Prognostic implications of polycomb proteins ezh2, suz12, and eed1 and histone modification by H3K27me3 in sarcoma

Background Polycomb repressive complex 2 (PRC2; formed by EZH2, SUZ12, and EED protein subunits) and PRC1 (BMI1 protein) induce gene silencing through histone modification by H3K27me3. In the present study, we characterized the PRC expression pattern and its clinical implication in sarcoma. Methods Using immunohistochemistry, we analyzed PRC expression in 105 sarcoma patients with 5 subtypes: synovial sarcoma (n = 18), rhabdomyosarcoma (n = 28), Ewing sarcoma (n = 15), osteosarcoma (n = 30), and others (n = 14). Results The median age at diagnosis in the patient cohort was 26.8 years (range: 1–78 years) and the male-to-female ratio was 1:4. Initial disease presentation was locoregional disease in 83% of patients and initial metastatic disease in the remaining 17%. PRC expression was not significantly different according to histologic subtype (P = 0.400). Overall survival (OS) was significantly poor for SUZ12 high (P = 0.001), EED1 high (P = 0.279), and H3K27me3 high (P = 0.009). Ultimately, patients with PRC2high had significantly inferior OS than the no expression group (P = 0.009). In the Cox proportional hazard model adjusted for stage, histologic grade, surgery, margin and initial metastasis, SUZ12 expression (P = 0.020, HR 29.069, 95% CI 1.690–500.007), H3K27me3 (P = 0.010, HR 3.743, 95% CI 1.370–10.228) expression was significantly associated with shorter OS. Conclusion We detected PRC expression in various sarcomas and demonstrated its independent negative prognostic role, suggesting the PRC axis as promising therapeutic target for treating sarcoma.


Background
Sarcomas are a rare and highly heterogeneous group of neoplasms originating from bone and soft tissue and account for < 1% of all human malignancies [1,2]. Although surgical resection is the primary treatment for localized disease, more than 40% of cases ultimately experience tumor recurrence, resulting in < 12 months of overall survival (OS) [3]. For patients with these tumors, mortality is high and the local and/or systemic tumor burden results in significant morbidity.
Gene silencing though histone deacetylation, histone methylation, and DNA methylation by deregulated histone deacetylase (HDAC), histone methyltransferase, and DNA methyltransferase has been widely investigated in oncology studies. Among them, polycomb group (PcG) proteins, as epigenetic chromatic modifiers, exhibit histone methyltransferase activity. These proteins regulate various physiologic processes, such as cell cycle, embryogenic development, genetic imprinting, and oncogenesis. The polycomb group protein consists of multiprotein complexes, including polycomb repressive complexes (PRC1) involved in the maintenance of gene silencing and PRC2, which initiates gene silencing. PRC2, containing the enhancer of zeste homologue 2 (EZH2), induces histone H3 trimethylation at lysine 27 (H3K27me3) through histone methyltransferase activity. PRC1, which contains BMI-1 (B lymphoma Mo-MLV insertion region 1 homolog), is recruited to DNA by binding to the H3K27me3 mark and mediates the ubiquitylation of histone 2A at lysine 119. Such coordinated action of PRC2 and PRC1 associated with H3K27me3 on chromatin results in transcriptional repression [4]. The clinical relevance of PRC protein expression has been studied in various solid tumors, such as colorectal cancer, stomach cancer, and lymphoma [5][6][7]. However, PRC expression and its clinical implications in sarcoma have not been widely investigated.
In this study, we investigated PRC expression in various subtypes of sarcoma and evaluated its clinical relevance. We then analyzed the prognostic potential to provide a practical guide as a diagnostic and therapeutic strategy.

Patients and tissue samples
This study was conducted in a consecutive cohort of patients who were pathologically diagnosed with synovial sarcoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, mesenchymal chondrosarcoma, and epithelioid sarcoma between 1994 and 2013 at the Yonsei Cancer Center. A total of 105 formalin-fixed, paraffin-embedded (FFPE) tissue blocks were available for examination of PRC expression. Fortunately, the samples obtained are all origin site specimens and the adjuvant therapy was not previously performed. All hematoxylin and eosin (H&E) slides were independently reviewed by two experienced pathologists (E.K.K. and S.H.K).
Clinicopathologic variables such as sex, age, tumor histology and grade, tumor location, tumor stage, initial presentation of disease, and status of the resection margin were reviewed retrospectively from electronic medical records. Tumors were graded according to the FNCLCC (French Federation Nationale des Centre de Lutte Contre le Cancer) [8]. Staging was determined using the 7th edition American Joint Committee on Cancer guideline of tumor, node, and metastasis (TNM) classification. The study was approved by the institutional review board of Severance Hospital.

