YAP1-mediated suppression of USP31 enhances NF-κB activity to promote sarcomagenesis

Abstract

To date, no consistent oncogenic driver mutations have been identified in most adult soft tissue sarcomas; these tumors are thus generally insensitive to existing targeted therapies. Here we investigated alternate mechanisms underlying sarcomagenesis to identify potential therapeutic interventions. Undifferentiated pleomorphic sarcoma (UPS) is an aggressive tumor frequently found in skeletal muscle where deregulation of the Hippo pathway and aberrant stabilization of its transcriptional effector yes-associated protein 1 (YAP1) increases proliferation and tumorigenesis. However, the downstream mechanisms driving this deregulation are incompletely understood. Using autochthonous mouse models and whole genome analyses, we found that YAP1 was constitutively active in some sarcomas due to epigenetic silencing of its inhibitor angiomotin (AMOT). Epigenetic modulators vorinostat and JQ1 restored AMOT expression and wild type Hippo pathway signaling, which induced a muscle differentiation program and inhibited sarcomagenesis. YAP1 promoted sarcomagenesis by inhibiting expression of ubiquitin-specific peptidase 31 (USP31), a newly identified upstream negative regulator of NF-κB signaling. Combined treatment with epigenetic modulators effectively restored USP31 expression, resulting in decreased NF-κB activity. Our findings highlight a key underlying molecular mechanism in UPS and demonstrate the potential impact of an epigenetic approach to sarcoma treatment.

Introduction

Soft tissue sarcomas are a heterogeneous group of mesenchymal malignancies arising in muscle, fat, cartilage and connective tissues (1, 2). Whereas loss or mutation of tumor suppressors (i.e. p53) occurs in ~50% of sarcomas (3, 4), sequencing of common adult sarcoma subtypes including fibrosarcoma, liposarcoma, and undifferentiated pleomorphic sarcoma (UPS) have produced no evidence of consistent oncogenic driver mutations (4). The lack of targetable oncogenes has stalled development of therapeutic modalities (5, 6). We focused our efforts on skeletal muscle UPS given its particularly aggressive nature, lack of non-surgical treatment strategies, and its high frequency of diagnoses relative to other adult subtypes.

We previously reported that deactivation of the Hippo pathway, a signaling cascade that negatively regulates cell proliferation, promotes sarcomagenesis in skeletal muscle-derived UPS (7). Furthermore, genome-wide analysis of The Cancer Genome Atlas (TCGA) sarcoma dataset confirmed that deregulated Hippo signaling is a contributing factor in sarcomagenesis (2). Inactivation of the Hippo pathway stabilizes the transcriptional effector, YAP1, allowing it to translocate to the nucleus and promote a pro-proliferation gene expression program. Inhibtion of YAP1 reduces proliferation in multiple sarcoma subtypes, including UPS, though no genetically engineered mouse model of Yap1-deficient UPS existed prior to this study. While YAP1 activity has been implicated in multiple sarcoma subtypes (7–10), its exact function, regulation and targets in this context context are unclear.

The precise cell of origin for UPS is not known (11). However, in skeletal muscle these tumors are thought to arise from muscle progenitor cells/satellite cells (12). Whereas investigation of the Hippo pathway in muscle-derived sarcomas has been limited to rhabdomyosarcoma (13), in normal myoblasts persistent elevated YAP1 and NF-κB signaling facilitate proliferation and inhibit differentiation (13–15).

In addition to Hippo pathway deregulation, several studies have recently shown that alterations in the epigenetic landscape can promote sarcomagenesis. Specifically, certain pediatric sarcomas are linked to chromosomal translocations of transcription factor loci encoding chromatin remodeling factors (16, 17). Copy number loss of chromatin modulators have also been found in some subtypes (15). Together, these studies suggest that disruption of chromatin architecture may be a common event in sarcomagenesis. Our recent work showed that treatment with the histone deacetylase (HDAC) inhibitor Vorinostat, also known as Suberoylanilide Hydroxamic Acid (SAHA), leads to re-expression of HIF2α and a corresponding 50% reduction in UPS sarcomagenesis in vivo (18). These findings support the hypothesis that epigenetic modulation can reduce tumorigenesis by returning key transcription factors to the expression and activity levels found in quiescent cells.

In the present study, we utilized human skeletal muscle UPS samples and our autochthonous mouse model of UPS to determine that YAP1 promotes proliferation and de-differentiation through persistent hyper-activation of NF-κB. Analysis of the chromatin state in human UPS revealed the presence of massive enhancers, or super enhancers, notably at NF-κB target gene loci. Using the epigenetic modulators Vorinostat/SAHA and JQ1 (BET bromodomain protein inhibitor), we found that tumor growth was reduced secondary to Hippo pathway reactivation and loss of YAP1 expression. Furthermore, we found that YAP1 suppresses expression of Ubiquitin Specific Peptidase 31 (USP31), a newly identified negative regulator of NF-κB signaling (19), resulting in uncontrolled proliferation and tumorigenesis. YAP1 inhibition restored expression of USP31, indicating that aberrant stabilization of YAP1 promotes NF-κB activity. We conclude that epigenetic therapy can reclaim control of YAP1-mediated NF-κB signaling and subsequently inhibit tumor cell proliferation, induce differentiation and decrease sarcomagenesis in vivo.

Materials and Methods

Mouse Models.

GEMM.

All experiments were performed in accordance with NIH guidelines and were approved by the University of Pennsylvania Institutional Animal Care and Use Committee. We generated Kras^G12D+; Trp53^fl/fl; YAP1^fl/fl (KPY) mice by crossing KP and YAP1^fl/fl animals. Tumors were generated by injection of a calcium phosphate precipitate of adenovirus expressing Cre recombinase (University of Iowa) into the right gastrocnemius muscle of 3- to 6-month-old mice.

Allograft mouse model.

For subcutaneous implanted tumors, 1 × 10⁶ KP230 cells were injected subcutaneously into the flanks of 6-wk-old nu/nu mice (Charles River Laboratories). Animals were euthanized after 14 days of JQ1 peritoneal injection. For Rela-mediated knockdown KP230 allografts, 1 × 10⁶ cells were injected into the flanks of mice with control tumor cells (scrambled shRNA) and experimental tumor cells (Rela shRNA). Tumor size was measured every other day, and animals were euthanized after 21 days post-tumor cells injection. Tumor volume was calculated by using the formula (ab²)π/6, where a is the longest measurement and b is the shortest.

In vivo drug treatment.

For in vivo drug studies, total 44 (n=11 per group) autochthonous KP mice were randomly divided into 4 groups to receive different treatments once tumors reached 100 mm³, and injected for up to 20 days. The mice were euthanized 24hrs after the tumor volume reached 2000 mm³). 1) Vehicle group (10% Hydroxypropyl-β-cyclodextrin plus DMSO was diluted daily in sterile 45% PEG/55% H₂O); 2). JQ1 group (JQ1 was diluted daily in 10% HP-β-CD injected into the peritoneal cavity 25 mg/kg twice daily); 3). SAHA group (SAHA was diluted in sterile 45% PEG/55% H₂O injected into the peritoneal cavity 50 mg/kg daily); 4). JQ1 and SAHA combination treatment group (drug were diluted in its vehicles respectively). Treatment method for drug combination group: 1). 25mg/kg SAHA+50mg/kg JQ1 for first 5 days. 2) 25mg/kg SAHA+25mg/kg JQ1 each other day for 10days. 3). 25mg/kg SAHA+50mg/kg JQ1 for 2 days. 4). Then mice with tumors received 25mg/kg SAHA with 25mg/kg JQ1 for 3 days. Mice without tumors received 5mg/kg SAHA and 5mg/kg JQ1 for 3 days). JQ1 was provided by Jun Qi (Dana-Farber Cancer Institute) and SAHA was purchased from Cayman Chemical. HP-β-CD and PEG400 were obtained from Sigma-Aldrich.

