Targeting BET Proteins Decreases Hyaluronidase-1 in Pancreatic Cancer

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by the presence of dense stroma that is enriched in hyaluronan (HA), with increased HA levels associated with more aggressive disease. Increased levels of the HA-degrading enzymes hyaluronidases (HYALs) are also associated with tumor progression. In this study, we evaluate the regulation of HYALs in PDAC. Methods: Using siRNA and small molecule inhibitors, we evaluated the regulation of HYALs using quantitative real-time PCR (qRT-PCR), Western blot analysis, and ELISA. The binding of BRD2 protein on the HYAL1 promoter was evaluated by chromatin immunoprecipitation (ChIP) assay. Proliferation was evaluated by WST-1 assay. Mice with xenograft tumors were treated with BET inhibitors. The expression of HYALs in tumors was analyzed by immunohistochemistry and by qRT-PCR. Results: We show that HYAL1, HYAL2, and HYAL3 are expressed in PDAC tumors and in PDAC and pancreatic stellate cell lines. We demonstrate that inhibitors targeting bromodomain and extra-terminal domain (BET) proteins, which are readers of histone acetylation marks, primarily decrease HYAL1 expression. We show that the BET family protein BRD2 regulates HYAL1 expression by binding to its promoter region and that HYAL1 downregulation decreases proliferation and enhances apoptosis of PDAC and stellate cell lines. Notably, BET inhibitors decrease the levels of HYAL1 expression in vivo without affecting the levels of HYAL2 or HYAL3. Conclusions: Our results demonstrate the pro-tumorigenic role of HYAL1 and identify the role of BRD2 in the regulation of HYAL1 in PDAC. Overall, these data enhance our understanding of the role and regulation of HYAL1 and provide the rationale for targeting HYAL1 in PDAC.


Background
Pancreatic ductal adenocarcinoma (PDAC), one of the most lethal malignancies, is projected to become the second leading cause of cancer-related death by 2030 [1]. PDAC is associated with a pronounced stroma that can account for up to 90% of tumor mass [2,3]. The PDAC stroma, which is enriched in collagen and hyaluronan (HA), can contribute to tumor progression and treatment resistance [4]. Increased HA accumulation is associated with more aggressive disease and therapy resistance in human tumors [5][6][7].
HA is a linear, soluble glycosaminoglycan (GAG) macromolecule consisting of repeated chains of disaccharides N-acetyl glucosamine and D-glucuronic acid [8]. In normal Quantitative real-time PCR-Cells were lysed in RLT buffer (Qiagen, Hilden, Germany). Primary human PDAC tumors and tumors from nude mice bearing PDAC cells were homogenized in RLT buffer using TissueRuptor (Qiagen). Total RNA was isolated using RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer's protocol. RNA concentration and purity were measured using a NanoDrop OneC spectrophotometer (Thermo Scientific, Waltham, MA, USA). First-strand cDNA was synthesized from 1.5 µg of total RNA using a high-capacity cDNA transcription kit (Applied Biosystems, Waltham, MA, USA). Quantitative gene expression was performed with gene-specific TaqMan primers, TaqMan Universal PCR Master Mix, and the 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The relative mRNA expression levels were calculated using the 2 −∆∆CT method.
Chromatin immunoprecipitation (ChIP) assay-ChIP analysis was performed using a Millipore Sigma kit according to the manufacturer's instructions. Ten million AsPC1 cells were seeded in 150 mm culture dishes and allowed to adhere overnight. The next day, cells were treated with JQ1 or DMSO overnight. The adherent cells were fixed and crosslinked in 1% formaldehyde at room temperature (RT) for 10 min. The unreacted formaldehyde was then neutralized with 125 mM glycine for 5 min. Cells were washed twice with ice-cold PBS. The cells were scraped using a sterile cell scraper in 2 mL of ice-cold PBS supplemented with Protease Inhibitor Cocktail II. After centrifugation at 800× g at 4 • C for 5 min, the cell pellet was resuspended in 800 µL ice-cold EZ-Zyme Lysis Buffer containing Protease Inhibitor Cocktail II. Chromatin fragments were prepared using the EZ-Zyme Chromatin Prep kit Millipore), and ChIP performed using the EZ-Magna ChIP A/G Chromatin Immunoprecipitation kit (17-10086, Millipore) and anti-BRD2 antibody (A302-583A, Bethyl Laboratories), or control IgG antibody (2729, Cell Signaling). DNA was purified after reverse crosslinking. Purified DNA was then analyzed by PCR using KiCqStart SYBR Green qPCR ReadyMix (KCQS02, Sigma) and primers specific for the HYAL1 promoter: forward 5 -AACCAAGATCCCTTTGCCAG-3 and reverse 5 -TCCAAATTTCCTGACCCCAG-3 [35].
