Pharmacological Targeting of BET Bromodomains Inhibits Lens Fibrosis via Downregulation of MYC Expression

Abstract

Purpose: Lens fibrosis involves aberrant growth, migration, and transforming growth factorβ (TGFβ)-induced epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs). In this study, we investigated the role of the bromo- and extra-terminal domain (BET) inhibitor in lens fibrotic disorder to identify drug-based therapies.

Methods: Rat lens explants, rabbit primary lens epithelial cells (rLECs), human lens explants and human SRA01/04 cells were treated with TGFβ2 in the presence or absence of the BET bromodomain inhibitor JQ1 or the MYC inhibitor 10058-F4. Proliferation was determined by MTS assay. Cell migration was measured by wound healing and transwell assays. The expression levels of fibronectin (FN), α-smooth muscle actin (α-SMA), E-cadherin, and phosphorylated downstream Smads were analyzed by Western blot, qRT-PCR, and immunocytochemical experiments. Transcriptome analysis was conducted to explore the molecular mechanism.

Results: Blockage of BET bromodomains with JQ1 significantly suppressed rLECs proliferation by inducing G1 cell cycle arrest. Furthermore, JQ1 attenuated TGFβ2-dependent upregulation of mesenchymal gene expression and phosphorylation of Smad2/3 during the progression of EMT, whereas E-cadherin expression was preserved. JQ1 repressed MYC expression, which was dose- and time-dependently upregulated by TGFβ2. Inhibiting MYC with either the small-molecule inhibitor 10058-F4 or genetic knockdown phenocopied the effects of JQ1 treatment. MYC overexpression partially reversed the JQ1-regulated EMT-related alteration of gene expression. Both JQ1 and 10058-F4 blocked the expression of TGFβ receptor II and integrin αv in rLECs and abolished TGFβ2-induced opacification and subcapsular plaque formation in rat lens explants.

Conclusions: Our results demonstrate the antifibrotic role of JQ1 in maintaining the epithelial characteristics of LECs and blocking TGFβ2-induced EMT, possibly by downregulating MYC, thereby providing new avenues for treating lens fibrosis.

Fibrosis, which is characterized by excessive accumulation of extracellular matrix (ECM), is the common pathologic feature of many chronic diseases, such as liver cirrhosis, idiopathic pulmonary fibrosis, renal fibrosis, and ocular fibrosis.^1–3 During the development of fibrotic disorder, ECM-secreting myofibroblasts are activated in response to various stimuli, including persistent infections, exposure to chemical insults, radiation, and mechanical injury, and produce connective tissue elements such as collagen and fibronectin (FN), which progressively distort normal tissue architecture and impair organ function.⁴ Although as high as 45% mortality is attributed to fibrosis, therapeutic options for fibrotic diseases are limited.⁵ Lens fibrosis, such as anterior subcapsular cataract (ASC) and posterior capsule opacification (PCO), is an important subtype of cataract, which is associated with several complications and places significant personal and financial strain on the patients.⁶ Owing to its unique biologic properties, the lens acts as a powerful tool for investigating the molecular mechanisms of fibrosis. In addition, lens epithelial cells (LECs) have been identified as the predominant precursors of myofibroblasts and allow fibrotic modifications to be directly monitored.⁷ Therefore, identifying and understanding the molecular basis of fibrosis using lens as a model are not only beneficial for developing novel therapeutic strategies for lens fibrosis, but may also yield clues for targeting other fibrotic diseases. 

Existing data show that the pathogenesis of lens fibrosis involves processes such as hyperproliferation, migration, and the epithelial–mesenchymal transition (EMT) of LECs.^8–11 Transforming growth factor β, particularly TGFβ2, which is the major isoform in the aqueous humor, acts as the predominant pathogenic factor in the development of EMT.¹² During EMT, LECs at the equator and under the anterior lens capsule proliferate and transdifferentiate into spindle-shaped myofibroblastic cells that elongate and migrate across the lens capsule. Migration of these cells centrally toward the visual axis, myofibroblast transdifferentiation, and the excessive deposition of collagen and other ECM components such as FN, COL-I, and COL-IV, change light scattering and eventually contribute to the development of lens fibrosis. Considering that EMT, proliferation, and migration result in fibrosis of the lens, preventative strategies targeting these processes may suppress disease progression, and even restore lens transparency if sufficiently potent. 

