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The chromatin regulator Brg1 suppresses formation of intraductal papillary mucinous neoplasm and pancreatic ductal adenocarcinoma

Nature Cell Biology volume 16, pages 255–267 (2014)Cite this article

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Abstract

Pancreatic ductal adenocarcinoma (PDA) develops through distinct precursor lesions, including pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasia (IPMN). However, genetic features resulting in IPMN-associated PDA (IPMN–PDA) versus PanIN-associated PDA (PanIN-PDA) are largely unknown. Here we find that loss of Brg1, a core subunit of SWI/SNF chromatin remodelling complexes, cooperates with oncogenic Kras to form cystic neoplastic lesions that resemble human IPMN and progress to PDA. Although Brg1-null IPMN–PDA develops rapidly, it possesses a distinct transcriptional profile compared with PanIN-PDA driven by mutant Kras and hemizygous p53 deletion. IPMN–PDA also is less lethal, mirroring prognostic trends in PDA patients. In addition, Brg1 deletion inhibits Kras-dependent PanIN development from adult acinar cells, but promotes Kras-driven preneoplastic transformation in adult duct cells. Therefore, this study implicates Brg1 as a determinant of context-dependent Kras-driven pancreatic tumorigenesis and suggests that chromatin remodelling may underlie the development of distinct PDA subsets.

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Figure 1: Loss of Brg1 leads to reduced pancreas size and duct dilations.
Figure 2: Loss of Brg1 cooperates with Kras to form neoplastic cystic lesions.
Figure 3: Cystic neoplastic lesions resemble human IPMNs but not MCNs.
Figure 4: Molecular characterization of neoplastic cystic lesions.
Figure 5: IPMN lesions progress to form PDA with short latency but carry a much better prognosis for survival than PanIN-PDAs.
Figure 6: IPMN–PDA cells are intrinsically less proliferative than PanIN-PDA cells and carry a distinct molecular profile.
Figure 7: Brg1 ablation abrogates PanIN formation from adult acinar cells.
Figure 8: Brg1 ablation sensitizes adult duct cells to Kras-driven initiation of IPMN-like lesions.

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Acknowledgements

We thank C. Wright for sharing Ptf1a–Cre and Ptf1a–CreER mice, D. Tuveson for KrasG12D mice, and P. Chambon and D. Reisman for Brg1flox mice, respectively. We thank C. Austin and D. Ngow for tissue processing and excellent technical assistance and all M.H. laboratory members for helpful discussion. Work in M.H.’s laboratory was supported by a grant from the NIH (CA112537). G.v.F. was supported by a post-doctoral Research Fellowship from the Deutsche Forschungsgemeinschaft (DFG, FI 1719/1-1) and a Klein Family Foundation Fellowship. A.F. was supported by a post-doctoral Research Fellowship from the Japan Society for the Promotion of Science, a Fellowship from the US National Pancreas Foundation, and a Fellowship from the Kato Memorial Biosciences Foundation. M.E.L. was supported by CIRM training grant TG2 01153. W.F.M., A.B. and K.J.H. were supported by a grant from the NIH (CA149548). Image acquisition was supported by the imaging core of the UCSF Diabetes and Endocrinology Research Center (DERC) NIH grant P30DK63720.

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Author notes

  1. Guido von Figura, Akihisa Fukuda, Nilotpal Roy and Muluye E. Liku: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Medicine, Diabetes Center, University of California, San Francisco, 513 Parnassus Avenue, HSW 1116, Box 0540 San Francisco, California 94143, USA,

    Guido von Figura, Akihisa Fukuda, Nilotpal Roy, Muluye E. Liku, John P. Morris IV, Holger A. Russ & Matthias Hebrok

