BET inhibitors block pancreatic stellate cell collagen I production and attenuate fibrosis in vivo
Krishan Kumar, … , Kazumi Ebine, Hidayatullah G. Munshi
Krishan Kumar, … , Kazumi Ebine, Hidayatullah G. Munshi
Published February 9, 2017
Citation Information: JCI Insight. 2017;2(3):e88032. https://doi.org/10.1172/jci.insight.88032.
View: Text | PDF
Research Article Gastroenterology Oncology

BET inhibitors block pancreatic stellate cell collagen I production and attenuate fibrosis in vivo

Abstract

The fibrotic reaction, which can account for over 70%–80% of the tumor mass, is a characteristic feature of human pancreatic ductal adenocarcinoma (PDAC) tumors. It is associated with activation and proliferation of pancreatic stellate cells (PSCs), which are key regulators of collagen I production and fibrosis in vivo. In this report, we show that members of the bromodomain and extraterminal (BET) family of proteins are expressed in primary PSCs isolated from human PDAC tumors, with BRD4 positively regulating, and BRD2 and BRD3 negatively regulating, collagen I expression in primary cancer-associated PSCs. We show that the inhibitory effect of pan-BET inhibitors on collagen I expression in primary cancer-associated PSCs is through blocking of BRD4 function. Importantly, we show that FOSL1 is repressed by BRD4 in primary cancer-associated PSCs and negatively regulates collagen I expression. While BET inhibitors do not affect viability or induce PSC apoptosis or senescence, BET inhibitors induce primary cancer-associated PSCs to become quiescent. Finally, we show that BET inhibitors attenuate stellate cell activation, fibrosis, and collagen I production in the EL-KrasG12D transgenic mouse model of pancreatic tumorigenesis. Our results demonstrate that BET inhibitors regulate fibrosis by modulating the activation and function of cancer-associated PSCs.

Authors

Krishan Kumar, Brian T. DeCant, Paul J. Grippo, Rosa F. Hwang, David J. Bentrem, Kazumi Ebine, Hidayatullah G. Munshi

×

Figure 7

BET inhibitors induce primary cancer-associated PSCs to become quiescent.

Options: View larger image (or click on image) Download as PowerPoint
BET inhibitors induce primary cancer-associated PSCs to become quiescent...
(A) Primary cancer-associated PSCs growing on glass coverslips were treated with the BET inhibitors JQ1 or I-BET151 (1 μM) for 72 hours, and the effect on α-SMA filaments was determined by immunofluorescence staining. Original magnification, ×400 (top); ×1,000 (bottom). These results are representative of 3 (n = 3) independent experiments. (B) Primary cancer-associated PSCs were treated with the BET inhibitors JQ1 (1 μM) or I-BET151 (1 μM) for 72 hours, and the effect on α-SMA mRNA was determined by qRT-PCR (n = 3). ***P < 0.001. Data were analyzed by 2-tailed unpaired Student’s t test. The effect on α-SMA protein expression was determined by Western blotting (n = 3). These results are representative of 3 (n = 3) independent experiments. (C) Primary cancer-associated PSCs were treated with thymidine (2 mM) for 24 hours to synchronize PSC cycle. The cells were then washed with PBS to remove thymidine and grown in fresh media in the presence of vehicle control (DMSO) or JQ1 (1 μM) with or without Click-iT EdU (2 μM) for additional 40 hours. The cells were then labeled using the Click-iT Alexa Fluor 488 Assay kit and costained with propidium iodide, and the percentage of S-phase cells was detected by flow cytometry (n = 4). **P < 0.01. Data were analyzed by 2-tailed paired Student’s t test. (D) Primary cancer-associated PSCs growing on glass coverslips were treated with the BET inhibitors JQ1 (1 μM) or I-BET151 (1 μM) for 72 hours, and the effect on lipid droplet accumulation was determined using Bodipy 493/503 fluorophore. Original magnification, ×400 (top); ×1,000 (bottom). The percentage of lipid-containing PSCs was quantified in 5 separate (n = 5) experiments. ****P < 0.0001. Data were analyzed by 2-tailed unpaired Student’s t test. BET, bromodomain and extraterminal; COL, collagen; EdU, 5-ethynyl-2 deoxyuridine; PDAC, pancreatic ductal adenocarcinoma; PSCs, pancreatic stellate cells.