Chromatin remodeling through histone acetylation plays a pivotal role in the regulation of gene expression. The BET family proteins, including BRD2, BRD3, BRD4, and BRDT, act as epigenetic readers that recognize acetylated histones and transduce signals by facilitating transcriptional activation. While the bromodomain at the N-terminal binds to acetylated lysine residues, the extra-terminal and C-terminal domain of BET proteins can interact with chromatin histone modification enzymes and transcriptional activators, thereby modulating gene expression. Cell growth, hematopoietic development, inflammation, and maintenance of higher-order chromatin structure are among the physiologic processes affected by BETs.^13–15 JQ1, a selective small-molecule inhibitor of BET bromodomains, competitively binds to the acetyl-lysine recognition pocket and disassociates BET proteins from chromatin.¹⁶ BET inhibition is widely studied in oncology as it exerts promising anti-tumor effects in hematologic malignancies, lung cancer, pancreatic cancer, and other solid tumors.^16–20 Recently, several studies showed that JQ1 can regulate tissue fibrosis, such as pressure overload-induced cardiac fibrosis, unilateral ureteral obstruction-induced fibrosis, and radiation-induced lung fibrosis^21,22; however, its function in lens fibrosis has not yet been established. 

MYC is a helix–loop–helix–leucine zipper (HLH–ZIP) protein that performs a broad spectrum of functions by regulating the transcription of its target genes. It is one of the most potent regulators of cell cycle progression and also participates in the regulation of various biological functions, including cell proliferation, apoptosis, and differentiation.²³ 

In this study, we focused on BET bromodomain inhibitor JQ1 to investigate its role in EMT using lens as a model and further investigated the therapeutic effect of JQ1 in the treatment of fibrotic forms of cataract. Notably, we uncovered the critical mechanism underlying the anti-fibrotic function of JQ1 by showing that it inhibited cell proliferation, migration, and TGFβ2-induced EMT in lens epithelial cells in an MYC-dependent manner. Furthermore, the specific MYC pathway inhibitor, 10058-F4, could abolish the progression of EMT in both primary LECs and the whole lens ex vivo system. Our findings indicate that BET proteins and MYC may be potential pharmacological targets for preventative and therapeutic strategies against lens fibrosis and other fibrotic disorders. 

Materials and Methods

Lens Epithelial Explant Collection, Cell Culture, and Treatment

All experiments were performed in accordance with the tenets of the Declaration of Helsinki and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All investigations were approved by the Institutional Animal Care and Use Committee of Zhongshan Ophthalmic Center. Rabbit primary lens epithelial cells (rLECs) were prepared as previously described.²⁴ Briefly, the eyeball was enucleated from 2-month-old New Zealand white rabbits and washed twice in PBS containing antibiotics. After removal of the iris, the lens capsule with attached epithelial cells was carefully peeled out and cut into 1  ×  1 mm² pieces and cultured in minimum essential media (MEM, Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Life Technologies), NEAA (Life Technologies), and antibiotics (Life Technologies). Lens capsule pieces showed cell outgrowth within 1 week, cells were passaged at approximately 80% confluence, and cells that were passaged a second or third time were used for experiments. Human lens explants were collected from organ donors provided by the Eye Bank of Zhongshan Ophthalmic Center. The ages of the donors ranged from 30 to 40 years. Lens explants were cultured in MEM containing 1% FBS with 1% NEAA. The human LEC line SRA01/04 was kindly provided by Professor Fu Shang (Tufts University, Boston, MA, USA) and grown in Dulbecco's modified Eagle's medium (DMEM, Life Technologies) containing 10% FBS. 

Ex Vivo Lens Culture

Lenses of 21-day-old Sprague-Dawley rats were cultured as described previously.²⁵ Briefly, whole lenses of 21-day-old rats were collected and cultured in serum-free medium 199 with Earle's salts supplemented with 0.1% bovine serum albumin (BSA), 0.1 μg/ml L-glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin. After isolation, lenses were kept in fresh medium overnight, and the cloudy lenses caused by technique were excluded for further studies. TGFβ2 (5 ng/mL; R&D Systems, Minneapolis, MN, USA), JQ1 (0.5 μM; catalogue no. S7110, Selleckchem, Houston, TX, USA), or 10058-F4 (75 μM; catalogue no. S7153, Selleckchem) were added to the culture medium. The culture medium was changed every other day. Lenses were cultured for up to 4 days and photographed before being harvested. The results are representative of three different repetitions. 