HYAL1 ELISA-Measurements of HYAL1 Concentrations in tissue culture media of the cell-free supernatants were determined using a Human Hyaluronidase 1/HYAL1 DuoSet ELISA kit (R&D Systems, Inc., Minneapolis, MI, USA) according to manufacturer's instructions with slight modification. For low HYAL1 expressing cells, Panc1 and stellate tissue culture media supernatants were concentrated two times using Amicon Ultra-2 mL Centrifugal filters Ultracel-10K (Millipore). Quantification was performed by measuring the absorbance at 450 nm with 570 as a reference using a microplate reader. A curve of absorbance versus concentration of HYAL1 in the standard wells was determined by interpolation from a standard curve.
In vivo study-All animal studies were completed per NIH guidelines on the care and use of laboratory animals for research purposes. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Northwestern University (Chicago, IL, USA). Six-to eight-week-old athymic nude female mice were obtained from Charles River and maintained in a specific pathogen-free facility. Five million Panc1 cells were implanted subcutaneously in the flank of nude mice as 100 µL cell suspensions with an equal volume of Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) [36]. Tumors were measured twice a week with a digital caliper, and volumes (V) were calculated using the formula V = 1 2 Length × Width 2 . Once the tumor volume reached approximately 200 mm 3 , mice were randomized into two treatment groups: control (DMSO) and JQ1 (50 mg/kg). Treatments were administered by intraperitoneal (i.p.) injections five days per week (Monday-Friday) for three weeks in a suspension containing 10% hydroxypropylβ-cyclodextrin in double-distilled water. At the end of the study, mice were euthanized by CO 2 inhalation and cervical dislocation, and the tumors were excised, weighed, and photographed. Tumor fragments were either processed by formalin fixation before paraffin embedding for IHC or frozen for later RNA extraction. The number of Ki67+ cells in each section was calculated by ImageJ. At least four different sections were taken for each tumor. Statistical analysis-In vivo and in vitro results were compared using a two-tailed t-test analysis or Mann-Whitney U test. Error bars represent SD or SEM as specified. All statistical analyses were done using Microsoft Excel and GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA). A p-value of less than 0.05 was considered significant.

Results
Expression of Hyaluronidase-1 (HYAL1) in PDAC specimens and cell lines. Human PDAC tumors have increased fibrosis, as demonstrated by trichrome staining, and increased hyaluronan (HA) expression, as demonstrated by increased staining for HA binding protein (HABP) ( Figure 1A). Since HA undergoes dynamic regulation by HYALs [37][38][39], we evaluated the expression of HYAL1, HYAL2, and HYAL3 by RT-qPCR in three normal pancreatic tissue samples and seven PDAC tumor specimens. Variable levels of HYAL1 expression were seen in all tumor specimens, with the highest HYAL1 expression observed in PDAC specimen #3. There was~25-fold higher expression of HYAL1 in PDAC specimen #3 compared to the adjacent normal pancreatic tissue sample #1 ( Figure 1B). Overall, there was increased expression of HYAL1 in human PDAC tumors compared to adjacent normal pancreatic tissue samples. In contrast, there was not a significant difference in HYAL2 and HYAL3 expression between human PDAC tumors and adjacent normal tissue (Supplementary File: Figure S1A,C).
(Monday-Friday) for three weeks in a suspension containing 10% hydroxypropyl-β-cyclodextrin in double-distilled water. At the end of the study, mice were euthanized by CO2 inhalation and cervical dislocation, and the tumors were excised, weighed, and photographed. Tumor fragments were either processed by formalin fixation before paraffin embedding for IHC or frozen for later RNA extraction. The number of Ki67+ cells in each section was calculated by ImageJ. At least four different sections were taken for each tumor.
Statistical analysis-In vivo and in vitro results were compared using a two-tailed t-test analysis or Mann-Whitney U test. Error bars represent SD or SEM as specified. All statistical analyses were done using Microsoft Excel and GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA). A p-value of less than 0.05 was considered significant.

