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. 2018 Aug 1;128(8):3475-3489.
doi: 10.1172/JCI94287. Epub 2018 Jul 16.

The BRG1/SOX9 Axis Is Critical for Acinar Cell-Derived Pancreatic Tumorigenesis

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Free PMC article

The BRG1/SOX9 Axis Is Critical for Acinar Cell-Derived Pancreatic Tumorigenesis

Motoyuki Tsuda et al. J Clin Invest. .
Free PMC article

Abstract

Chromatin remodeler Brahma related gene 1 (BRG1) is silenced in approximately 10% of human pancreatic ductal adenocarcinomas (PDAs). We previously showed that BRG1 inhibits the formation of intraductal pancreatic mucinous neoplasm (IPMN) and that IPMN-derived PDA originated from ductal cells. However, the role of BRG1 in pancreatic intraepithelial neoplasia-derived (PanIN-derived) PDA that originated from acinar cells remains elusive. Here, we found that exclusive elimination of Brg1 in acinar cells of Ptf1a-CreER; KrasG12D; Brg1fl/fl mice impaired the formation of acinar-to-ductal metaplasia (ADM) and PanIN independently of p53 mutation, while PDA formation was inhibited in the presence of p53 mutation. BRG1 bound to regions of the Sox9 promoter to regulate its expression and was critical for recruitment of upstream regulators, including PDX1, to the Sox9 promoter and enhancer in acinar cells. SOX9 expression was downregulated in BRG1-depleted ADMs/PanINs. Notably, Sox9 overexpression canceled this PanIN-attenuated phenotype in KBC mice. Furthermore, Brg1 deletion in established PanIN by using a dual recombinase system resulted in regression of the lesions in mice. Finally, BRG1 expression correlated with SOX9 expression in human PDAs. In summary, BRG1 is critical for PanIN initiation and progression through positive regulation of SOX9. Thus, the BRG1/SOX9 axis is a potential target for PanIN-derived PDA.