MTS Assay

The proliferative abilities of rabbit primary LECs and human lens epithelial SRA01/04 cells were determined using the cell proliferation assay (CellTiter; Promega, Madison, WI, USA) according to the manufacturer's instructions. Briefly, LECs were seeded into 96-well plates at a density of 10³ cells/well. The next day, the cells were treated with increasing concentrations of JQ1 (0.25 μM, 0.5 μM, and 0.75 μM) or 10058-F4 (25 μM, 50 μM, and 75 μM), and the control groups were treated with equivalent volumes of DMSO. Cells were collected daily after treatment. We added 25 μL CellTiter 96 AQueous One Solution directly to the culture wells containing 100 μL culture medium and incubated for another 2 hours before collection. Absorbance was measured by spectrophotometry at 490 nm. 

Cell Cycle Analysis

rLECs were treated with the indicated chemicals for 24 hours and fixed in cold 70% ethanol for 1 hour at 4°C. The fixed cells were washed in PBS, resuspended in 400 μL propidium iodide/RNase A (BD Biosciences, Franklin Lakes, NJ, USA) solution and incubated for 20 minutes at room temperature. Cell cycle distribution was measured using flow cytometry (BD Biosciences). 

Cell Viability Analysis

Cell viability were tested in rLECs treated with DMSO or JQ1 or 10058-F4 for 48 hours. rLECs were collected and stained with an FITC Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer's instructions. The percentage of apoptotic cells (Annexin V+) were determined using flow cytometry (BD Biosciences). 

Transwell Migration Assay

rLECs or SRA01/04 cells (2 × 10⁴, after pretreatment with DMSO or JQ1 or 10058-F4 at indicated concentrations for 24 hours) in 100 μl serum-free medium were added to the upper chamber of 24-well transwell plates (Corning, NY, USA) and allowed to migrate through the 8-μm pores. Medium (600 μL) containing 10% fetal bovine serum was added to the lower chamber. After 24 to 36 hours of incubation, the cells on the top of the inserts (nonmigrated) were removed, and those on the lower surface (migrated) were fixed with 4% paraformaldehyde and stained with crystal violet (Beyotime, Shanghai, China). The number of migrated cells was counted in five randomly selected fields. 

Wound Healing Assay

rLECs were allowed to grow to confluence in 12-well plate (after pretreatment with DMSO or JQ1 or 10058-F4 at the indicated concentrations for 24 hours). After starvation for 12 hours, the cell monolayers were scratched with a 20-μL pipet tip and washed with PBS. Then, the cells were cultured in serum-free medium supplied with increasing doses of JQ1 (0.25 μM, 0.5 μM, and 0.75 μM) or 10058-F4 (25 μM, 50 μM, and 75 μM) or an equivalent volume of DMSO as a control. Wound closure was measured using a microscope (Zeiss Axio Observer; Carl Zeiss Microscopy, White Plains, NY, USA) at 0 and 8 hours after scratching. The distance migrated by the cells was determined and analyzed using ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). All experiments were repeated independently at least three times. 

Immunoblot Analysis

rLECs or SRA01/04 cells were lysed with radioimmunoprecipitation assay buffer (Beyotime), and the protein concentration was determined using a bicinchoninic acid assay (Beyotime). Proteins were separated using sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with 5% fat-free milk, followed by incubation with primary antibodies (1:1000) overnight at 4°C and subsequently with secondary antibodies (1:2000) for 2 hours at room temperature. The following antibodies were used for immunoblotting analysis. Antibodies against total SMAD2/3, pSMAD2/3, Ki67, PCNA, MMP2, ITGAV, TGFBR2, α-SMA, GAPDH, E-cadherin, and HRP-conjugated secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against α-SMA and FN were from Abcam, Cambridge, UK. Antibodies against COL-IV were purchased from Merck Millipore (Burlington, MA, USA), and antibodies against MYC were purchased from Santa Cruz Biotechnology, TX, USA. 

Immunofluorescence Staining

After the cells are attached to the dished and treated as described. rLECs were fixed with 4% paraformaldehyde and blocked with 3% BSA in phosphate-buffered saline-Tween 20 (PBST) for 1 h. Then, the cells were incubated with primary antibodies (1:100) overnight at 4°C, followed by incubation with fluorochrome-conjugated secondary antibodies (1:1000, Cell Signaling Technology). The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich Corp., St. Louis, MO, USA). Slides were mounted with antifade mounting medium, and images were captured using a fluorescence microscope (Leica DM4000; Leica Microsytems, Wetzlar, Germany). 

Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)

At the end of each culture treatment, total RNA from rLECs or SRA01/04 cells was purified using an RNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and quantified with a spectrophotometer (NanoDrop; Thermo Fisher Scientific, Waltham, MA, USA). Total RNA (1 μg) was converted to cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Quantitative RT-PCR was performed using Universal qPCR master mix (New England Biolabs, Ipswich, MA, USA) on a real-time PCR system (StepOnePlus; Applied Biosystems) with a 10-μl reaction system. The thermocycler parameters were 94°C for 5 minutes, followed by 40 cycles of 95°C for 10 seconds, 60°C for 20 seconds, and 72°C for 20 seconds. The expression levels were normalized to that of GAPDH. The sequences of oligonucleotide primers used in this study are listed in Supplementary Table S1. 

Transfection in SRA01/04

To knockdown MYC, SRA01/04 cells were transfected with siRNAs specific to MYC with nontargeting siRNAs (Life Technologies) as a negative control for 48 hours using reagent (Lipofectamine 3000; Life Technologies). AlexaFluor Red Positive Control was used to detect the transfection efficiency, and all siRNAs were used at a concentration of 50 nM. Real-time PCR was conducted to confirm the effects of siRNA transfection. 

To overexpress MYC, SRA01/04 cells in 6-well culture plates were transfected with either control plasmid or plasmid overexpressing MYC using the reagent (Life Technologies), and the medium was changed after 8 hours. After 48 hours of transfection, the cells were subjected to various treatments. 

RNA Sequencing

Human lens explants were isolated and cultured in DMSO, 5 ng/ml TGFβ2, 5 ng/ml TGFβ2 in combination with 0.5 μM JQ1 or 50μM 10058-F4 for 48 hours. Samples from three individual treatments were pooled. Total RNA was isolated, and RNA integrity was assessed using a commercial kit and system (RNA Nano 6000 Assay Kit of the Bioanalyzer 2100; Agilent Technologies, Santa Clara, CA, USA). Sequencing libraries were generated using a prep kit for Illumina (NEBNext Ultra RNA Library Prep Kit; NEB). RNA-seq was conducted with a high sequence platform (HiSeq; Illumina), and 150-bp paired-end reads were generated. Raw sequencing reads were aligned onto the human reference genome using HISAT2 and read summarization was conducted with featureCounts function of the Subread package. EdgeR R package was performed for determining differential expression genes with P < 0.05 and log2|FC| >1. The P values were adjusted using the Benjamini & Hochberg method. Gene Ontology (GO) and KEGG enrichment analysis were enriched by R statistical software. 

Statistics

All data are expressed as the means ± standard deviations (SDs). Statistical analyses were performed using GraphPad Prism software. Significance of difference was determined using the two-tailed unpaired Student's t-test or 1-way ANOVA, and P < 0.05 (*P < 0.05, **P < 0.01, and ***P < 0.001) was considered statistically significant. 

Results

BET Inhibition With JQ1 Suppresses Cell Growth by Inducing G1 Cell Cycle Arrest

As cell proliferation is the fundamental step for fibrotic processes, we first investigated the role of BET bromodomains in cell proliferation with JQ1. rLECs were treated with increasing doses of JQ1 (0.25, 0.5, and 0.75 μM) for 24 to 72 hours. Indeed, JQ1 strongly inhibited the growth of rLECs in a concentration-dependent manner (Fig. 1A). A decrease in the number of Ki67-positive proliferative cells and proliferating cell nuclear antigen (PCNA) expression levels were observed in rLECs treated with JQ1 (Figs. 1B, 1D). To further explore the underlying mechanisms that contribute to cell proliferation inhibition, we tested whether JQ1 could regulate the cell cycle progression of rLECs using flow cytometry. As shown in Fig. 1C, treatment with JQ1 significantly induced G1 cell cycle arrest with impaired S phase entry. Furthermore, annexin V staining detection of cell viability showed that only high dose of JQ1 (0.75 μM) triggered apoptosis (Fig. 1E). Taken together, BET inhibition could suppress the cell growth of rLECs by inducing G1 cell cycle arrest. 

Figure 1

 

BET inhibition with JQ1 suppresses cell growth and migration. (A) Proliferation of rLECs treated with increasing concentrations of JQ1. (B) Immunofluorescence staining of Ki67 in rLECs treated with or without JQ1 (0.5 μM) for 48 hours. Scale bar, 50 μm. (C) Flow cytometric evaluation of PI staining for cell cycle analysis in JQ1-treated rLECs. (D) The expression of PCNA and MMP2 in rLECs treated with increasing doses of JQ1 were detected by western blotting. (E) Annexin V staining detection of cell viability in JQ1-treated rLECs. (F, G) The effect of JQ1 on rLECs migration was determined using wound-healing (F) and transwell assays (G). Representative images are shown. Scale bar: 200 μm. All data are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus the DMSO-treated control group. NS, not significant.