Cell lines.

Human HT-1080 (Fibrosarcoma), RD (Rhabdomyosarcoma), LPS224 and LPS863 (Liposarcoma), SKLMS1 and SKUT1 (Leiomyosarcoma) and HEK-293T cell lines were purchased from ATCC (Manassas, VA, USA). STS-109 and STS-48 cell lines were derived from human UPS patients. KP230 and KIA cell lines were derived from UPS mouse tumors as described in Eisinger-Mathason et al. (4). STR analysis was performed at the time of derivation and confirmed in April 2015. Cells were purchased, thawed, and then expanded in the laboratory. Multiple aliquots were frozen down within 10 days of initial resuscitation. For experimental use, aliquots were resuscitated and cultured for up to 20 passages (4–6 wk) before being discarded. Cells were cultured in DMEM with 10% (vol/vol) FBS and 1% penicillin/streptomycin. All cell lines were confirmed to be negative for mycoplasma contamination.

Lentiviral Transduction.

shRNA-mediated knockdown of AMOT TRCN: 0000166812, 0000165373, 0000162009, 0000159177, 0000162010; Amot TRCN: 00001266880, 0000126883, 0000349515, 0000126881, 0000317410. Yap1 TRCN: 0000095864, 0000095867, 0000095868; YAP1 TRCN: 0000107266, 0000107267, 0000015547. Usp31 TRCN: 0000092218, 0000092219, 0000092220, 0000092221, 0000092222, Rela (p65 NF-κB) TRCN: 0000055343, 0000055344, 0000055346, 0000055347 and Scramble shRNA was obtained from Addgene. shRNA plasmids were packaged by using the third-generation lenti-vector system (VSV-G, p-MDLG, and pRSV-REV) and expressed in HEK-293T cells. Supernatant was collected at 24 and 48hrs after transfection and subsequently concentrated by using 10-kDa Amicon Ultra-15 centrifugal filter units (Millipore). Lenti GFP-AMOT p130 (plasmid 32828), Lenti GFP-AMOT p130 Y242/287A (plasmid 32829), YAP1-V5 (plasmid 42555), YAP1 (S6A)-V5 (plasmid 42562) were purchased from Addgene. pLX304 vector was obtained from Addgene. Lenti-pcdh-EF1 and Lenti-pcdh-GFP were used as the empty vector control in our lab.

Immunohistochemistry.

Twenty human UPS paraffin embedded tissues from obtained from the Surgical Pathology group at Univ. of Pennsylvania were stained for both p-p65 and Yap1. IHC was performed on 5 micron tissue sections according to standard protocols. Slides were digitally scanned by the Pathology Core Laboratory at the Research Institute at the Children’s Hospital of Philadelphia. The software of Aperio ImageScope (Leica Biosystem, USA) were used for slides quantification. Modified macro (Nuclear v9 parameter) were used to distinguish intensity of staining. The following antibody concentrations were used: rabbit anti-YAP1 (4912; 1:100) (Cell Signaling Technology), anti-phospho p65 (Abcam) (86299; 1:250) rabbit anti-Ki-67 (15580; 1:100) (Abcam), rabbit anti-MYOD (18943–1-AP; 1:100) (Proteintech). IHC of human soft tissue sarcoma and smooth/skeletal muscle samples was performed by using core biopsy arrays (US Biomax, Rockville, MD, USA #SO801a). Images were taken by Leica 500 microscope and analyzed by using Photoshop CS3 (Adobe systems). For quantification: 5 areas of tumor and 5 areas of adjacent muscle (identified by H&E staining) were captured per section and averaged to determine the mean % positive nuclei/section.

Immunoblots.

Protein lysate was prepared in SDS/Tris (pH7.6) lysis buffer, separated by electrophoresis in 8–10% SDS/PAGE gels, transferred to nitrocellulose membrane, and probed with the following antibodies: rabbit anti-YAP1 (4912; 1:1000), rabbit anti-p-Yap (Ser397) (13619; 1:1000), rabbit anti-GAPDH (2118; 1:1000), mouse anti-AMOT (60156–1-lg 1:500), rabbit anti-V5-tag (13202; 1:1000), rabbit anti-p65 (8242; 1:500), rabbit anti-p-p65 (Ser536) (3033; 1:500), rabbit anti-Caspase-3 (9662; 1:1000), rabbit anti-HSP90 (4875; 1:1000) (Cell Signaling Technology), rabbit USP31(12076–1-AP; 1:1000), rabbit anti-MYOD1 (18943–1-AP; 1:500) (Proteintech), rabbit FOXM1 (sc-502; 1:500) (Santa Cruz Biotechnology), c-Myc (32072; 1:2000), p57 Kip2 (75974; 1:500) (Abcam), mouse anti-Lamin B2 (D18) (University of Iowa Developmental Studies Hybridoma Bank). SuperSep Phos-tag™ gel was purchased from Wako. Rabbit anti-LATS1 (3477; 1:1000), rabbit anti-MST1 (3682; 1:1000), rabbit anti-MST2 (3952; 1:1000) (Cell Signaling Technology) were used to detect phosphorylated proteins.

Luciferase assay:

plasmid pHAGE NF-κB-TA-LUC-UBC-GFP-W (49343; Addgene) was transfected into 293T cells (ATCC) to generate lentiviral particles in the supernatant. Viral supernatant was harvested and then concentrated by centrifugal filter units (Amicon Ultra-15, Millipore). NF-κB reporter virus was transduced into KP230 cells. GFP positive NF-κB reporter cells were sorted generating an ~85% pure GFP+ cell line. For shRNA assays the NF-κB the reporter cell line was transduced with lentivirus expressing control, Usp31 or Yap1 shRNA. For drug studies, the NF-κB reporter cells were treated with SAHA (2μM)/JQ1 (0.5μM) and BAY 11–7085 (1.5 μM, Selleckchem) 48hrs. Twelve hours prior to detecting luciferase activity, TNFα (10ng/ml, R&D) was added. Luciferase activity was assayed using the Dual Luciferase Assay System (E2920, Promega) according to the manufacturer’s protocol on a Luminometer (GLOMAX, Promega). Results were calculated as fold induction.

Oncomine and TCGA survival analysis:

We used the publically available database Detwiller et al. through Oncomine Research Premium edition software (version 4.5, life Technologies) to query AMOT and MYOD1 expression and survival. YAP1 and USP31 gene expression in overall survival from MFH/UPS and DDLS patients, respectively in TCGA sarcoma. Kaplan-Meier analyses were performed for overall survival of patients.

Microarray based gene set enrichment analysis.

Differential gene expression was tested using Significance Analysis of Microarrays (SAM, samr v2.0), yielding fold change, q-value (false discovery rate) and d-score for each gene. We observed a small number of genes meeting our cutoffs for differential expression and so proceeded to GSEA. Log₂-transformed RMA-sst expression values were used as input to GSEA(20) where enrichment was tested against the hallmark gene sets from the Molecular Signatures Database (MSigDB, v5.1, http://software.broadinstitute.org/gsea/msigdb/index.jsp)

C2C12 growth and differentiation.