  2. II. Medizinische Klinik, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany

    Guido von Figura

  3. Department of Gastroenterology and Hepatology, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan

    Akihisa Fukuda

  4. Department of Pathology, University of California, San Francisco, San Francisco, California 94143, USA

    Grace E. Kim

  5. Department of Surgery,

    Matthew A. Firpo & Sean J. Mulvihill

  6. Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84115, USA

    Matthew A. Firpo & Sean J. Mulvihill

  7. Department of Pathology, University of California, Los Angeles, California 90095, USA

    David W. Dawson

  8. Department of Medicine, Imperial College London, W12 ONN London, UK

    Jorge Ferrer

  9. Department of Microbiology & Molecular Genetics, University of California, Irvine, California 92697, USA

    William F. Mueller, Anke Busch & Klemens J. Hertel

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Contributions

G.V.F., A.F., N.R. and M.E.L. contributed to equal parts. G.V.F., A.F., N.R. and M.E.L. carried out all experiments and were involved together with M.H. in design and analysis of the experiments. G.V.F., A.F., N.R., M.E.L. and M.H. drafted the manuscript. J.P.M.I.V. generated cell lines, contributed to the survival analysis, was involved in experimental analysis, and critically reviewed the manuscript. G.E.K. performed the histopathological analysis including IPMN and tumour identification and tumour grading. H.R. performed quantification of tumour proliferation. J.F. generated the HNF1b–CreERT2 mice. D.W.D. analysed Brg1 expression on human samples. M.A.F., S.J.M. and J.F. provided intellectual contribution to this study. M.F.W., A.B. and K.J.H. carried out deep sequencing analyses. M.H. conceived the study.

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Correspondence to Matthias Hebrok.

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Integrated supplementary information

Supplementary Figure 1 Cystic lesions in Ptf1a-Cre; KrasG12D; Brg1f/f pancreata are marked by thin stroma and are reminiscent of human pancreatobiliary IPMN.

(a) Representative H&E staining of a cystic lesion in Ptf1a-Cre; KrasG12D; Brg1f/f pancreata reveals thin underlying stroma (S = stroma, E = epithelium). Despite some variability the majority of cystic lesions in Ptf1a-Cre; KrasG12D; Brg1f/f mice presented with thin stroma lacking cells with wavy nuclei. (b) H&E staining of representative fibrovascular bundle in Ptf1a-Cre; KrasG12D; Brg1f/f pancreata. (c) Cystic lesions of Ptf1a-Cre; KrasG12D; Brg1f/f and PanINs of Ptf1a-Cre; KrasG12D mice stain positive for Muc5AC (a’–e’), and Muc1 (a”’–e”’), but are negative for Muc2 (a”–e”). In contrast, ducts of control mice do not express Muc5AC and Muc2 (a’, a”, a”’). The mucin expression pattern of the cystic lesions in Ptf1a-Cre; KrasG12D; Brg1f/f pancreata (positivity for Muc1, and Muc5AC and negativity for Muc2) matches that of human IPMNs of the pancreatobiliary type (e’, e”, e”’). (a) and (c) scale bar 50 μm, (b) scale bar 100 μm.

Supplementary Figure 2 Brg1 is lost in neoplastic epithelium of Ptf1a-Cre; KrasG12D; Brg1f/f mice and characterization of Brg1 null PDA cell lines.

