loss of Brg1 expression was observed in 5 of the 60 (8.3%) lesions. Finally, a decrease in Brg1 protein expression was furthermore found in a low-passage non-invasive IPMN cell line by Western blot analysis. We did not observe correlation between Brg1 expression and IPMN subtype or with location of the cyst. We provide first evidence that Brg1 expression is lost in noninvasive cystic precursor lesions of pancreatic adenocarcinoma.
The expanding usage of abdominal imaging technologies is uncovering an increasing number of asymptomatic pancreatic cystic lesions, including cystic precursors of pancreatic cancer. In this scenario, Intraductal Papillary Mucinous Neoplasms (IPMNs) represent the most frequent cystic precursor lesions of pancreatic cancer and they have been associated with malignancy in about one third of patients 1–4. Although the malignant potential of IPMNs is unequivocal, the natural history of this disease is still incompletely understood.
Numerous molecular alterations that are thought to contribute to the development and progression of IPMNs have been described 5. Those include activating KRAS2 mutations 6 as well as inactivation of CDKN2A/p16 and TP53 7;8. Interestingly SMAD4/DPC4 inactivation is not usually observed in non-invasive IPMN, as opposed to pancreatic adenocarcinoma (PDAC) 9. Somatic mutations of the STK11/LKB1 gene, which is responsible for Peutz-Jeghers Syndrome, are also reported in IPMN, as well as mutations of PIK3CA, which represents a finding rarely found in classic PDAC 10;11. Furthermore, IPMN gene expression profiling studies show differential up-or down regulation of numerous genes, compared to PDAC 12. Chromosomal imbalances are also a common feature of IPMN and have been shown by global profiling approaches to accumulate gradually, in parallel with the increasing grades of dysplasia. Particular chromosomal losses (5q, 6q, 11q) are more commonly observed in high-grade IPMN compared to PDAC, whereas others (18p, 15q, 18q, 22q) occur in both entities at a similar rate 13–16.
A facilitative chromatin state, which allows for ready access to transcription factors, is essential for gene expression. Consequently, It has been shown that deregulation of the chromatin structure may have a severe impact on cellular processes and may contribute to cancer development 17
Brg1 is an ATPase/helicase and constitutes the catalytic subunit of the SWI/SNF chromatin remodeling complex. This complex has been shown to disrupt the adhesion of histone components and DNA, thereby giving transcription factors access to their target genes. Several studies have demonstrated the tumor suppressive nature of BRG1 in different types of cancer. Disruption of BRG1 was observed in several human cancer cell lines, including lung, breast, brain, colon, ovarian, and less frequently, pancreas 18. In particular, the involvement of BRG1 in lung cancers is well documented19–22.
Brg1 protein may play a role in the development of breast cancer, since it has been shown to promote growth suppression mediated by the estrogen receptor and BRCA1 interaction 23;24 Moreover, BRG1 haploinsufficient mice were shown to develop mammary tumors25.
Bi-allelic inactivating mutations in the BRG1 gene have recently been reported in a minor subset (~2%) of pancreatic cancer, suggesting that BRG1 may play a role in its development and progression 26.
Based upon this evidence, we immunolabeled a series of IPMNs for the Brg1 protein to determine if this protein may have a role in its development.
The study was approved by the Johns Hopkins Institutional Review Board. For the analysis of Brg1 protein expression we used tissue microarrays (TMA) created from 66 independent archival surgically resected IPMNs 27. These 66 IPMNs are representative of the spectrum of IPMN subtypes and grades of dysplasia, and were classified according to the most recent WHO guidelines 28. Hence, with respect to the predominant characteristics of the epithelium, all IPMNs were defined as gastric-foveolar, pancreatobiliary, intestinal or oncocytic, and, based on the maximum cytoarchitectural atypia in the intraductal component, were graded as having low-, intermediate-, or high-grade dysplasia. The presence or absence of an associated invasive adenocarcinoma was also documented. Matched non-neoplastic pancreatic tissue cores were available for the IPMNs arrayed on the TMA.
Unstained 4μm sections were cut from each tissue microarray. Immunolabeling for the Brg1 protein was performed with a 1:100 dilution of murine monoclonal anti-BRG1 antibody (clone G-7, catalog #17796, Santa Cruz Biotechnology). Labeling was performed on a Leica-Bond autostainer using biotin free polymer detection reagents (Leica Microsystems). Briefly, after deparaffinization and hydration of the tissue microarray sections, antigen retrieval was achieved in EDTA buffer (pH 9.0) for 20 minutes. Tissues were incubated with the primary antibody for 15 minutes at room temperature, followed by incubation with postprimary and polymer detection for 8 minutes each. Chromogenic detection was performed using 3,3-diaminobenzidine and the slides counterstained with hematoxylin. Slides were then dehydrated and mounted permanently for evaluation.