JQ1 Inhibits Migration of Lens Epithelial Cells

To further determine other biological effects of BET inhibition, we assessed cell migratory capacity using the wound healing assay. Whereas rLECs in the control group had largely covered the wound area, cell migration was clearly decreased in the JQ1-treated groups (Fig. 1F). To confirm this observation, we tested the effect of JQ1 using a transwell assay, which also demonstrated reduced migration of JQ1-treated cells compared to the migration of control cells (Fig. 1G). Furthermore, the expression level of matrix metalloprotease-2 (MMP2), which is related to cell migration, is suppressed by JQ1 (Fig. 1D). These results indicate that the migratory capacity of rLECs is markedly inhibited by JQ1. 

JQ1 Negatively Regulates TGFβ2-Induced EMT

As TGFβ2 is a potent driving force behind the transdifferentiation of epithelial cells, we stimulated rLECs with TGFβ2 to investigate the role of JQ1 in EMT. Cells treated with 5 ng/mL TGFβ2 for 48 hours exhibited a notable change from a cobblestone-like to an elongated spindle-like morphology, indicative of the induction of EMT (Fig. 2A). However, JQ1 abolished cellular elongation, and the cells retained their epithelial-like appearance as well as the expression and membrane localization of E-cadherin (Figs. 2B, 2C). Consistently, JQ1 treatment repressed the TGFβ2-induced increase in the expression of mesenchymal markers, FN and α-SMA (Figs. 2B–D). To further investigate whether JQ1 can terminate established fibrosis, rLECs were treated with TGFβ2 for 24 hours first to induce EMT, followed by another 48-hour incubation in the presence or absence of JQ1 (Fig. 2E). Similarly, JQ1 conspicuously abrogated the induction of mesenchymal markers. These findings indicate that JQ1 suppresses the EMT progression of LECs. 

Figure 2

 

JQ1 negatively regulates TGFβ2-induced EMT. (A) Morphologic alteration in rLECs treated with TGFβ2 (5 ng/ml) and JQ1 alone or together for 48 hours. (B) Immunofluorescent staining analysis of E-cadherin, α-SMA and FN in rLECs. (C) The protein expression levels of E-cadherin, α-SMA and FN were detected by western blotting analysis. (D) The mRNA expression levels of α-SMA and FN. (E) Schematic diagram and western blot results for TGFβ2-pretreated rLECs. TJ_0.25, TGFβ2+JQ1 0.25 μM; TJ_0.5, TGFβ2+JQ1 0.5 μM. Expression data from the qRT-PCR analysis are presented relative to the GAPDH expression data and are shown as the mean ± SD of triplicates. All results are representative of 3 independent experiments. ***P < 0.001. Scale bar: 100 μm.

MYC is Implicated in JQ1-Mediated Inhibition of EMT

Previous studies have demonstrated that JQ1 regulates MYC expression in malignancies.^16,19,26,27 Thus, to further identify the underlying mechanism responsible for the inhibitory effect of JQ1 during fibrotic progression, we evaluated the expression of MYC and its downstream molecules. Interestingly, we found that MYC was induced upon TGFβ2 stimulation in a dose- and time-dependent manner in rLECs (Figs. 3A, 3B). The expression of MYC and NCL, a transcriptional target of MYC, was significantly suppressed by JQ1 treatment in the presence or absence of TGFβ2 (Fig. 3C). Based on these findings, we inhibited MYC with a selective small-molecule inhibitor, 10058-F4, to determine whether MYC blockage could phenocopy the biologic activities of JQ1 treatment. Consistent with BET inhibition, rLECs in the TGFβ2 and 10058-F4 cotreatment group retained the cuboidal monolayer appearance with a characteristic cobblestone-packed arrangement, indicative of their epithelial phenotype (Fig. 3D). The expression of mesenchymal markers, FN and α-SMA, increased dramatically when stimulated with TGFβ2, which was notably blocked by 10058-F4 (Figs. 3E–G). Notably, forced overexpression of MYC in SRA01/04 cells partially rescued JQ1-mediated FN and α-SMA downregulation (Fig. 3H), indicating that JQ1 might exert inhibitory effects on EMT by regulating MYC expression. 