C2C12 murine myoblast cells were obtained from ATCC (Manassas, VA, USA). The cells grow as undifferentiated myoblasts in growth medium (20% FBS with 1% penicillin/streptomycin), and were passaged every 2–3 days at 50% sub-confluence. To induce differentiation cells were grown overnight to ~80% confluence in growth medium, and then switched to DMEM supplemented with 2% horse serum. Differentiation media was refreshed every 2 days.

Chip-seq and RNA-seq.

ChIP-seq

For tumor samples resected from UPS patients at the Hospital of the University of Pennsylvania, approximately 100 mg of tissue was minced into 1–2 mm pieces and incubated in 1% formaldehyde for 15 minutes. Formaldehyde was quenched with glycine at 0.125 M. Fixed tissue was homogenized for 60 seconds with a Tissue Tearor Homogenizer (Biospec) at 30,000 RPM. Homogenized tissue was washed with ice-cold PBS with 1X HALT protease inhibitor. For cell-line ChIP-RX, samples were fixed for 10 minutes in 1% formaldehyde quenched with glycine and washed with PBS as above. 5e6 S2 cells (Drosophila Melanogaster) were added to each sample of 2.5e7 for ChIP-RX normalization in downstream analysis.

RNA-seq

Tissue pre-processed via mortar and pestle homogenization or 2.5e5 cells were homogenized via Qiashredder column (Qiagen). Samples were then processed via mRNeasy Mini Kit (Qiagen). RNA-seq datasets in fastq format were fed into RSEM for mRNA sequence alignment and quantification (21). Gene counts generated by RSEM were fed into edgeR (22) to compute logFC and p-value. FDR is used for multiple comparison correction. The GSEA software and Hallmark gene sets (20) were used for GSEA. Genes were ranked by rank score –log(p-value) * sign(logFC).

Statistical Analysis:

Statistical analysis was performed using Prism (Graph Pad Software). Data are shown as mean± SEM or SD. Data are reported as biological replicates, with technical replicates indicated in figure legends. Student t-tests (unpaired two tailed) were performed to determine whether a difference between two values is statistically significant different, with a P-value <0.05 considered significant. In vitro assays were performed in triplicate unless otherwise stated.

Accession Codes

Sequencing data reported in this paper have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE97295, GSE97296, GSE97297, GSE109920, and GSE109923.

Results

YAP1 regulates NF-κB target expression in murine UPS

Deregulation of the Hippo pathway is associated with muscle-derived sarcoma subtypes but the downstream mechanisms are unclear (7, 8, 23). Though we have access to only a small number of TCGA patient samples, which prevents our analysis from reaching statistical significance, expression of YAP1 strikingly correlates with long-term survival in human UPS patients (Figure 1A). To determine the relevance of these findings we evaluated YAP1 expression in a variety of sarcomas by immunohistochemistry (IHC) of a human biopsy tissue microarray. YAP1 expression is particularly high in muscle UPS (Figure 1B Group 2, Boxed), compared to normal mesenchymal tissues including skeletal and smooth muscle (Figure 1B, Group 1). The clinical details of each sample can be found in Figure S1A. To test the role of YAP1 in UPS, we introduced Yap1^fl/fl alleles into our LSL-Kras^G12D/+; Trp53^fl/fl (KP) genetically engineered mouse model of skeletal muscle UPS to generate LSL-Kras^G12D/+; Trp53^fl/fl; Yap1^fl/fl (KPY) animals. Tumors are generated by injection of adenovirus expressing Cre recombinase into the right gastrocnemius muscle. The Cre recombinase activates expression of oncogenic Kras and deletes p53 expression in infected muscle progenitor cells (24, 25). Though Kras mutation is rare in sarcomas, Trp53 mutation and deletion are very common (2). Furthermore, hyperactivation of the MAPK pathway, downstream of KRAS activation, is common in UPS and is an excellent prognostic indicator for recurrence (26). This model and a similar one, Kras^G12D/+; Ink4a/arf^fl/fl (KIA), generate sarcomas that are histologically, transcriptionally, and morphologically identical to UPS and are thus the standard GEMMs in UPS studies (24, 25). Yap1 protein expression is stabilized in KP tumors providing further rationale for the study of Yap1 function in sarcoma using this model (Figure 1C,D). Loss of Yap1 in KPY tumors was confirmed by Western blot and IHC (Figure 1C,D and Figure S1B). KP tumors were initially palpated at day 45 post Adeno-cre injections and were collected when they reached 2 cm³ (max. tumor volume). KPY tumors were harvested either when they reached 2cm³ or 60 days post Adeno-cre injection, the time-point at which all KP mice had reached max. tumor volume. Deletion of Yap1 delayed tumor initiation (Median latency KP:57 days; KPY:59 days, P<0.0001) and growth (Figure 1E-H). Several KPY mice never developed visible tumors and those animals are represented in Figure 1E with a data point at day 65. Proliferation was reduced by ~50% in KPY tumors as defined by Ki67 positivity (Figure 1D and Figure S1C). To determine the functional role of Yap1 in UPS we performed microarray analysis of 5 individual KP and KPY tumors. Gene Set Enrichment Analysis (GSEA) revealed that “TNFα-induced NF-κB” signaling is significantly reduced in KPY tumors (Figure 1I,J). Furthermore, using the TCGA sarcoma dataset we determined that the YAP1-dependent NF-κB target PHLDA1 is associated with poor survival in UPS (Figure 1K). The specific effects of YAP1 on Tnfα-mediated signaling suggest high levels of Tnfαcytokine production in the UPS tumor microenvironment, which activates downstream NF-κB signaling. To validate Tnfα expression in UPS tumor tissue, and to determine the source of Tnfα production, we fractionated KP tumors and isolated tumor-associated macrophages (TAM), which are known to produce significant amounts of Tnfα. We compared Tnfα expression in isolated KP tumor cells and TAMs. Tnfα mRNA expression was ~45 fold higher in TAMs than in tumor cells (Figure 1L; left). To determine if the relative exprssion of Tnfα in KP TAMs was physiologically significant, we compared this in vivo induction to that of the classical in vitro model of LPS stimulated murine bone marrow derived macrophages (BMDMs). LPS stimulated BMDMs express ~100 fold more Tnfα than untreated BMDMs (Figure 1L; right). Together, these data suggest that KP TAMs produce a physiologically relevant amount of Tnfα, which could easily activate NF-κB signaling. However, NF-κB signaling may be activated by additional mechanisms in this context.

Figure 1.

YAP1 promotes sarcomagenesis and NF-κB activity. (A) Kaplan-Meier curve of overall survival of MFH/UPS patients (n=29; TCGA sarcoma dataset). (B) IHC of patient tissue microarray; Group 1=normal skeletal muscle, smooth muscle, arterial tissue etc.; Group 2=sarcomas black box indicates UPS. (C) Kras^G12D/+; Trp53^fl/fl model of UPS; Western blot of YAP1 from 3 independent KP and KPY tumors. (D) IHC of KP and KPY tumors (Scale bar: 100 μm). (E) Yap1 deletion in KP (n=20 mice per group from two independent cohorts). (F) Images of KP and KPY tumors. (G) Tumor volume and (H) tumor weight in KP and KPY. SEM, 1 true outlier (GraphPad Quick Calcs: outlier calculator) removed from each group. (I) Heat map of microarray analysis of 5 independent KP and KPY tumors. (J) GSEA of microarray from (I) using the”Hallmark TNF-a signaling via NF-κB” gene set. (K) Kaplan-Meier curve of overall survival of UPS patients based on PHLDA1 expression. (L) (left) mRNA from macrophages (MACs) and tumor cells (TCs) from KP tumors (right) LPS stimulated (100ng/mL; 24hrs) BMDM in vitro right SD. (M) (left) NF-κB luciferase assay in KP cells; SD, (top right) KP cells expressing control or 2 independent Yap1 shRNAs (bottom) cell sorting report indicating that the GFP-luciferase construct was present in ~86% of cells.