Immunohistochemical staining and assessment
Tissue microarray blocks were prepared as follows: the hematoxylin-eosin slides were reviewed and representative formalin-fixed, paraffin-embedded (FFPE) archival blocks were selected for each case. Tumors were confirmed and marked on the slides under a microscope. Two or three different representative tumor areas per case were selected and used to prepare the tissue microarrays. Core tissue biopsies (3 mm in diameter) were taken from individual FFPE blocks (donor blocks) and arranged in recipient paraffin blocks (tissue array blocks) using a trephine apparatus. All tissue microarray blocks were confirmed to contain suitable sarcoma lesions with more than 50% of the core area after hematoxylin and eosin staining. The original H&E slides were reviewed by two pathologists (E.K.K and S.H.K).

Statistical methods
SPSS version 18.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analyses. The correlation between PRC expression and clinicopathologic variables was analyzed using independent sample t-test for continuous variables and chi-square test for discrete variables. For survival analysis, OS was defined as the time interval between the diagnosis of metastatic/recurrent disease and death or last follow-up. Survival analysis was performed using the Kaplan-Meier method with the log-rank test. Multivariate analyses for overall survival were performed using the Cox proportional hazards model. The accepted level of statistical significance was P < 0.05.

PRC expression status and clinicopathologic features
For PRC2 (EZH2, SUZ12, and EED1 protein subunits) and H3K27me3, representative images of PRC-positive and PRC-negative cases are shown in Fig. 1. In Fig. 1. a-b, c-d, e-f and g-h are combinations of samples showing the high and low expression of the same polycomb proteins in the same pathologies. In detail, the pleomorphic tumor cells produce osteoid describing irregular trabeculae in Fig. 1a. The specimen obtained from the patient diagnosed with osteosarcoma has high grade expression of EZH2 staining. Fig. 1b. shows the specimen obtained from patients diagnosed with osteosarcoma with low grade expression of EZH2. c-d, e-f and g-h were diagnosed as osteosarcoma but showed high grade and low grade expression in SUZ12, EED1 and H3K27me3 expression, respectively. The high expression rates of EZH2, SUZ12, and EED1 were 77.1% (n = 81), 5.7% (n = 6), and 77.1% (n = 81), respectively. Each PRC expression level was significantly different according to the histologic subtype of sarcoma (Fig. 2). As seen in Fig. 2a, SUZ12 and H3K27me3 are relatively low. Fig. 2b. shows that EZH2 and H3K27me3 are low at the same time, one of them is high or both are high, and EZH2 high or H3K27me3 high occupies a high ratio as shown by the relatively low H3K27me3. As shown in Fig. 2c. SUZ12 and H3K27me3 are low at the same time, one of them is high, and both of them are high, and SUZ12 non-high and H3K27me non-high occupies a high ratio. Finally, Fig. 2d shows that EED1 and H3K27me3 are low at the same time, one of them is high and both of them are high. For the same reason, EED1 high or H3K27me3 high seems to occupy a high percentage.The expression patterns of EZH2, SUZ12, EED1, and H3K27me3 were most distinctive; EZH2 high and EED1 high were frequently observed in all subtypes. SUZ12 high and H3K27me3 high were uniformly infrequent across all subtypes, whereas H3K27me3 high was frequent in Ewing's sarcoma. There was no significant difference in the PRC2 expression of each protein with sex (P = 0.965), primary site of diagnosis (P = 0.512), histologic grade (P = 0.853), stage at diagnosis (P = 0.205), initial distant metastasis (P = 0.531), surgery (P = 0.259), resection margin (P = 0.636), and adjuvant treatment (chemotherapy; P = 0.081, radiotherapy; P = 0.144). The combined expression of all PRC2 proteins was not significant for clinicopathologic features.