Figure 3

 

MYC is implicated in the JQ1-mediated inhibition of EMT. (A, B) The dose- and time-dependent induction of MYC in rLECs upon TGFβ2 stimulation was determined using Western blot analysis and qRT-PCR. (C) MYC and NCL were downregulated by JQ1. (D) Representative images showing morphological changes in rLECs. Scale bar: 100 μM. (E–G) Expression of EMT-related molecules was detected using western blotting, qRT-PCR and immunofluorescence staining (10058-F4, 75 μM, 48 hours). Scale bar: 50 μM. (H) SRA01/04 cells were transfected with MYC overexpression plasmids or vectors and treated with JQ1 (0.5 μM, 24 hours) or the DMSO control. All three groups were treated in combination with 5 ng/mL TGFβ2. The expression of FN and α-SMA were evaluated. *P < 0.05, ***P < 0.001.

Pharmacological Inhibition of MYC With 10058-F4 Suppresses the Proliferation and Migration of LECs

Based on these findings, we next determined whether pharmacological targeting MYC could phenocopy other inhibitory effects of JQ1 on rLECs. As shown in Figure 4A, 10058-F4 suppressed cell growth in a dose-dependent manner, strongly indicating its antiproliferative activity. Furthermore, 10058-F4 induced G1 cell cycle arrest and retarded S-phase entry (Fig. 4C). The expression of proliferative markers, Ki67 and PCNA, was blocked by the MYC inhibitor (Figs. 4B, 4D), while cell viability was not affected (Fig. 4E). Microscopic examination of wound-healing and transwell assays revealed a significant reduction in cell migration (Figs. 4F, 4G) with MMP2 downregulation (Fig. 4D). These results demonstrate that the pharmacological inhibition of MYC with 10058-F4 markedly inhibits the proliferative and migratory capacities of LECs. 

Figure 4

 

Pharmacological inhibition of MYC with 10058-F4 suppresses the proliferation and migration of rLECs. (A) Normalized proliferative activity of rLECs treated as indicated, and an equivalent volume of DMSO was used as a control. (B) Immunofluorescence staining of Ki67 in rLECs treated with or without 10058-F4 (75 μM) for 48 hours. Scale bar: 50 μm. (C) Flow cytometric evaluation of PI staining for cell cycle analysis in 10058-F4-treated rLECs. (D) The expression of PCNA and MMP2 in rLECs treated with increasing doses of 10058-F4 were detected by western blotting. (E) Annexin V staining detection of cell viability in 10058-F4-treated rLECs. (F) Wound healing assays were used to determine the roles of 10058-F4 in the regulation of rLECs migration. (G) The migratory ability of rLECs was further confirmed by a transwell assay. Scale bar: 200 μm. Data are representative of three independent experiments. *P < 0.05, ***P < 0.001 versus control group.

Knockdown of MYC Inhibits the Proliferation, Migration, and TGFβ2-Dependent EMT of LECs

To determine whether the genetic ablation of MYC recapitulates the pharmacological inhibition, we then transfected siRNAs into SRA01/04 cells. As expected, real-time qPCR analyses revealed that siRNA-mediated knockdown of MYC in SRA01/04 cells was able to reduce the expression of MYC and attenuate TGFβ2-dependent MYC induction (Fig. 5A). The levels of MYC downstream targets, NCL and ODC1, were also significantly decreased in the MYC siRNA-transfected cells compared to those in the control siRNA-transfected cells. As shown in Fig. 5B, the depletion of MYC exerted potent inhibitory effects on the proliferation of SRA01/04 cells. Furthermore, CDK4, a key modulator of cell cycle progression, was reported to be transcriptionally activated by MYC in diverse cell types. Consistently, we found that knockdown of MYC significantly suppressed TGFβ2-induced CDK4 induction at the mRNA level, whereas CDK4 expression under normal conditions was not significantly affected (Fig. 5C). MYC inhibition strongly reduced cell migration, which was reflected by the transwell assay (Fig. 5D). Furthermore, we found that TGFβ2-induced upregulation of FN and α-SMA expression was partially abrogated by MYC knockdown (Fig. 5E). These findings further support that MYC is required for rLEC proliferation, migration and TGFβ2-induced EMT. 

Figure 5

 

Knockdown of MYC inhibits the proliferation, migration, and TGFβ2-dependent EMT. (A) MYC and downstream NCL, ODC1 expression in SRA01/04 cells transfected with control siRNA (siNC) or MYC-specific siRNA (siMYC) with or without TGFβ2 (5 ng/mL, 24 hours). (B) Normalized proliferative activity of SRA01/04 cells measured by MTS assay. (C) Expression of CDK4 determined by qRT-PCR analysis. (D) SRA01/04 cells were transfected with siNC or siMYC and subjected to transwell assays. Scale bar: 200 μm. (E). The effect of MYC knockdown on FN and α-SMA was measured by qRT-PCR and Western blotting. Data in qRT-PCR analysis are presented relative to GAPDH and shown as the mean ± SD of triplicates. All results are representative of 3 independent experiments. **P < 0.01, ***P < 0.001.