We tested the ability of Yap1 to control NF-κB activity in vitro using an established luciferase reporter assay (27). Sarcoma cells derived from a KP tumor were infected with a GFP labeled NF-κB -reporter construct, sorted to ~86% purity, then infected with lentivirus expressing control or Yap1 shRNA. NF-κB activity is reduced by ~50% in Yap1 shRNA-expressing cells, treated with Tnfα, compared to treated control shRNA-expressing cells (Figure 1M). From these, we conclude that YAP1 promotes NF-κB activity, which may enhance sarcomagenesis.

Hyperactivation of NF-κB super enhancers in human UPS

To determine the extent of NF-κB signaling in human UPS, we performed multiple genome wide analyses of these tumor samples directly obtained from patients including acetylated histone 3 lysine 27 (H3K27Ac) chromatin immunoprecipitation with massively parallel DNA sequencing (ChIP-seq) using 3 independent UPS patient samples originating in skeletal muscle tissue (Figure S2A). Acetylation of lysine residues on histone tails is linked to active euchromatin transcription, especially at H3K27Ac, which is associated with active cis-regulatory elements (28). Therefore, high levels of H3K27Ac at the enhancer regions of NF-κB target genes are directly associated with elevated NF-κB activity. The observed genome-wide binding of H3K27Ac antibody was consistent in all three UPS samples. We then conducted focused epigenomic analysis on regions of dramatic H3K27Ac enrichment, frequently referred to as super enhancer (SE) analysis, across all three samples to map the regions of highly active enhancers across the whole genome, and identify regions of differential hyperacetylation (SEs) (Figure 2A). We identified activation of multiple SE regions associated with NF-κB from the set of overall SEs. The SEs associated with NF-κB pathway genes correlated well with average H3K27Ac signal density in representative human UPS at SEs (Figure 2B,C). More convincingly, NF-κB pathway components, such as RELA, BCL3 and RELB showed enhanced H3K27Ac signal in all three UPS samples (Figure 2D-F), the input data are listed in Figure S2B-D, and the identified SEs were most significantly associated with the hallmark M5890 gene set referred to as, “TNFA signaling via NF-κB “ (Figure 2G-I) in all three human samples. As a control assay for H3K27Ac ChIP-Seq we performed RNA sequencing (RNA-seq) human UPS tumor sample#1 compared to normal adult skeletal muscle. Using differential gene expression analysis (29) we observed that TNFα and NF-κB pathway genes, including RELA itself, were significantly upregulated in UPS, while the muscle differentiation markers, MEF2C and MYOD1, were contrastingly downregulated (Figure 2J, enlarged in Figure S2E). Likewise, we noted high expression of NF-κB targets in UPS, including many of those identified as YAP1-regulated in the KP/ KPY microarray (i.e. CCL2 (30), BCL3 (31), PHLDA1(32)) (Figure 1I). Furthermore, GSEA confirmed that genes of the “TNFA signaling via NF-κB “ pathway are significantly and increasingly differentially expressed in UPS compared to skeletal muscle (Figure 2K), whereas “myogenesis” associated gene expression in inhibited in these tumors (Figure S2F). These RNA-Seq observations validate the utility of the H3K27Ac ChIP-Seq predicting the transcriptional profile of UPS. Thus, we have identified a “pro-proliferation” gene signature in human UPS that correlates well with our observations in the KP GEMM.

Figure 2.

ChIP-seq (H3K27Ac) and super-enhancer analysis of human undifferentiated pleomorphic sarcoma (UPS) patient samples. (A) Ranked plots of enhancers defined in three primary human UPS tumors ranked by increasing H3K27Ac ChIP-seq signal (average reads). SE-associated genes from the “Hallmark TNF-a signaling via NF-κB” gene set are highlighted in red. (B) Meta gene representation of H3K27Ac ChIP-seq read density (rpm/bp) at NF-κB associated SEs (red line) compared to all SEs (black line). The x-axis shows the start and end of the SE regions flanked by ± 10 kb of adjacent sequence. (C) Boxplot quantification of genes (reads) closest to NF-κB associated SEs compared to all other SEs. P=4.25×10e6 (Welch’s two-tailed t-test). (D-F) Gene tracks of H3K27ac ChIP-seq signal (rpm/bp) for H3K27ac at the (D) RELA, (E) BCL3 and (F) RELB regions. (G-I) Pathway enrichment analysis (via Metascape) of genes associated with SEs called via meta-analysis of 3 primary human UPS samples. Shades of red indicate significance by –log10 p-value (J) Volcano plot representation of fold change in gene expression values (logFC) from RNA-seq in normal adult skeletal muscle versus UPS patient sample#1. (K) GSEA of gene expression changes from human UPS sample #1 compared with normal human skeletal muscle tissue. Pathway analysis was performed with genes passing differential expression analysis (FDR < 0.25) and pre-ranked by logFC.

NF-κB activity promotes UPS sarcomagenesis.

The ChIP-seq and RNA-seq experiments described above were performed on a limited number of fresh frozen patient samples (n=3) due to the paucity of available UPS tissues. Therefore, we sought a larger scale approach to evaluate NF-κB activity in human UPS tissues. We performed IHC of 20 independent human skeletal muscle UPS tumor sections provided by Dr. Kumarasen Cooper (University of Pennsylvania, Dept. of Pathology& Lab Medicine). We evaluated active NF-κB (phospho-p65/p-p65) and YAP1 protein levels and found that both proteins are highly expressed in ~80% of tumors (Figure 3A) relative to normal adjacent muscle tissue (Figure 3B,C, and D) and the expression is specifically nuclear. To confirm the functional importance of NF-κB signaling in UPS we inhibited Rela (the gene encoding p65) with two independent and specific shRNAs in KP cells. Rela shRNA #1 reduced expression by ~50%. shRNA #2 was less effective, reducing expression by only ~25% (Figure 3E, F). Control and Rela shRNA expressing KP cells were implanted subcutaneously into nude mice. Importantly, Loss of Rela significantly reduced final tumor volume (Figure 3G, H) and weight (Figure 3I) and these reductions directly correlated with the degree of knockdown provided by each shRNA. Based on these findings we postulated that sarcoma cells are sensitive to NF-κB inhibitors. We compared proliferation of KP cells treated with two independent NF-κB inhibitors caffeic acid phenethyl ester (CAPE) (33, 34) and BAY 11–7085 (35) to similarly treated HCT-116 colorectal cancer cells. HCT-116 cells are thought to be particularly sensitive to NF-κB inhibition (36). KP cells are 2X as sensitive to CAPE inhibition and 3X as sensitive to BAY 11–7085 as HCT-116 cells (Figure 3J,K). Together these findings clearly show that NF-κB activity promotes UPS growth and that YAP1 expression correlates with NF-κB upregulation in human UPS.