(a) (a’) Immunohistochemistry staining for Brg1 on neoplastic epithelium of a 9 weeks old Ptf1a-Cre; KrasG12D; Brg1f/f mouse. Low grade dysplasia marked by the presence of abundant mucin, undulating base, nuclear enlargement, or papillary or very dilated structures, tended to be negative for Brg1. In contrast, intermediate to high-grade dysplasia was uniformly negative for Brg1. (b’) Higher magnification of a low-grade dysplastic epithelium. (c’) Higher magnification of an intermediate to high-grade dysplastic epithelium. Scale bar 250 μm. (b) (a”) PCR analysis of the KrasG12D (Kras PCR: 1–4) and Brg1f/f (Brg1 PCR: 5–9) alleles in cancer cell lines. Murine genomic DNA was isolated from the following sources 1: Kras+/+ (embryonic fibroblasts isolated from a wild type mouse). 2: unrecombined KrasG12D/+ (embryonic fibroblasts isolated from a KrasG12D/+ mouse). 3: Ptf1a-Cre; KrasG12D; p53f/+ cancer cell line. 4: Ptf1a-Cre; KrasG12D; Brg1f/f cancer cell line. 5: Brg1+/+ (tail of a wild type mouse). 6: unrecombined Brg1f/+ (tail of Brg1f/+ mouse). 7: unrecombined Brg1f/f (tail of Brg1f/f mouse). 8: Ptf1a-Cre; KrasG12D; p53f/+ cancer cell line. 9: Ptf1a-Cre; KrasG12D; Brg1f/f cancer cell line. wt = wt allele, flox = unrecombined floxed allele, rec = recombined floxed allele. (b”) Western blot analysis of Brg1 in cancer cell lines derived from IPMN- and PanIN-PDAs. (c) Anoikis analysis of PanIN-PDA (n = 3), IPMN–PDA (n = 3 independent experiments) and Ptf1a-Cre; KrasG12D (n = 3 independent experiments) derived cancer cells by Annexin V/PI staining. 200,000 Cells were seeded onto poly-hema coated petri dishes to inhibit cell adhesion. After 48 h, detachment induced cell death or anoikis was assayed by measuring both early and late apoptosis. Total apoptosis is measured by counting both Annexin V single positive cells (early apoptotic) and Annexin V/PI double positive cells are (late apoptotic). Values are shown mean + /− SD. p value for total apoptosis was calculated by one way ANOVA between three sets of cell lines.

Supplementary Figure 3 Tumour suppressor gene expression in PDA and PDA precursor lesions.

(a) Immunohistochemistry staining for p53, p21, and p16 on pancreatic tissue isolated from PanIN-PDA and IPMN–PDA in mice. Scale bars 50 μm. (b) Immunohistochemistry for p53, p21, and p16 in ADM/PanIN and IPMN neoplastic precursor lesions on pancreatic sections derived from Ptf1a-Cre; KrasG12D and Ptf1a-Cre; KrasG12D; Brg1f/f mice. Insets show higher magnification pictures of PanIN or IPMN lesions. Scale bars 50 μm. (c) Quantification of p16 positive PDA cells in PanIN- versus IPMN–PDA (n = 7 tumours; values are shown as mean ± s.e.m. unpaired t-test was used for calculating p values). (d) Summary of tumour suppressor gene expression in cancer and precursor lesions of the respective genotypes. (e) Real-time PCR (RT-PCR) for Hmga2 relative to Cyclophilin A in murine pancreas containing PanIN (from Ptf1a-Cre; KrasG12D mice;n = 3) or IPMN (from Ptf1a-Cre; KrasG12D; Brg1f/f mice;n = 3) lesions. Values are shown mean ± s.e.m. Unpaired t-test was performed to calculate the p value.

Supplementary Figure 4 Brg1 null PDA cells display a gene pathway signature indicative of lower malignant potential.

Gene pathway/function analysis displaying the deep sequencing results of PanIN-PDA versus Brg1 null IPMN–PDA using Ingenuity®; software. The analysis was performed by focusing on those genes with significantly altered expression levels (p < 0.05) between PanIN- and IPMN–PDA. (a) Depicted is the heatmap clustering of the affected genes grouped into categories of cellular function. Highlighted in green are gene signatures with a z-score <= −2. The z-score reflects the significance and direction of the deviation of the individual gene signature from the mean. Category 1 = Cellular Movement, 2 = Hematological System Development and function, 3 = Cell to cell signalling and interaction, 4 = Tissue Development, 5 = Immune Cell Trafficking, 6 = Cancer, 7 = Cardiovascular system development and function, 8 = Inflammatory response, 9 = Cellular growth and proliferation, 10 = Cellular development, 11 = Organismal injury and abnormalities, 12 = Tissue morphology, 13 = Skeletal and muscular system development and function, 14 = Gastrointestinal diseases, 15 = Antigen presentation, 16 = Hepatic system disease, 17 = Infectious disease. (b) List of the 15 most significantly down-regulated pathways in IPMN–PDA.