Inhibition of BET/MYC Blocks SMAD2/3 Activation

It is well established that TGFβ2 signaling is mediated through a canonical, SMAD2/3-dependent pathway. Hence, we investigated whether the anti-EMT effect observed post-BET protein or MYC blockage was associated with the activation of SMAD proteins. As expected, TGFβ2 stimulation activated the SMAD2/3 signaling pathway, as shown by the increased phosphorylation of SMAD2/3. However, both JQ1 and 10058-F4 significantly attenuated SMAD2/3 phosphorylation (Figs. 6A, 6B). These findings were also corroborated by the results of immunofluorescence analysis identifying the cellular localization of SMAD2/3 (Figs. 6C, 6D). SMAD2/3 predominately accumulated in the cytoplasm of the control group and translocated to the nucleus when stimulated with TGFβ2, indicating SMAD2/3 activation. However, the addition of JQ1 or 10058-F4 reduced SMAD2/3 localization to the nuclei and increased the cytoplasmic signal. 

Figure 6

 

Inhibition of BET/MYC blocks SMAD2/3 activation. rLECs were cultured in the absence or presence of 5 ng/ml TGFβ2 with increasing concentrations of JQ1 (0.25 μM, 0.5 μM, and 0.75 μM) or DMSO for 48 hours. The phosphorylation level of SMAD2/3 was detected by western blotting analysis (A). The cellular localization of SMAD2/3 was detected by immunofluorescence staining ([C], 0.5 μM JQ1). The effect of 10058-F4 (37.5 μM, 50 μM, and 75 μM) on TGFβ2-induced activation of SMAD2/3 was also evaluated by Western blotting (B) and immunofluorescence staining ([D], 75 μM 10058-F4). Scale bar: 100 μm. Data are representative of three independent experiments.

Transcriptome Analysis of the Downstream Targets of BET/MYC in LECs

To gain further insight into potential molecular connections between BET/MYC and EMT, we next conducted RNA-seq to compare the transcriptome of human lens epithelial explants treated with DMSO, TGFβ2, and TGFβ2 in combination with JQ1 or 10058-F4 (Fig. 7A). Heatmap analysis revealed that most of the TGFβ2-responsive genes were negatively affected in their induction following JQ1 or 10058-F4 incubation (Fig. 7B). Furthermore, we found that genes differentially regulated in response to either inhibitor were associated with pathways controlling ECM accumulation, receptor regulation, growth factor binding, integrin and cell adhesion, pathways predominantly associated with EMT (Figs. 7C, 7D). In total, 86.2% of TGFβ2-upregulated genes were suppressed by JQ1 or 10058-F4, and 45.8% of genes were negatively regulated by both inhibitors, such as ITGAV, TGFBI, MMP2 and TGFBR2 (Fig. 7E). We further confirmed that integrin αv and TGFβ receptor II, two novel mediators of signal transduction by TGFβ,^8,28,29 were inhibited by both JQ1 and 10058-F4 in rLECs (Figs. 7F, 7G). 

Figure 7

 

Transcriptome analysis of the downstream targets of BET/MYC in LECs. Human lens explants were cultured in DMSO, 5 ng/mL TGFβ2, 5 ng/mL TGFβ2 in combination with 0.5 μM JQ1 or 50 μM 10058-F4 for 48 h for RNA-seq analysis. (A) Differential gene expression is shown in the volcano plots. (B) Heat map analysis of the EMT signature in human lens explants. (C, D) GO analysis of inhibitor regulated genes was performed and top 10 enriched molecular functions are shown. (E) Venn diagrams of differential and shared altered genes. (F, G) The effect of JQ1 and 10058-F4 on the expression of ITGAV and TGFBR2 in rLECs was evaluated by Western blotting.

Therapeutic Benefits of Inhibiting BET Bromodomains and MYC in Lens Fibrosis

Based on this mechanistic rationale, we evaluated the therapeutic opportunity of using pharmacological inhibition of BET bromodomains and MYC using a whole-lens culture ex vivo model. Lenses from 21-day-old rats were incubated with 5 ng/mL TGFβ2 for 4 days, and obvious opacities beneath the lens capsule were observed, whereas lenses cotreated with JQ1 or 10058-F4 largely abrogated TGFβ2-induced lens fibrosis and were transparent (Fig. 8A). The clumps in TGFβ2-treated lenses showed an accumulation of FN and an increased expression level of α-SMA (Fig. 8B). In contrast, cotreatment with JQ1 or 10058-F4 reduced the induction of mesenchymal transition-related proteins and preserved E-cadherin expression. Taken together, these results demonstrate that BET proteins are important for the development of lens fibrosis by regulating MYC expression and that pharmacological inhibition by JQ1 or 10058/F4 can abrogate TGFβ2-induced opacification and subcapsular plaque formation in rat lenses. 