Figure 3.

NF-κB activity is upregulated in human UPS. (A) IHC of UPS tumors. Scale bar=50μM. n=20 (B) IHC of UPS tumor sections at the invading edge of normal adjacent muscle Scale bar=50μM. n=20 (C) 20 tumor sections were quantified. (D) Positive nuclei were stratified into low-moderate, moderate-high, and high expression for YAP1 and p-P65. (E) Western blot and (F) qRT-PCR of KP cells expressing shRNAs targeting p65/RelA (NF-κB). (G) Images of subcutaneous KP tumors. (H) Tumor volume and (I) weights from shRNA expressing KP tumors. (J) KP and HCT116 cell proliferation after treatment with CAPE and (K) BAY 11–7085 for 72hrs; SD.

Dual epigenetic therapy inhibits YAP1-mediated sarcoma proliferation

Based on our identification of YAP1 as a mediator of tumorigenesis we sought inhibitory small molecule approaches in vivo. The existing YAP1 inhibitor (i.e. Verteporfin) has non-specific effects as well as cell permeability challenges. Recent findings from our group and others suggest a strong epigenetic basis for soft tissue sarcoma development and efficacy of HDAC inhibitors in pre-clinical UPS models (18, 37–39). Similarly, Mazur et al. published an RNA-seq dataset of KP driven PDAC that suggested epigenetic modulation increased expression of the key YAP1 inhibitor AMOT (40). Therefore, we queried whether inhibitors of chromatin modifiers/readers would be effective against muscle-derived UPS in a YAP1-dependent manner. We hypothesized that the chromatin landscape in UPS is permissive with regard to NF-κB signaling, which promotes constitutive activation of NF-κB SEs. Therefore, we focused on inhibitors that modify the SE landscape, such as the bromodomain inhibitor, JQ1 (41). To test this hypothesis we initially treated 3 independent sarcoma cell lines with increasing doses of JQ1, including human fibrosarcoma cells, KP cells, and a cell line derived from a second sarcoma muscle UPS GEMM, the KIA model. Though not specifically a muscle-derived tumor, some fibrosarcomas are now thought to be genetically indistinguishable from UPS (2). Consistent with this observation, HT-1080 fibrosarcoma cells require YAP1 for proliferation and share critical proliferation mechanisms with UPS cells (7). Therefore, we include HT-1080 cells in our present studies. All 3 cell lines were sensitive to JQ1 (Figure S3A). Therefore, we tested the effect of 50 mg/kg daily JQ1 on our KP-derived subcutaneous allograft sarcoma model in nude mice. JQ1 dramatically reduced tumor progression (Figure 4A) with a 5-fold reduction in final tumor volume (Figure 4B) and a 6-fold reduction in final tumor weight (Figure 4C) compared to DMSO-treated controls. Interestingly, we also found that JQ1 inhibits expression of Yap1 in vivo (Figure 4D, Figure S3B). Given JQ1’s many potential molecular effects, a rescue assay was performed to determine whether JQ1-mediated inhibition of proliferation is YAP1-dependent. We used HT-1080 cells, which are sensitive to 250 nM JQ1, easily transduced, and proliferate under control conditions at a less rapid pace than the other sarcoma cell lines. This last parameter is important because it allowed us to ectopically express a constitutively nuclear/active YAP1 mutant, YAP1(S6A), and observe the expected significant increase in proliferation (Figure 4E, blue lines), a necessary positive control for the rest of the experiment. YAP1(S6A) rescued ~40% of the JQ1-induced proliferation deficit by day 5 (Figure 4E, red lines). To confirm that the role of YAP1 in proliferation is dependent on its nuclear functions, we performed a similar rescue assay using WT-YAP1, which is shuttled back and forth from the nucleus to the cytoplasm. WT-YAP1 also rescued proliferation but the effects were less substantial as predicted (Figure 4F). Similar, but less dramatic results were observed in the more proliferative KP cells (Figure S3C). Western blot of mutant and WT YAP1 expressing cells shows the level of expression of V5-tagged constructs in each condition (Figure S3D). Consistent with the above findings we observed that 250–500 nM JQ1 treatment lowers YAP1 mRNA levels by 50% in HT-1080, KIA, and KP cells (Figure S3E,F).

Figure 4.

Epigenetic modulation by SAHA/JQ1 inhibits sarcoma growth in vivo. (A) Representative tumors from DMSO (n=10) and JQ1 (50mg/kg per day, n=10) treated mice. (B) Tumor volume and (C) weight from DMSO and JQ1 cohorts; SEM. (arrow indicates beginning of treatment at tumor volume=100mm³) (D) Western blots of DMSO and JQ1 treated tumors. (E) Proliferation assay of HT-1080 cells expressing mutant YAPS6A or (F) WT-YAP with or without 250nM JQ1; SEM. (G) 72hr MTT proliferation assay of KP and (H) KIA cells treated with SAHA/JQ1; SEM. (I) Western blot of KP cells treated with SAHA (2μM), JQ1 (0.5μM) or both for 48hrs. (J) qRT-PCR of HT-1080 and (K) KP cells treated as in (I); SEM. (L) Western blots of HT1080, KIA, STS-48, and STS-109 cells treated as in (I). (M) NF-κB luciferase reporter assay performed in KP cells treated with SAHA (2μM)/JQ1 (0.5μM) and BAY11–7085 for 12hrs or 48hrs. 12hrs prior to detection of luciferase activity, we treated with PBS or TNFα (10ng/ml); SEM (N) Western blots of KP cells.

In some cancer contexts the efficacy of JQ1 is dependent on its ability to inhibit the MYC oncogene. However, that is not the case in the sarcoma subtypes we studied (Figure S4A-D). Our previously published work showed that the pan HDAC inhibitor SAHA inhibited sarcoma proliferation in vitro and tumor growth in vivo by 50% (18) and we hypothesized that combination SAHA/JQ1 treatment would be more effective than either drug individually. MTT proliferation studies comparing dose escalation of SAHA and JQ1 separately and in combination showed that the combination of both drugs at lower doses is more effective than either drug alone in vitro (Figure 4G,H, Figure S4E). Consistent with our proliferation findings, combination therapy (SAHA (2μM)/JQ1 (0.5μM)) most effectively inhibited expression of both YAP1 and its transcriptional target Foxm1 in KP cells (Figure 4I). We saw similar effects on YAP1 and FOXM1 in 6 additional human sarcoma cell lines representing multiple subtypes including rhabdomyosarcoma, liposarcoma, Leiomyosarcoma, and UPS (Figure S4F). These findings suggest potential broader applicability of SAHA/JQ1 against human sarcomas.

We sought to determine how SAHA/JQ1 inhibits YAP1 activity so effectively. In addition to AMOT-mediated inhibition of YAP1, its activity is regulated by Hippo kinase LATS1/2-dependent phosphorylation (42). SAHA/JQ1 treatment does not change expression of the upstream hippo kinases MST1/2 or LATS1 (Figure S4G,H). However, we did observe a modest increase in LATS1 phosphorylation suggesting a possible but likely minor increase in activity ( Figure S4G*). Importantly, YAP1 phosphorylation and cytoplasmic localization increased substantially in response to SAHA/JQ1 (Figure S5A,B). Therefore, we focused on AMOT-mediated YAP1 inhibition.