Keywords: Cancer; Epigenetics; Gastroenterology; Mouse models; Oncology.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Acinar-specific ablation of Brg1 attenuates oncogenic KRAS-driven ADM and PanIN formation.
(A) Immunohistochemistry for BRG1 in adult Ptf1a-Cre; KrasG12D; Trp53R172H mice. Scale bars: 50 μm. (B) The genetic strategy for determining the efficiency of acinar cell–specific Brg1 deletion following tamoxifen (Tam) induction and the experimental design for tamoxifen administration and analysis. (C) Deletion rate of BRG1 in Ptf1a-CreER; Brg1fl/fl mice at 3 weeks after tamoxifen administration. n = 3 mice. Data are shown as mean ± SEM. (D) The genetic strategy used to delete Brg1 and activate oncogenic Kras in adult pancreatic acinar cells and the experimental design for tamoxifen administration and analysis. (E) H&E staining and immunohistochemistry for BRG1 with Alcian blue and phospho-ERK staining in Ptf1a-CreER; KrasG12D; Brg1fl/fl mice with littermate controls. Scale bars: 50 μm. (F) Quantification of Alcian blue–negative ADM-like lesions and Alcian blue–positive late ADMs and PanINs in Ptf1a-CreER; KrasG12D; Brg1fl/fl mice with littermate controls. Red bars show incidence of BRG1-negative late ADMs and PanINs. n = 3–4 mice per genotype. Data are shown as mean ± SEM. *P < 0.05, Student’s t test. (G) Quantification of PanINs in Ptf1a-CreER; KrasG12D; Brg1fl/fl mice with littermate controls. Claudin-18–positive area was counted. Red bars show incidence of BRG1-negative PanINs. n = 4 mice per genotype. Data are shown as mean ± SEM. *P < 0.05, Student’s t test.
Figure 2
Figure 2. Acinar-specific ablation of Brg1 attenuates acute pancreatitis–induced PanIN formation in an oncogenic KRAS background.
(A) Schematic showing experimental design for tamoxifen administration and caerulein-induced acute pancreatitis in Ptf1a-CreER; KrasG12D; Brg1fl/fl mice. (B) H&E and Alcian blue staining and immunohistochemistry for BRG1 and phospho-ERK in Ptf1a-CreER; KrasG12D; Brg1fl/fl mice and littermate controls at 21 days after caerulein induction. Scale bars: 50 μm. (C) Immunohistochemistry for BRG1 in Ptf1a-CreER; KrasG12D; Brg1fl/fl mice at 21 days after caerulein induction. Representative BRG1-positive and BRG1-negative PanINs. Scale bars: 50 μm. (D) Quantification of Alcian blue–positive area in Ptf1a-CreER; KrasG12D; Brg1fl/fl mice with littermate controls. Red bars show incidence of BRG1-negative late ADMs and PanINs. n = 4 mice per genotype. Data are shown as mean ± SEM. *P < 0.05, Student’s t test.
Figure 3
Figure 3. Acinar-specific ablation of Brg1 attenuates spontaneous PanIN formation in a background of oncogenic KRAS and mutant p53.
(A) The genetic strategy used to delete Brg1 and activate oncogenic Kras and mutant p53 in adult pancreatic acinar cells in addition to experimental design for tamoxifen administration and analysis. (B) H&E and Alcian blue staining and immunohistochemistry for phospho-ERK and p53 in Ptf1a-CreER; KrasG12D; Trp53R172H; Brg1fl/fl mice and littermate controls. Scale bars: 50 μm. (C) Immunohistochemistry for BRG1 in Ptf1a-CreER; KrasG12D; Trp53R172H; Brg1fl/fl mice. Representative BRG1-positive and BRG1-negative PanINs. Scale bars: 50 μm. (D) Quantification of Alcian blue–positive late ADMs and PanINs in Ptf1a-CreER; KrasG12D; Trp53R172H; Brg1fl/fl mice with littermate controls. Red bars show incidence of BRG1-negative late ADMs and PanINs. n = 3 mice per genotype. Data are shown as mean ± SEM. *P < 0.05, Student’s t test. (E) Quantification of claudin-18–positive PanIN area in Ptf1a-CreER; KrasG12D; Trp53R172H; Brg1fl/fl mice with littermate controls. Red bars show incidence of BRG1-negative PanINs. n = 3 mice per genotype. Data are shown as mean ± SEM. *P < 0.05, Student’s t test.
Figure 4
Figure 4. BRG1 promotes Sox9 expression in murine ADMs and PanINs.
(A) ChIP qPCR of isolated acinar cells from WT mice on the Sox9 promoter regions. n = 3 mice per genotype. Relative fold enrichment of BRG1 over IgG control on the Sox9 promoter regions. Numbers in primer names denote distance from transcription start site (TSS) of the Sox9 gene. Data are shown as mean ± SEM. *P < 0.05, Student’s t test. (B) ChIP qPCR of isolated acinar cells from WT and Ptf1a-CreER; Brg1fl/fl mice that were treated with tamoxifen (KO). n = 3–4 mice per genotype. Relative fold enrichment of PDX1 over IgG control on the Sox9 promoter and enhancer. Numbers in primer names denote distance from TSS of the Sox9 gene. Data are shown as mean ± SEM. *P < 0.05, Student’s t test. (C) Immunohistochemistry of serial sections for BRG1 and SOX9. Left: BRG1-positive and BRG1-negative ADMs in Ptf1a-CreER; KrasG12D; Brg1fl/fl mice at 2 months after tamoxifen administration. Right: BRG1-positive and BRG1-negative PanINs in Ptf1a-CreER; KrasG12D; Brg1fl/fl mice at 2 months after tamoxifen administration. Scale bars: 50 μm. (D) Quantification of the relationship between expression of BRG1 and SOX9 in ADM and PanIN in Ptf1a-CreER; KrasG12D; Brg1fl/fl mice. BRG1-positive ADMs or PanINs and SOX9-positive ADMs or PanINs are defined as the lesions in which more than half of the cells are positive for BRG1 and SOX9, respectively. n = 4 mice. ***P < 0.001, Fisher’s exact test.
Figure 5
Figure 5. Sox9 overexpression cancels the decrease in KRAS-driven PanIN formation following Brg1 deletion in mice.
(A) The genetic strategy used to delete Brg1 and activate oncogenic Kras and Sox9 expression in adult pancreatic acinar cells in addition to the experimental design of tamoxifen administration and analysis. (B) H&E staining and immunohistochemistry for BRG1 with Alcian blue staining, SOX9, and the HA-tag in Ptf1a-CreER; KrasG12D; Sox9OE; Brg1fl/fl mice and Ptf1a-CreER; KrasG12D; Sox9OE; Brg1fl/fl mice. Scale bars: 50 μm. (C) Quantification of PanINs in Ptf1a-CreER; KrasG12D; Sox9OE; Brg1fl/+ mice and Ptf1a-CreER; KrasG12D; Sox9OE; Brg1fl/fl mice. Red bars show incidence of BRG1-negative PanINs. n = 3 per genotype. Data are shown as mean ± SEM. Student’s t test.
Figure 6
Figure 6. BRG1 is required for PanIN maintenance.
(A) The genetic strategy used to activate oncogenic Kras at the embryonic stage and delete Brg1 at a subsequent adult stage and experimental design for tamoxifen administration and analysis. (B) H&E and Alcian blue staining and immunohistochemistry for BRG1 and SMA in Pdx1-Flp; FSF-KrasG12D and Pdx1-Flp; FSF-KrasG12D; FSF-R26CAG-CreERT2; Brg1fl/fl mice. Scale bars: 200 μm (H&E, Alcian blue, and SMA): 50 μm (BRG1). (C) Quantification of PanINs in Pdx1-Flp; FSF-KrasG12D (green; n = 7) and Pdx1-Flp; FSF-KrasG12D; FSF-R26CAG-CreERT2; Brg1fl/fl (red; n = 6) mice. Data are shown as mean ± SEM. ***P < 0.001, Student’s t test. (D) Quantification of BRG1-positive/negative PanINs in Pdx1-Flp; FSF-KrasG12D; FSF-R26 CAG-CreERT2; Brg1fl/fl mice. n = 22 mice.
Figure 7
Figure 7. BRG1 expression correlates with SOX9 expression in human PDAs.
(A) Immunohistochemistry for BRG1 in human PanINs and PDAs. Scale bars: 50 μm. (B) BRG1 immunohistochemistry score from patient samples with PanINs (n = 26) and PanIN-derived PDAs (n = 27). PDA samples containing PanINs are selected and BRG1 expression is scored using an IHC score ranging from 0 to 8 (low to high). Means are shown. (C) Quantification of SOX9 expression in BRG1lo or BRG1hi human PDA samples. The cut-off IHC score is 0 to 6 for low and 7 to 8 for high expression. Left: PanIN (n = 26); right: PDA (n = 27). **P < 0.01, Fisher’s exact test. (D) Plots of mRNA expression of BRG1 and SOX9 from a cohort of 150 patients in the TCGA data set. (E) The box-and-whisker plot demonstrates the differential expression for SOX9 between the BRG1hi (n = 112, the higher 75%) and BRG1lo groups (n = 38, the lower 25%) from a cohort of 150 patients in the TCGA data set. In the box-and-whisker plots, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers indicate minimum to maximum without outlier. ***P < 0.001, Student’s t test.

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