Discussion
We examined the frequency and prognostic impact of PRC expression in various sarcomas. This is the first study to evaluate the prognostic impact of PRC expression in sarcoma subgroups. We found that PRC2 proteins and H3K27me3 were frequently expressed and that overexpression was as an independent prognostic factor for OS. Accumulating evidence has suggested the controversial values of PRC expression in solid tumors. Wang et al. reported that high EZH2 expression was associated with poor prognosis in colorectal cancer [10], whereas EZH2 expression was associated with relapse-free survival in another study [11]. Similarly to PRC2, BMI1 was also associated with good prognosis in breast cancer and poor prognosis in colorectal cancer [12,13]. Clinicopathologic heterogeneity including primary tumor, pathologic stage, and chemotherapy or radiotherapy may have contributed to these controversial results. Furthermore, conclusions based on one protein marker may lead to inconsistent results.
In addition to individual markers, combinations of PRC proteins have been reported by several groups. Coexpression of EZH2 and BMI1 was found to be a poor prognostic factor. Coactivation of PRC2 proteins (EZH2, SUZ12, and EED) were reported to be associated with inferior OS in NK T cell lymphoma [14]. Esophageal squamous cell carcinoma, which exhibits high expression of BMI1 and EZH2, showed poor OS and diseasefree survival. In our study of sarcoma, all PRC proteins were consistently poor prognostic factors. To obtain additional prognostic information regarding epigenetic pathways, we performed multivariate analyses using combined expression of PRC2 proteins and the association with histone modification H3K27me3. By simultaneously examining the protein complex, we clarified the prognostic value of PRC expression in sarcoma patients.
Synovial sarcoma has been widely studied to determine its association with sarcoma-specific genetic aberration. Synovial sarcoma is defined by a characteristic translocation t(X;18)(p11.2;q11.2), which is observed in > 95% of cases and results in the fusion of SS18 to the SSX1, SSX2, or SSX4 genes [15]. Thus, SS18-SSX fusion leads to disease development by disrupting the epigenetic regulation of gene expression. Soulez et al. demonstrated that the polycomb group proteins RNG1 or BMI are co-localized with SSX focal staining in synovial sarcoma [16]. In addition, poorly differentiated synovial sarcoma showed high expression of EZH2, which was predictive for distant metastasis [17]. Recently, the SS18-SSX fusion protein was reported to be associated with the recruitment of PRC, suggesting polycomb-mediated epigenetic gene regression as a mechanism of oncogenesis in synovial sarcoma cell lines [18].
In a study by Schaefer et al. loss of H3K27 trimethylation was shown in 51% (51/100) of malignant peripheral nerve sheath tumors and in another study by Prieto-Granada et al., loss of H3K27 tri-methylation was found in 69% (47/68) of malignant peripheral nerve sheath tumors [19,20]. Arjen et al. reports that malignant peripheral nerve sheath tumors with loss of H3K27 tri-methylation showed inferior survival compared to malignant peripheral nerve sheath tumors with intact H3K27 tri-methylation [21]. Schaefer et al. demonstrated increased loss of H3K27 with increasing histological grade [19]. Combinational effect of genetic aberration, heterogeneity of primary tumor and treatment modality, the role of genetic aberration in the progression of sarcoma can be considered as reasons for these paradoxical findings in sarcoma. Anthracyclines and ifosfamide, either alone or in combination, are the gold standard treatments for advanced sarcoma [3]. After the failure of conventional first-line cytotoxic chemotherapy, available treatment options such as gemcitabine, trabectedin, and pazopanib have been assessed for the subsequent treatment of sarcoma. However, these treatments are severely limited because of their high toxicities and modest survival benefits; thus, the availability of less toxic agents with novel mechanisms that can improve progression-free survival or OS are crucial. Currently, most studies have focused on the prognostic role of PRC, while the therapeutic role of PRC has not been widely examined. The initiation of gene silencing by polycomb activity has been shown to involve HDAC [22]. The HDAC inhibitor FK228 shows growth inhibitory effects in synovial sarcoma cell lines and in vivo models [23]. Introduction of SYT-SSX cDNA enhanced the sensitivity to FK228, and the growth of synovial sarcoma tumor graft was markedly inhibited by FK228 treatment. Puppe et al. reported that BRCA-1-deficient breast cancer, which overexpress EZH2, was 20-fold more effective compared to BRCA-proficient breast cancer cell lines when the H3K27me3 selective inhibitor DZNep was  used [24]. The S-adenosyl-methionine (SAM)-competitive inhibitors of EZH2, including tazemetostat (EPZ-6438, E7438) [25], (R)-N-((4-Methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-1 H-indole-3-carboxamide (CPI-1205) [26], GSK2816126, are effective and selective small molecular against EZH2. Tazemetostat, CPI-1205, and GSK2816126, are currently performed in the clinical trials in different cancer types, including lymphomas, kidney tumors, synovial sarcoma, epitheliod sarcoma, mesothelioma, advanced solid tumors, and ovarian cancer. Therefore, PRC are putative therapeutic targets in sarcoma.
The main limitations of our study include its retrospective nature and patient selection. Therefore, our findings should be validated in an independent sarcoma cohort and the response data to HDAC inhibitors should be examined in future clinical trials. Higher magnification of IHC staining and normal tissue comparing may have been better understood, but in this study it is an important limitation that we could not prepare it beforehand.

Conclusion
We determined PRC expression in various sarcomas and demonstrated the independent negative prognostic role of these proteins. Our results suggest the PRC axis as a promising therapeutic target for the treatment of sarcoma.