Importantly, AMOT is highly expressed in human skeletal muscle (43, 44) but is lost in all commonly diagnosed sarcomas according to the Detwiller et al. sarcoma dataset (Figure S6A) (45). Our RNA-seq of human UPS confirmed this finding (Figure 2J), suggesting that AMOT may function as a tumor suppressor in sarcoma. To test whether Amot is the predominant inhibitor of Yap1 in our cells we used the Tankyrase inhibitor XAV939, which stabilizes Amot protein levels (46). Increasing doses of XAV939 clearly result in higher levels of YAP1 phosphorylation and loss of total YAP1 (Figure S6B). To confirm that expression of Amot suppresses proliferation in sarcoma we transduced human HT-1080 cells with GFP tagged WT-AMOT. We sorted the GFP-AMOT+ population to 85% purity (Figure S6C). By Immunofluorescence (IF) we observed that GFP-AMOT appropriately localizes to the perinuclear region as has been previously reported (47) (Figure S6D). Control vector and GFP-AMOT expressing cells were pulsed with fluorescent CellTrace dye. CellTrace levels were appropriately depleted during each control cell division as dye molecules are distributed to daughter cells. GFP-AMOT expressing cells quantified by flow cytometry contained ~50% more CellTrace than control cells, indicating that cell division in experimental cells was reduced by half (Figure S6E). Furthermore, analysis of the control GFP+ and GFP-AMOT+ cells at Day 5 revealed that AMOT-GFP+ cells only are reduced from ~85% to ~45% of the population, having been overtaken in culture by GFP- cells (Figure S6F). Our findings show that expression of AMOT is a strong YAP1 inhibitor in our cells and accordingly suppress sarcoma cell proliferation.

Together, our studies revealed an AMOT-dependent mechanism of YAP1 degradation in SAHA/JQ1 treated sarcoma cells. Consistent with this observation, loss of Yap1 transcription, together with increased expression of AMOT, resulted in near ablation of YAP1 targets FOXM1 and BIRC5 in SAHA/JQ1 treated cells (Figure 4I-K). We expanded our observations to include additional murine (KIA) and human (STS-48, STS-109) UPS cell lines (Figure 4L). Next we asked whether SAHA/JQ1 treatment altered NF-κB signaling. Using the NF-κB luciferase system in KP cells we evaluated NF-κB activity after 12 or 48hrs of SAHA/JQ1 treatment. The NF-κB inhibitor BAY 11–7085 was used as a control and reduced NF-κB activity by 50% at both time points. We observed that NF-κB activity doubled after 12hrs of SAHA/JQ1 treatment and then plummeted to 50% of the TNFα treated positive control at 48hrs (Figure 4M). This finding is consistent with published observations that NF-κB transcriptional activity is extremely dynamic and oscillates over time in response to stimuli (48). However, we observed that phosphorylation of p65, a key readout of NF-κB activity remains high after 12hrs of SAHA/JQ1 treatment (Figure 4N). We hypothesize that elevated NF-κB activity after 12hrs of treatment is due to increased nuclear localization of p-p65. By 48hrs of treatment p-p65 levels were substantially reduced. These observations, suggest that SAHA/JQ1 may induce oscillation but that treatment ultimately reduces NF-κB signaling

SAHA/JQ1 mediates loss of YAP1 and induces a differentiated muscle transcriptional program

Combination SAHA/JQ1 treatment inhibits Yap1 and dramatically reduces proliferation of sarcoma cells (Figure 4G-I). One possible explanation for this finding is induction of apoptosis or differentiation. Importantly, persistent YAP1 and NF-κB signaling are central components of myoblast proliferation and are suppressed during differentiation (14, 16, 49–51). Therefore, we tested the possibility that epigenetic therapy differentiates sarcoma cells into a less malignant muscle-like cell. First we evaluated NF-κB signaling in the C2C12 murine model of normal myoblast differentiation. We treated undifferentiated C2C12 myoblasts with differentiation media (DM) for up to 6 days and evaluated differentiation markers, NF-κB targets, and myotube formation. Differentiation markers (Myh1, Myh2, Mef2c, and Myh3) were all upregulated dramatically by day 6 (Figure 5A), coincident with myotube formation (Figure 5B). We also observed that expression of the YAP1-dependent NF-κB targets Ccl2 and Hbegf oscillated over time (Figure 5C), consistent with our NF-κB luciferase activity observations (Figure 4M) and published findings (48). Furthermore, YAP1-mediated NF-κB targets/regulators (Ccl2, Hbegf, Areg, and Phlda1) did not oscillate in proliferating C2C12 cells in growth media (GM) for 3 days (Figure 5D). Beyond 3 days confluence initiates expression of differentiation markers, and we can no longer accurately measure proliferation-associated gene expression. We next evaluated expression of these targets in KP cells treated with SAHA/JQ1 and observed the oscillation associated with myoblast differentiation (Figure 5E). Interestingly, NF-κB targets do not oscillate synchronously either in SAHA/JQ1 treated KP cells or in differentiating C2C12 cells; (Figure 5C) suggesting that timing of target expression may be dependent on production of specific NF-κB cofactors (52). The temporal oscillation of individual targets will have to be studied in depth to fully understand the role of these patterns in differentiation and proliferation. Our data suggest that epigenetic therapy of sarcoma cells restores the NF-κB oscillation observed in differentiating myoblasts. Consistent with this hypothesis SAHA/JQ1 treated KP cells have a significantly different morphology than control or individually drug treated cells, though they do not form myotubes, many cells appear flat, multinucleated, and in some cases seem to be merging (arrows) (Figure 5F). To determine if a muscle differentiation program is induced transcriptionally we analyzed differential changes in gene expression by microarray of DMSO and SAHA/JQ1 treated KP cells. Metascape analysis of genes enriched in DMSO treated cells showed most significant association with cell cycle and cell division (Figure 5G). However, genes enriched in SAHA/JQ1 treated cells were associated with numerous processes linked to normal muscle function, including endoplasmic reticulum (ER) stress, autophagy, catabolic metabolism, mitochondrial function, and lipid processing (Figure 5H). (53). Similarly, Ingenuity Pathway Analysis (IPA) of our KP/KPY tumor microarray revealed that Yap1 loss induces ER stress and unfolded protein response (UPR), as well as the expected changes in TNF receptor signaling suggesting that YAP1 contributes specifically to these processes during muscle differentiation (Figure S7A). The lack of induction of pathways associated with terminal skeletal muscle differentiation including hallmark “Myogenesis” suggests that SAHA/JQ1 promotes incomplete differentiation. To validate upregulation of a muscle phenotype in SAHA/JQ1 treated cells we evaluated expression of the muscle differentiation markers MEF2C, MYOD1, and CDKN1C (p57). Both MEF2C and CDKN1C mRNA levels were substantially increased due to SAHA/JQ1 treatment in KP and HT-1080 cells (Figure 5I,J). However, MYOD1 mRNA levels were unaffected (Figure 5I). MYOD is regulated at the protein level by its physical interaction with p57 (54), therefore we tested expression of Myod and p57 by Western blotting of murine (Figure 5K) and human (Figure 5L) UPS cells and saw both proteins were increased in drug treated cells. Importantly, expression of YAP1 and its transcriptional target FOXM1 (7) were nearly abolished (Figure 4I-L). We saw similar effects in HT-1080 cells. Together, these findings show that combination SAHA/JQ1 treatment promotes differentiation, potentially due to inhibition of YAP1-mediated transcriptional regulation.