Results
Expression of Hyaluronidase-1 (HYAL1) in PDAC specimens and cell lines. Human PDAC tumors have increased fibrosis, as demonstrated by trichrome staining, and increased hyaluronan (HA) expression, as demonstrated by increased staining for HA binding protein (HABP) ( Figure 1A). Since HA undergoes dynamic regulation by HYALs [37][38][39], we evaluated the expression of HYAL1, HYAL2, and HYAL3 by RT-qPCR in three normal pancreatic tissue samples and seven PDAC tumor specimens. Variable levels of HYAL1 expression were seen in all tumor specimens, with the highest HYAL1 expression observed in PDAC specimen #3. There was ~25-fold higher expression of HYAL1 in PDAC specimen #3 compared to the adjacent normal pancreatic tissue sample #1 ( Figure 1B). Overall, there was increased expression of HYAL1 in human PDAC tumors compared to adjacent normal pancreatic tissue samples. In contrast, there was not a significant difference in HYAL2 and HYAL3 expression between human PDAC tumors and adjacent normal tissue (Supplementary File: Figure S1A,C). Similarly, we evaluated HYAL1, HYAL2, and HYAL3 mRNA expression in immortalized human pancreatic duct epithelial (HPDE) cells, in five PDAC cell lines, and in an immortalized pancreatic stellate cell line. Varying levels of HYAL1 expression were seen in PDAC cell lines, with the AsPC1 cell line exhibiting the highest HYAL1 mRNA expression. There was over 50-fold higher HYAL1 mRNA in the AsPC1 cell line compared to HPDE cells ( Figure 1C). Overall, there was increased HYAL1 expression in human PDAC cell lines compared to HPDE cells. In contrast, there was minimal to no difference in the expression of HYAL2 and HYAL3 expression between HPDE cells and the five pancreatic cancer cell lines (Supplementary File: Figure S1B,D).
Targeting BET proteins decreases HYAL1 expression. Since we previously showed that BET inhibitors can decrease fibrosis [27], we evaluated the effects of targeting BET proteins on HYAL expression in pancreatic cancer cells (Panc1 and AsPC1) and in the pancreatic stellate cell line. The cells were treated with the well-established BET inhibitor JQ1, and the effect on HYAL1, HYAL2, and HYAL3 was determined. JQ1 consistently decreased HYAL1 expression at mRNA and protein levels in Panc1, AsPC1 and the stellate cell line (Figure 2A). In contrast, JQ1 had variable effects on HYAL2 and HYAL3 mRNA expression (Supplementary File: Figure S2). As JQ1 treatment qualitatively recapitulates the phenotype of siRNA-mediated knockdown of BET protein [40,41], we evaluated the effects of co-knockdown of BRD2, BRD3, and BRD4 with siRNAs on HYAL expression. The efficiency of the knockdown of BRD2, BRD3, and BRD4 was confirmed with RT-qPCR and western blotting ( Figure 2B). Consistent with our findings with JQ1 treatment, siRNA-mediated co-knockdown of the BET proteins significantly decreased HYAL1 mRNA and protein concentration in all three cell lines ( Figure 2C). In contrast, co-knockdown of BRD2, BRD3 and BRD4 had minimal to no effects on HYAL2 and HYAL3 mRNA levels (Supplementary File: Figure S2).
Regulation of HYAL1 expression by BRD2, BRD3 and BRD4. We next evaluated the role of the different BRD proteins in regulating HYAL1 expression by transfecting Panc1, AsPC1 and stellate cells with individual siRNAs against BRD2, BRD3, or BRD4. The efficiency of knockdown was confirmed by Western blotting, and the effect on HYAL1 protein levels was determined with ELISA ( Figure 3A-C). Targeting BRD2 decreased HYAL1 protein concentration most efficiently and consistently in all three cell lines. In contrast, BRD3 and BRD4 knockdown showed modest effects on HYAL1 protein concentration.