Figure 8

 

Therapeutic benefits of BET bromodomains and MYC inhibition in lens fibrosis. (A) Representative images of lenses treated as indicated for 4 days. (B) Immunofluorescence staining for FN, α-SMA and E-cadherin. Scale bar: 50 μm.

Discussion

Considering that current therapies have limited clinical benefits, we investigated the underlying mechanisms of lens fibrosis to identify drug-based therapies. In the present study, we demonstrated for the first time that inhibition of BET bromodomains using a selective small-molecule inhibitor, JQ1, attenuated TGF-β2-induced EMT of the lens epithelium in a MYC-dependent manner, establishing the therapeutic rationale for BET protein/MYC inhibition in lens fibrotic diseases (Supplementary Fig. S1). 

Epigenetic mechanisms have been implicated in the progression of organ fibrotic diseases. BET family members recognize histone acetylation and facilitate assembly of the transcription machinery. Recently, epigenetic screening identified BRD4, which binds to the enhancer of certain pro-fibrotic genes, as a potent driver of the fibrotic response in liver.³⁰ Several studies have highlighted the contribution of BET proteins to cancer progression, liver fibrosis, and kidney fibrosis, reinforcing their value as potent therapeutic targets for fibrotic diseases.^21,22 However, the role of BET on lens fibrosis remains unclear. 

In our study, we observed that inhibition of BET bromodomains by JQ1 significantly suppressed proliferation and migration of LECs in a dose- and time-dependent manner. Furthermore, JQ1 attenuated the induction of TGF-β2–induced EMT with downregulation of FN and α-SMA in LECs. JQ1 also prevented the formation of TGF-β2-stimulated ASC in the whole lens culture ex vivo model. Thus, our results indicate that BET inhibition by JQ1 is highly effective at suppressing the development of lens subcapsular plaque. 

Compelling evidence shows that BET inhibition preferentially represses MYC transcription.^19,26 However, the role of MYC in fibrotic disorders is controversial. Previous findings indicate that TGFβ plays a growth inhibitory role via transcriptional inhibition of MYC, which is supported by the identification of a SMAD-binding element within the MYC promoter region.³¹ In contrast, other studies have shown that MYC is overexpressed during renal fibrosis and participates in ECM component production by inducing ITGAV-mediated TGFβ signaling.³² MYC knockdown impaired TGFβ-induced EMT in cancer cells by regulating SNAIL.³³ In this study, we demonstrated that MYC is dose- and time-dependently upregulated upon TGFβ2 treatment, and elucidated that MYC upregulation is a requirement for the anti-fibrotic effects of JQ1 in LECs. Pharmacological and genetic inhibition of MYC significantly suppressed the proliferation, migration, and EMT of LECs, exerting effects similar to those exerted by JQ1 treatment on LECs. Notably, we also demonstrated that blocking of the MYC pathway with a small-molecule inhibitor, 10058-F4, completely abrogated the onset of TGFβ2-induced subcapsular plaque formation and lens fibrosis in whole lens explants. Collectively, the results suggest that the anti-fibrotic effect of JQ1 is MYC-dependent during lens fibrosis. 

Taken together, our findings demonstrated a novel inhibitory effect of JQ1, a potent BET bromodomain inhibitor, on cell proliferation, migration, and TGFβ2–induced EMT in LECs, which is mediated via downregulation of MYC. It is possible that other undefined functions of BET proteins may contribute to lens fibrosis. Although further investigation is required, we present the first evidence regarding the application of JQ1 as a protective agent against lens fibrosis, which may provide a valid option for the clinical treatment of lens fibrotic diseases and also yield clues for other proliferative and fibrotic ocular diseases. 

Acknowledgments

Supported by National Natural Science Foundation of China (No. 81300749 and 81530028), Guangdong Province Natural Science Foundation (2018A030313628), 973 program (2015CB964600), and the State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University. The authors alone are responsible for the content and writing of the paper. 

Disclosure: X. Wang, None; B. Wang, None; N. Zhao, None; C. Wang, None; M. Huang, None; B. Chen, None; J. Chen, None; Y. Sun, None; L. Xiong, None; S. Huang, None; Y. Liu, None