Immunolabeling was evaluated by four of the authors (AM, MDM, SMH, HM) on a multi-headed microscope with consensus reached in all cases. Each tissue core was assessed independently without knowledge of the patient group. Based on the degree of nuclear labeling in the neoplastic epithelium, a three-tier intensity score (0: absent or minimal; 1: weak; 2: intense) was used for evaluation. In cases with heterogeneous labeling intensity, the respective tissue core was classified according to the predominant pattern of expression in the neoplastic cells. Neoplasms were given an area score from 0 to 4 based on the proportion of cells with labeling (“0”: <5%, “1”: 5–25%, “2”: 26–50%, “3”: 51–75%, “4”: 76–100% of the cells labeled). The scores from different tissue cores of the same patient were averaged in order to obtain one case specific value. A total labeling score was calculated for each patient by multiplying intensity score and area score yielding values from 0 to 8. A total score > 3 was considered as positive whereas a total labeling score of ≤3 was regarded as negative. This cutoff was selected because the highest labeling observed in the normal pancreatic epithelium was a score of 4.
The derivation of low passage IPMN 1T cells has recently been described 29. The cells were maintained in MEM with 20% FBS (GIBCO), 5 ng/ml EGF (Promega), 0.2 U/ml human recombinant insulin (Invitrogen), and 1% penicillin streptomycin (Invitrogen). The hTERT immortalized Human Pancreatic Nestin Expressing epithelial cell line (HPNE cell line) was maintained according to established protocols 30. The human pancreatic cancer cell line Pa03C was maintained in DMEM containing 10% FBS and 1% Penicillin Streptomycin 26.
Cells grown to confluence were scraped in RIPA buffer and sonicated at high power to lyse cells. 50 μg of protein lysate was resuspended in sample buffer, separated by SDS-PAGE and transferred to nitrocellulose membranes. The membrane was blocked in TBST containing 5% powdered milk for 30 min and incubated in anti-Brg1 antibody (H-88; Santacruz) for 1 h to overnight. Blots were probed with HRP-conjugated antirabbit secondary antibody (Santacruz). HRP activity was detected using western lighting chemiluminescence reagents (Pierce).
Statistical computations were performed using SPSS version 17 (SPSS Inc., Chicago, IL, USA). Comparison of categorical expression parameters were examined using Fisher’s exact tests. Comparison of means was performed using Mann-Whitney test. A P-value of less than 0.05 was considered statistically significant.
Of the 66 IPMN lesions 60 (90.1%) were suitable for evaluation of Brg1 immunohistochemical labeling. This included 14 (23.3%) lesions with low-grade dysplasia, 29 (48.3%) with intermediate-grade dysplasia, and 17 (28.3%) with high grade dysplasia. Representative examples for each labeling category are shown in Figure 1. Eight IPMNs presented with an associated invasive pancreatic ductal adenocarcinoma, all tubular subtype. Both the IPMN and the associated infiltrating cancer were available for analysis.
Matched normal pancreatic ductal epithelium was available for 51 IPMNs (83.3%) and all normal epithelial samples were positive for Brg1 nuclear expression. A full labeling score of 8 was seen in nearly all evaluable “normal” tissues (n=45; 88.2%) (Figure 2). In contrast, Brg1 labeling was reduced in 32 of the 60 (53.3%) IPMN lesions. This loss was observed significantly more often in high grade IPMN lesions (13 of 17; 76%) compared to low grade (4 of 14; 29%) and intermediate-grade IPMNs (15 of 29; 48%; p=0.03). A complete loss of expression was noted in 5 (8.3%) lesions comprising 4 IPMNs with intermediate-grade and 1 IPMN with high-grade dysplasia We did not observe a statistical difference in Brg1 labeling scores among different histological IPMN subtypes. Furthermore, total labeling score did not correlate with tumor location within the ductal tree (main-duct vs. branch-duct vs. mixed) (Table 1). Brg1 labeling was negative in 2 out of 8 invasive cancers (25%). However, no significant difference was observed, when the mean labeling score of the cancers was compared with the mean value of their matched non-invasive IPMNs (Table 2).
To further substantiate our immunohistochemical findings in tissue sections, Western blot analysis was subsequently performed, to evaluate Brg1 expression in the low passage non-invasive IPMN cell line, IPMN 1T 29. As a positive control for a cancer with known somatic mutation of BRG1, we used the Pa03C line, as recently described in the pancreatic cancer genome sequencing effort 26. When compared with HPNE cell lines, both Pa03C and the IPMN 1T cells showed a marked and comparable decrease in Brg1 protein expression (Figure 3).
Pancreatic cancer continues to be one of the most lethal gastrointestinal malignancies 31. Currently, surgical resection of lesions detected at the earliest possible stage represents the greatest chance for cure. However, very few cancers are diagnosed at an early stage, and known precursor lesions of pancreatic cancer, such as IPMN or Mucinous Cystic Neoplasms (MCNs) do not harbor the same malignant potential in every single case, rendering their appropriate management difficult to assess. Therefore, there is a great need to better characterize the molecular biology of cystic precursor lesions such as IPMNs, in order to better stratify the management for individual patient needs.