Supplementary Figure 5 Sequence alignment of promoter regions.

Sequence alignment of promoter regions from mouse and human. Peak heights indicate degree of homology. Pink horizontal lines indicate evolutionary conserved regions. +1 indicates the start site. Black boxes are regions analyzed by ChIP. Blue: Coding exons, Yellow: Untranslated region, Red: Promoter elements, Salmon: Intronic region.

Supplementary Figure 6 ChIP analysis of promoter regions in PanIN- and IPMN–PDA cells.

(a) Relative fold enrichment of H3K4Me3 and H3K27Me3 (over IgG control) on promoter regions in PanIN-PDA cells (1 × 106 cells/ ., n = 3 independent experiments). Decreases in the solid color bars (H3K27) indicate a relative increase in active chromatin marks. Increases in the solid bars point to a relative increase in repressive marks. Each panel indicates individual cell lines. Values are shown as mean ± s.e.m. (b) Relative fold enrichment of H3K4Me3 and H3K27Me3 (over IgG control) on promoter regions in IPMN–PDA cells (1 × 106 cells/ ChIP; n = 3 independent experiments). Decreases in the solid color bars (H3K27) indicate a relative increase in active chromatin marks. Increases in the solid bars point to a relative increase in repressive marks. Each panel indicates individual cell lines. Values are shown as mean ± s.e.m.

Supplementary Figure 7 Brg1 ablation abrogates PanIN formation from adult acinar cells and does not induce duct cell atypia in the absence of oncogenic Kras.

(a) H&E and Alcian blue stainings of pancreata derived from Ptf1a-CreER; Kras, Ptf1a-CreER; Kras; Brg1f/+ (= Brg1 het) and Ptf1a-CreER; Kras; Brg1f/f (= Brg1 KO) mice 4 months after tamoxifen induction. Note the strong reduction of Alcian blue PanIN lesions in Ptf1a-CreER; Kras; Brg1f/f (= Brg1 KO) mice. (b) A representative image of a pancreatic duct of an Hnf1b-CreERT2; Brg1f/f; R26REYFP mouse 6 weeks after tamoxifen induction. A total of, 3 Hnf1b-CreERT2; Brg1f/f (± R26REYFP) mice were analyzed 6 weeks (n = 1) or 12 weeks (n = 2) after tamoxifen induction. None of the mice showed duct cell atypia on histological examination (c) YFP staining confirmed recombination upon tamoxifen administration in both the large (arrow) and small (asterisks) duct system. (a) and (b) Scale bar 100 μm, (c) scale bar 50 μm.

Supplementary Figure 8 Brg1 expression is associated with progression of human PanIN- and IPMN–PDA.

(a) Kaplan-Meier survival curve of PanIN-PDA patients with low or high Brg1 expression in tumour cells (n = 36 for low Brg1 and n = 106 for high Brg1). Brg1 expression was scored using a histoscore ranging from 0–8 (low to high expression). The cut off histoscore was 0–6 for low and 7–8 for high Brg1 expression. Log rank test, p = 0.007. Median survival was for low BRG1 = 15.1 months (95% CI 12.3-18.0) and for high BRG1 = 28.1 months (95% CI 24.3–31.8). (b) Brg1 labeling score from matched patient samples with IPMN and associated IPMN–PDA. The Brg1 expression was scored on the same section of a patient sample that contained an IPMN precursor and its associated IPMN–PDA. p value was calculated using the paired t-test; n=11 samples for IPMN precursors and n = 12 samples for IPMN–PDA, values are shown as mean ± s.e.m.

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von Figura, G., Fukuda, A., Roy, N. et al. The chromatin regulator Brg1 suppresses formation of intraductal papillary mucinous neoplasm and pancreatic ductal adenocarcinoma. Nat Cell Biol 16, 255–267 (2014). https://doi.org/10.1038/ncb2916

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