Figure 5.

SAHA/JQ1 decreases sarcomagenesis by inducing muscle differentiation. (A) qRT-PCR of C2C12 cells treated with differentiation media (DM) for 0, 1, 3, or, 6 days. SD; (Days 1, 3, and 6, are compared to Day 0 for each target). (B) Images of C2C12 cells forming myotubes. Scale bars= 50μM (C) qRT-PCR of C2C12 cells treated as in (A) SD. (D) qRT-PCR of C2C12 cells treated with growth media (GM); SD. (E) qRT-PCR of KP. (SAHA/JQ1) are compared to control treated cells at each time point); SD. (F) Images of KP cells treated as in (D). Scale bars=100μM (G) Pathway enrichment analysis (via Metascape) of genes identified by microarray and enriched in KP cells treated with DMSO or (H) SAHA/JQ1 for 48hrs. Shades of red indicate significance by –log10 p-value. (I) qRT-PCR of KP and (J) HT-1080 cells treated as in (D); SEM. (K) Western blot of murine (KP, KIA) and (L) human (HT1080 and STS-48) sarcoma cells treated as in (D).

YAP1 promotes NF-κB signaling by suppressing USP31 expression

Next we sought to determine if Yap1 inhibition via SAHA/JQ1-mediates the differentiation phenotype and identify the associated mechanism. First, we investigated whether SAHA (2μM)/ JQ1 (0.5μM) treatment induced apoptosis. We performed Annexin V/ Propidium Iodide (PI) assays by flow cytometry using 1μM Staurosporin as a positive control. Whereas Staurosporin treatment dramatically increased Annexin/PI positivity compared to vehicle control, SAHA/JQ1 did not alter apoptotic levels (Figure 6A,B). Consistent with these findings, we observed no change in cleaved caspase 3 levels due to SAHA/JQ1 treatment whereas Staurosporin induced the predicted increase in caspase 3 cleavage (Figure 6C and Figure S7B). Next, we tested the prediction that dampening NF-κB signaling recapitulates the effect of SAHA/JQ1 on the cell cycle. We treated sarcoma cells with the NF-κB inhibitor CAPE and performed BrdU analysis by flow cytometry and compared the results to SAHA/JQ1 treated cells. Both treatments induced cell cycle arrest in G2 phase, though SAHA/JQ1 is significantly more effective (Figure 6D,E). To determine whether NF-κB inhibition alone is sufficient to induce Myod expression and differentiation we investigated Myod levels in sarcoma cells treated with the NF-κB inhibitor BAY117085 and found that NF-κB inhibition alone could not induce MYOD1 protein expression (Figure 6F). Additional epigenetic changes due SAHA/JQ1 are necessary to facilitate the muscle differentiation program.

Figure 6.

YAP1 regulates NF-κB signaling pathway through USP31. (A) Representative flow cytometry plots and (B) quantification of percentage of Annexin V/PI positive HT-1080 cells treated with SAHA (2μM)/JQ1 (0.5μM) for 48hrs. 6hrs prior to harvest positive control cells were treated with 1μM Staurosporin to induce apoptosis; SEM. (C) Western blot of KP cells treated as in (A). (D) Representative flow cytometry plots and quantitation of BrdU incorporation in HT-1080 cells treated as in (A). (E) Representative flow cytometry plots and quantitation of BrdU incorporation in HT-1080 cells treated with CAPE (15 μM), for 48hrs. (F) Western blots of SAHA/JQ1 (2μM)/JQ1 (0.5μM) and BAY 11–7085 (1.5μM) treated KP cells. (G) qRT-PCR of KP cells treated as in (A); SD. (H) Western blot of KP cells treated as in (A). (I) qRT-PCR of human UPS (STS-109) and HT-1080 cells as in (A); SD. (J) Western blot of STS-109 cells treated as in (A). (K) qRT-PCR after 4 days expressing shRNA; SD. (L) qRT-PCR of C2C12 cells treated with differentiation media for 0–8 days SD. (L) qRT-PCR of C2C12 cells treated with growth media for 0–8 days.

Given the widespread effects of YAP1 signaling on NF-κB activity (Figure 1I-K,M) we hypothesized that a critical upstream regulator of the NF-κB pathway is modulated by YAP1. Recently identified as a critical upstream negative regulator of NF-κB, the peptidase USP31 controls ubiquitination of the TRAF molecules, which convey signals initiated by TNFα receptor engagement downstream to p65/NF-κB (19). Usp31 levels increase in response to SAHA/JQ1 in murine KP cells (Figure 6G). Interestingly, the time point at which USP31 induction is maximal varies between cell lines according to rate of proliferation. Rapidly growing KP cells require 72hrs of treatment. Consistent with this observation, 72hrs of drug treatment in KP cells decreased expression of NF-κB targets we identified as upregulated in human UPS (Litaf; Figure 2A) and as Yap1-mediated in KP tumors (Phlda1; Figure 1I,K) (Figure 6H). The importance of this observation will be defined in later studies. Slower growing human fibrosarcoma (HT-1080) and UPS (STS-109) cells only need 12–48hrs to induce maximal USP31 (Figure 6I,J). Importantly, these findings support the conclusion that SAHA/JQ1 inhibits NF-κB activity in multiple cell lines and sarcoma subtypes. Furthermore, genetic inhibition of Yap1 via specific shRNA also induces Usp31 expression indicating that this phenotype is mediated by YAP1 (Figure 6K). To ascertain the relevance of Usp31 expression in muscle cell differentiation we evaluated differentiating C2C12 cells and observed that Usp31 expression increases during the 8-day course of differentiation (Figure 6L). Amot levels increase during precisely the same time period in C2C12 cells indicating that loss of Yap1 activity occurs simultaneously with induction of Usp31. Importantly, Usp31 levels remained consistent in proliferating C2C12 cells (Figure 6M). Together, these findings support the hypothesis that YAP1 suppresses USP31 and in doing so maintains persistently high levels of NF-κB activity, signaling and proliferation.

USP31 mediates effects of SAHA/JQ1 and YAP1 loss on NF-κB activity and tumorigenesis

To determine the functional outcome of Usp31 expression we silenced it with specific shRNA. 50% loss of Usp31 expression significantly increased in vitro KP cell proliferation (Figure 7A). These data are consistent with increased NF-κB activity. Moreover, in the luciferase reporter system Usp31 shRNA expression rescued SAHA/JQ1-mediated suppression of NF-κB activity. This finding clearly shows that Usp31 mediates the effects of SAHA/JQ1 on NF-κB activity (Figure 7B). To confirm that drug treatment inhibits NF-κB signaling through upregulation of Usp31 we evaluated the early NF-κB regulator and direct target of Usp31 peptidase activity, Traf2. K63 linked ubiquitination of Traf2 is dependent upon USP31 (19). Interestingly, Traf2 protein expression is lost in SAHA/JQ1 treated cells (Figure 7C, top), whereas our microarray analysis of KP cells showed that Traf2 mRNA levels were not significantly altered relative to Yap1 and its transcription targets Foxm1 and Birc5 (Figure 7C, bottom). These data suggest that Usp31-mediated K63-linked ubiquitination affects Traf2 protein stability. However, this hypothesis and the nature of YAP1-mediated suppression of USP31 expression (direct vs. indirect) require additional investigation. To show that YAP1’s effect on proliferation is predominantly due to its control of Usp31 expression we performed an in vivo rescue allograft assay. KP cells were infected with control or Yap1 shRNA expressing lentivirus, puromycin selected for 48hrs, then infected with control or Usp31 shRNA lentivirus. Western blot of Yap1 and qRT-PCR expression of Usp31 confirms inhibition of expression in the appropriate cells (Figure 7D). As we have previously reported, expression of Yap1 shRNA significantly reduced tumor growth and weight compared to Scr shRNA control (7). Consistent with our in vitro proliferation findings (Figure 7A), Usp31-specific shRNA increased tumor growth (Figure 7E,F). Most importantly, loss of Usp31 expression rescued the Yap1 shRNA-mediated reduction in tumor growth. We therefore conclude that Usp31 suppression is critical for YAP1-dependent tumorigenesis.