Recently, high throughput sequencing of various human cancers have uncovered recurrent mutations in genes involved in chromatin remodeling 32;33. Vogelstein B. et al., have demonstrated that mutations of MEN1, which encodes the histone methyltransferase component menin, occur at a frequency of 44% in pancreatic neuroendocrine tumors (PanNETs). Another 43% of PanNETs harbor mutations in DAXX (death-domain–associated protein) and ATRX (a thalassemia/mental retardation syndrome X-linked) whose protein products are also involved in chromatin remodeling 34.
Brg1 is an ATPase/helicase and as such constitutes the catalytic subunit of the SWI/SNF chromatin remodeling complex. This complex disrupts the adhesion of histone components to DNA thereby giving transcription factors access to their target genes 35;36. In mice, developmental homozygous deletion of BRG1 results in peri-implantation lethality, whereas BRG1 heterozygotes are predisposed to exencephaly and apocrine like tumors 37.
Abnormalities in the expression of BRG1 have been reported in a number of different cancer types such as lung, breast, brain, colon, ovarian, and less frequently, pancreas 18;20;21;23;24;38. Functional studies have reiterated the putative tumor suppressive effects of Brg1 protein in human cancer cells. For example, knock-in of functional BRG1 in a breast cancer cell line with a mutant BRG1 allele induces growth inhibition. This inhibition appears to be at least partly mediated by the suppression of cyclin E as an E2F target gene, and the overexpression of transcripts of cyclin-dependent kinase inhibitors p21 and p15 36. It has also been shown that Brg1 itself interacts with tumor suppressor proteins (e.g. RB1, BRCA1) and components involved in Wnt signaling 24;39–45, suggesting a role for this gene not only in chromatin remodeling, but also in cell-cycle regulation and in the activity of tumor-suppressor factors.
The role of BRG1 in pancreatic carcinogenesis is poorly defined. The recent sequencing of the pancreatic cancer genome elucidated that somatic mutations of BRG1 occur in ~2% of pancreatic cancers 26. Rosson et al., found that reduction of BRG1 expression in pancreatic cancer cell lines by lentiviral short hairpin shRNA mediated RNA interference resulted in morphologic changes; however, no significant impact on in vitro growth of cultured cell lines could be observed 46.
In this study, we investigated the putative role of BRG1 in IPMNs, the most common cystic precursor lesion of pancreatic cancer, by performing immunohistochemical evaluation of BRG1 expression on IPMN tissue microarrays.
We found a progressive loss of Brg1 expression associated with increasing degrees of dysplasia in IPMNs. Only 28% of low-grade IPMNs showed loss of Brg1 expression, compared to 76% of high-grade lesions (p=0.03) (Table 1). Intermediate-grade IPMNs displayed an intermediate percentage of loss (52%), suggesting a progressive decrease of expression from low- towards high-grade lesions. Loss of Brg1 expression was found in 2 out of 8 invasive cancer present in our TMA (25%) and this pattern was highly concordant with their non-invasive counterpart. This finding substantiates the progressive loss of Brg1 during tumor progression from lesions with mild dysplasia to invasive cancer. Interestingly we did not observe a difference in Brg1 expression among different histologic subtypes nor among different locations within the ductal system. It is known that main duct IPMNs are more likely to exhibit pancreaticobiliary or intestinal features and high-grade dysplasia and are more commonly associated with an invasive carcinoma compared to branch duct lesions. However, loss of Brg1 does not appear to be involved in a phenotype differentiation nor to a preferential location within the ductal tree.
In order to extrapolate the tissue studies to cultured cell lines, especially in a recently described in vitro IPMN model 29, we assessed Brg1 expression in IPMN and PDAC cells. When compared with non-neoplastic HPNE cells, IPMN 1T showed a marked decrease in Brg1 expression, which was comparable to the “positive” control Pa03C cells, that are known to harbor a somatic mutation of BRG1. This finding corroborates our results and provides additional support to the role of Brg1 in IPMN development and progression. Further, the Pa03C and IPMN 1T cells provide a novel in vitro platform to study the functional consequences of BRG1 restitution in pancreatic neoplasia for the first time.
Our study shows that a progressive loss of immunolabeling for the Brg1 protein, confirmed by Western blot analysis on cell lines, is associated with increasing degrees of dysplasia in IPMNs. These results suggest an involvement in tumor progression and therefore contribute to the growing body of literature focusing on chromatin remodeling complexes and their role in cancer formation.
Further biological and biochemical analyses are needed to better characterize and validate the biological significance of Brg1 in pancreatic carcinogenesis.
This work was supported by R01CA113669, P50CA62924, P01CA134292, the Sol Goldman Pancreatic Cancer Research Center and the Michael Rolfe Foundation for Pancreatic Cancer Research. Hanno Matthaei is supported by a grant from the Mildred-Scheel-Stiftung, Deutsche Krebshilfe, Bonn, Germany.
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