BRD2 protein binds to the HYAL1 promoter. Given our results that targeting BET proteins, particularly BRD2, decreases HYAL1 levels, we evaluated BRD2 regulation of HYAL1 mRNA expression. Previous studies have shown that BRD2 regulates gene expression by binding to their promoters [42,43]. AsPC1 cells were treated with DMSO or JQ1, and the effect on BRD2 binding to the HYAL1 promoter was determined by ChIP assay. Compared to control IgG, there was~6-fold enrichment of the HYAL1 promoter with the BRD2 antibody in the ChIP assay. Importantly, JQ1 treatment decreased the binding of BRD2 protein to the HYAL1 promoter by~5-fold ( Figure 3D). These results demonstrate that BRD2 directly regulates HYAL1 gene expression by binding to its promoter. HYAL1 knockdown decreases proliferation and induces apoptosis. To investigate the functional role of HYAL1, we downregulated HYAL1 in Panc1, AsPC1 and stellate cells and evaluated the effect on proliferation (Figure 4). HYAL1 siRNA decreased HYAL1 mRNA and protein levels and significantly decreased proliferation (Figure 4). The effect of HYAL1 silencing on cell proliferation was confirmed using a different HYAL1 siRNA (Supplementary File: Figure S3). In contrast, siRNA targeting HYAL2 did not affect proliferation (Supplementary File: Figure S4), and siRNA targeting HYAL3 decreased proliferation only in AsPC1 cells (Supplementary File: Figure S5). We also evaluated the effects of targeting HYAL1 on apoptosis. HYAL1 siRNA decreased HYAL1 protein expression and increased caspase-3 and PAPR cleavage ( Figure 4A-C), indicating that HYAL1 knockdown results in increased apoptosis.   (DMSO). Mean ± SD. (B) Panc1, AsPC1 and Stl cell lines were transfected with control siRNA o with a combination of siRNAs against BRD2, BRD3, and BRD4 (siB2+3+4). The efficiency of BRD2 BRD3, and BRD4 knockdown was determined at the mRNA level after 48 h of transfection by qRT PCR (the mean ± SD) and at the protein level by western blotting after 72 h of transfection. (C) Th effect of siB2+3+4 on HYAL1 expression was determined by qRT-PCR and by ELISA after 96 h o transfection. Mean ± SD. The western blot results are representative of three independent experi ments. The qRT-PCR and ELISA results are the mean of three independent experiments. Statistica analysis of ELISA results was done by Student's t-test, * p < 0.05; ** p < 0.01.  cell line was with the BET inhibitor JQ1 (1 μM) overnight. The cells were subjected to ChIP analysis using an anti-BRD2 antibody, and an isotype-matched IgG was used as a negative control. The association with the HYAL1 gene promoter was quantified by qPCR and 2% agarose gel electrophoresis. The qPCR results are the mean of two independent experiments. Mean ± SD. The western blot results are representative of three independent experiments, and ELISA results are the mean of three independent experiments. Statistical analysis of ELISA results was done by Student's t-test, ns p > 0.05; * p < 0.05; ** p < 0.01. NC, no template control.

Targeting BET proteins decreases HYAL1 expression and proliferation in vivo.
Finally, we evaluated the effects of JQ1 on HYAL expression in vivo. Established Panc1 tumors were treated with JQ1 daily, and the effect on tumor growth was monitored. JQ1 reduced tumor growth, with a significant decrease in tumor weight at the end of the experiment ( Figure 5A). JQ1 also significantly decreased proliferation, as determined by , and stellate (Stl, C) cells were transfected with control siRNA or siRNAs against HYAL1. The effect on HYAL1 mRNA was determined after 48 h of transfection by qRT-PCR, and the effect of HYAL1 protein was determined by ELISA after 96 h of transfection. Mean ± SD. The effect on cell proliferation was analyzed with WST-1 assay (mean ± SD), and the effect on apoptosis was analyzed with western blotting for cleaved PARP and cleaved caspase-3 after 48 h of transfection. The western blot results are representative of three independent experiments. The qRT-PCR and ELISA assay results are the mean of three independent experiments. Statistical analysis of ELISA results was done by Student's t-test and relative proliferation by Mann-Whitney U test, ** p < 0.01 *** p < 0.001.
Targeting BET proteins decreases HYAL1 expression and proliferation in vivo. Finally, we evaluated the effects of JQ1 on HYAL expression in vivo. Established Panc1 tumors were treated with JQ1 daily, and the effect on tumor growth was monitored. JQ1 reduced tumor growth, with a significant decrease in tumor weight at the end of the experiment ( Figure 5A). JQ1 also significantly decreased proliferation, as determined by Ki67 staining ( Figure 5B). There was a significant decrease in HYAL1 mRNA levels in JQ1-treated tumors compared to control tumors ( Figure 5C), but not in HYAL2 and HYAL3 mRNA levels between DMSO-and JQ1-treated tumors (Supplementary File: Figure S6). The decrease in HYAL1 expression in JQ1-treated tumors was also confirmed at the protein level by IHC staining for HYAL1 ( Figure 5D).