Figure 7.

Yap-mediated suppression of USP31 promotes proliferation and opposes differentiation (A) left: Proliferation assay of Usp31 shRNA expressing KP cells. Right: qRT-PCR of shRNA treated cells; SD (B) NF-κB luciferase reporter in KP cells. shSCR+TNFα vs. shUsp31+TNFα, DMSO+shSCR+TNFα vs. SAHA/JQ1+shUsp31+ TNFα; SD (C) top: KIA cells treated with SAHA (2μM)/JQ1 (0.5μM) for 48hrs. bottom: Broad institute Morpheus analysis of microarray results from Figure 5G,H. FC= fold change. Q values: Traf2 (39.4), Yap1 (0), Foxm1 (0), Birc5 (0). (D) (left) Western blot and (right) qRT-PCR from KP cells prior to injection in. (E) Tumor volume and (F) tumor weights from subcutaneous allografts of KP cells treated with lentivirus expressing shRNAs; SEM. (G) Drug scheduling for KP GEMM beginning when tumors measure 100mm³. (H) Initial 10 day tumor volumes of SAHA and JQ1 treated KP tumors; SEM. (I) Survival curve (endpoint=tumor volume of 2cm³) of drug treated tumors (n=10 mice per group from 2 cohorts). Log-Rank Chi-sq. (J) Western blot of KP tumors harvested after 20 days of treatment. (K) IHC for Ki67 and MYOD1 (Scale bar=50μm). (L) Model of proposed molecular mechanism.

SAHA/JQ1 inhibits sarcomagenesis in the KP model of UPS

One of our major goals is to identify novel therapeutic approaches for the treatment of UPS and other sarcomas. To test the potential efficacy of SAHA/JQ1 as a therapeutic strategy we tested effects of the drug combination in vivo in KP GEMM tumors using 25mg/kg SAHA and 50mg/kg JQ1 (Figure 7G). These doses and the precise schedule were optimized in detail due to the loss of several animals in pilot experiments from drug toxicity. The schedule presented here was safe and no animals died from drug administration. SAHA/JQ1 treatment caused statistically significant tumor regression in this very aggressive autochthonous model (Figure 7H). Furthermore, the combined therapy delayed time to maximum tumor volume by 2-fold (Figure 7I). Median survival of animals bearing control treated tumors was 19 days relative to 25 days (SAHA) and 33 days (JQ1). The combination of SAHA and JQ1 was most effective with a median survival of 39 days (Chi Square P value< 0.0001). Importantly, Amot levels were significantly elevated in SAHA/JQ1 treated KP tumor tissue compared to controls (Figure 7J), which is consistent with our in vitro findings. Subsequent IHC of SAHA/JQ1 treated KP autochthonous tumors revealed that Myod levels were elevated while Ki67 positivity was lost in vivo (Figure 7K). Together, these findings show that epigenetic modulation of the Hippo pathway restores normal NF-κB activity, leads to decreased sarcomagenesis, and increased muscle differentiation in vivo via Usp31.

Discussion

Targetable oncogenic driver mutations are rare in sarcomas. As a result there are no effective targeted therapies. Our goal was to identify mutant oncogene-independent mechanisms of sarcomagenesis in the subtypes common to adults (i.e. UPS and fibrosarcoma) and potential therapeutic interventions. Our study highlights the efficacy of combined epigenetic inhibitors against UPS. Though not specific inhibitors, SAHA and JQ1 may provide a new option for these patients.

In the present study, we focused on understanding the pathways regulated by YAP1 in sarcomagenesis. Through genetic and epigenetic studies, we found that YAP1 contributes to hyper-activation of NF-κB, a signaling pathway with a crucial role in muscle progenitor cell division and differentiation. In fact, ChIP-seq for H3K27Ac and super enhancer analysis of human UPS samples led to the identification of NF-κB as the most transcriptionally active pathway in UPS. YAP1-mediated control of NF-κB target expression was confirmed in a genetically engineered mouse model of sarcoma. Based on our findings we postulate that NF-κB signaling is consistently up-regulated in human UPS and is enhanced by persistent YAP1 stabilization.

NF-κB signaling is complex and requires the expression and post-translational modification of multiple key effectors. Our studies revealed that YAP1 promotes NF-κB activity by controlling expression and stability of several key upstream regulators of NF-κB signaling (i.e. USP31 and TRAF2). Using our YAP1-deficient UPS GEMM, specific shRNAs, and SAHA/JQ1, we determined that NF-κB target expression, transcriptional activity, and phosphorylation/activation of p65 (the key dimer of the NF-κB transcription factor) are all decreased when YAP1 is lost. Importantly, our studies reveal two parallel alterations in NF-κB signaling found in human UPS 1) up-regulation of YAP1 alters expression USP31, a negative regulator of NF-κB and 2) chromatin modification at NF-κB target loci is modified to allow transcription of critical genes. Together these conditions promote NF-κB activity and sarcomagenesis.

Here we also report that the mechanism by which SAHA/JQ1 inhibits YAP1 expression is multi-pronged. The combination decreases YAP1 mRNA levels by ~50% and increases expression of the YAP1 inhibitor, AMOT, which is silenced in UPS compared to normal skeletal. SAHA/JQ1 restores AMOT expression, inhibiting YAP1 activity. SAHA/JQ1 treatment restores control of Hippo pathway signaling, inhibiting proliferation and simultaneously initiating a muscle differentiation transcriptional program.

The most critical YAP1 target we identified was the novel ubiquitin peptidase USP31. Virtually nothing is known about USP31 beyond its structure and connection to NF-κB signaling (19, 55). USP31 was induced in response to YAP1 inhibition and SAHA/JQ1. Importantly, we observed that Usp31 was upregulated in differentiating C2C12 myoblasts, suggesting that in sarcoma cells SAHA/JQ1 and Yap1 loss may restore differentiation (Figure 7L). Together our findings suggest that muscle-derived UPS cells behave like proliferating myoblasts incapable of undergoing differentiation due to persistent YAP1 and NF-κB signaling. SAHA/JQ1 inhibits YAP1 and restores NF-κB to patterns observed in differentiating myoblasts, forcing tumor cells to differentiate.

Overall, our work establishes NF-κB, a key regulator of normal muscle development, as a pathway that becomes persistently upregulated during sarcomagenesis at least in part through aberrant suppression of the YAP1 target USP31. Collectively, our data suggest that YAP1 stabilization, p65 phosphorylation and AMOT suppression could potentially serve as useful biomarkers for UPS and provide the mechanistic rationale for epigenetic therapy to restore Hippo pathway activity to normal levels in the treatment of this disease.