Ki67 staining ( Figure 5B). There was a significant decrease in HYAL1 mRNA levels in JQ1treated tumors compared to control tumors ( Figure 5C), but not in HYAL2 and HYAL3 mRNA levels between DMSO-and JQ1-treated tumors (Supplementary File: Figure S6). The decrease in HYAL1 expression in JQ1-treated tumors was also confirmed at the protein level by IHC staining for HYAL1 ( Figure 5D).

Discussion
Multiple studies have shown overexpression of HA and HYALs in various cancers, including PDAC [44]. HA overproduction and degradation are essential for PDAC tumor progression [45]. The HA molecule has a unique ability to imbibe a significant amount of water. Thus, high levels of HA increase interstitial pressure and impair drug entry in PDAC tumor cells, and consequently promote chemoresistance in tumors [7]. High levels of HYALs increase the low molecular weight HA during enzymatic degradation of high molecular weight HA [46]. The low molecular weight HA acts as a signaling molecule by interacting with specific cell surface receptors, such as the receptor for HA-mediated motility (RHAMM) and CD44, to modulate a variety of cellular processes [47]. In this study, we show that HYAL1, HYAL2, and HYAL3 are expressed in human PDAC tumors and in PDAC cell lines. We also show that BET inhibition decreases HYAL1 expression without significantly affecting the levels of HYAL2 and HYAL3.
HYAL1 gene transcription is dysregulated by epigenetic alterations, such as promoter methylation and histone acetylation [17]. In recent years, epigenetic readers, especially BET proteins, have become attractive targets for cancer therapeutics [21]. BET inhibitors are also being evaluated in combination with various therapies. For example, JQ1 has been reported to both radiosensitize PDAC cells and enhance the efficacy of gemcitabine, the standard first-line chemotherapy drug for PDAC [48][49][50]. In this study, we show that HYAL1 expression is primarily regulated by BRD2. In contrast, we previously showed that the expression of collagen in stellate cells was primarily regulated by BRD4 [27]. A number of studies have now shown that BET proteins have both overlapping and distinct functions in gene expression [51,52]. For example, BRD2 enhances epithelial-mesenchymal transition (EMT) in breast cancer cells, while BRD3 and BRD4 repress EMT [53]. BRD2, but not BRD3 or BRD4, regulates interleukin 8 production during keratinocyte inflammatory response [54]. Also, isoform switching of BRD2 and BRD4 regulates Smad2-dependent lineage specification of pluripotent stem cells [55].
While HYAL1 overexpression can induce migration, invasion, and metastasis in different cancer models [56][57][58], HYAL1 can also function as a tumor suppressor in some cancers [59]. For example, in prostate cancer cell lines, HYAL1 functions as an oncogene or a tumor suppressor, depending on the HYAL1 levels [60]. However, in PDAC cells, our results show a decreased proliferation in cells expressing high as well as low levels of HYAL1 upon HYAL1 silencing. In our study of five PDAC cell lines, we found that AsPC1 expresses the highest level of HYAL1 mRNA. Our findings are in agreement with a previous study demonstrating that HYAL1 mRNA is variably expressed in a panel of nine PDAC cell lines [17]. Four PDAC cell lines (AsPC1, BxPC3, CFPAC1 and NORP1) showed particularly high levels of HYAL1 mRNA [17]. We have also found that HYAL1 knockdown induces apoptosis in PDAC cells. Our findings are in contrast to the findings in prostate cancer, where HYAL1 overexpression was shown to induce apoptosis [60]. These results suggest that the role of HYAL1 may be dependent on the tumor type.

Conclusions
We show for the first time that BET inhibitors downregulate HYAL1 expression and that HYAL1 expression is primarily regulated by BRD2. We also show that HYAL1 downregulation decreases proliferation and enhances apoptosis. Thus, our study provides novel insights into the regulation and functional involvement of HYAL1 in PDAC and highlights HYAL1 as a promising therapeutic target for PDAC.
Author Contributions: K.K.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, and editing of the manuscript. D.K.: helped with data collection. D.J.B.: helped with procuring the human PDAC specimens and analyzed the data. R.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and approved by the In-stitutional Review Board of Northwestern University (protocol STU00007180, approval date 08/07/2015). The animal study protocol was approved by the Institutional Animal Care and Use Committee of Northwestern University (protocol IS00007481, approval date 01/09/2018).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study and samples were de-identified prior to use.

Data Availability Statement:
Uncropped and unprocessed images of western blots have been uploaded to the MDPI as part of this submission.