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Increased H3k9me3 Drives Dedifferentiated Phenotype Via KLF6 Repression in Liposarcoma

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Increased H3k9me3 Drives Dedifferentiated Phenotype Via KLF6 Repression in Liposarcoma The Journal of Clinical Investigation RESEARCH ARTICLE Increased H3K9me3 drives dedifferentiated phenotype via KLF6 repression in liposarcoma Emily Z. Keung,1,2,3 Kadir C. Akdemir,1 Ghadah A. Al Sannaa,4 Jeannine Garnett,2 Dina Lev,5 Keila E. Torres,2 Alexander J. Lazar,4 Kunal Rai,1 and Lynda Chin1 1Department of Genomic Medicine and 2Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. 3Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA. 4Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. 5Department of Surgery, Sheba Medical Center, Tel Aviv, Israel. Liposarcoma (LPS) can be divided into 4 different subtypes, of which well-differentiated LPS (WDLPS) and dedifferentiated LPS (DDLPS) are the most common. WDLPS is typically low grade, whereas DDLPS is high grade, aggressive, and carries a worse prognosis. WDLPS and DDLPS frequently co-occur in patients. However, it is not clear whether DDLPS arises independently from WDLPS, or whether epigenomic alterations underly the histopathological differences of these subtypes. Here, we profiled 9 epigenetic marks in tumor samples from 151 patients with LPS and showed elevated trimethylation of histone H3 at Lys9 (H3K9me3) levels in DDLPS tumors. Integrated ChIP-seq and gene expression analyses of patient-derived cell lines revealed that H3K9me3 mediates differential regulation of genes involved in cellular differentiation and migration. Among these, Kruppel-like factor 6 (KLF6) was reduced in DDLPS, with increased H3K9me3 at associated regulatory regions. Pharmacologic inhibition of H3K9me3 with chaetocin decreased DDLPS proliferation and increased expression of the adipogenesis-associated factors PPARγ, CEBPα, and CEBPβ, suggesting that increased H3K9me3 may mediate DDLPS- associated aggressiveness and dedifferentiation properties. KLF6 overexpression partially phenocopied chaetocin treatment in DDLPS cells and induced phenotypic changes that were consistent with adipocytic differentiation, suggesting that the effects of increased H3K9me3 may be mediated through KLF6. In conclusion, we provide evidence of an epigenetic basis for the transition between WDLPS and DDLPS. Introduction 12q14-15, the best-studied molecular aberration in LPS (2, 5–7), Liposarcoma (LPS) is one of the most common subtypes of soft which results in amplification of the MDM2 gene (located at 12q15) tissue sarcoma, accounting for 24% of extremity and 45% of in 100% of cases and CDK4 in 90% of cases (5). Amplification of retroperitoneal soft tissue sarcomas (1). There are 4 recognized 12q14-15 is associated with decreased apoptosis and increased cell LPS subtypes, with well differentiated (WD) and dedifferenti- proliferation, which is thought to be mediated by dysregulation of ated (DD) histologies being the most common. Whereas WDLPS the p53 pathway and the Rb-E2F cell cycle checkpoint. Currently, are low-grade tumors that typically have a more indolent course, MDM2 and CDK4 are being targeted by small-molecule inhibitors DDLPS are high-grade, aggressive tumors with a systemic met- (RG 7112, flavoperidol, PD0332991) which have shown promising astatic rate of 5% to 20% (1) and a considerably worse patient activity in a small number of preclinical and phase I and II clinical prognosis (2). Surgical excision remains the standard of care for studies (7–11). In the past decade, several groups have also under- localized disease, as these tumors are largely resistant to conven- taken gene expression profiling, DNA copy number profiling, tional cytotoxic therapies. Nevertheless, 58% to 80% of patients whole-exome sequencing, miR profiling, and RNA sequencing with DDLPS of the retroperitoneum will succumb to locally recur- approaches toward expanding our understanding of LPS patho- rent or metastatic disease within 5 years (3). Thus, gaining a bet- genesis (3, 12–15). These efforts have implicated a number of miRs ter understanding of the pathogenesis of WDLPS and DDLPS is (miRs 143, 155, and 193b), which constitute a mechanism of epi- a critical first step toward the development of effective targeted genetic gene regulation, and potential downstream pathways as therapies to improve outcomes in patients with this disease. being involved in liposarcomagenesis (3, 13, 15). Currently, the pathogenesis of WDLPS and DDLPS is poorly In recent years, it has become increasingly evident that understood. Whether DDLPS arises from WDLPS or indepen- epigenetic deregulation plays critical roles in human disease, dently is unknown. WDLPS and DDLPS often occur synchronously including cancers (16, 17). Epigenetic processes may play impor- or metachronously in patients and show similar cytogenetic fea- tant roles in liposarcomagenesis, as suggested by 2 recent studies tures (4). Both are characterized by amplification of chromosome focused on DNA methylation profiling of LPS (3, 18). Whereas Renner et al. (18) suggested NNAT (neuronatin) as a potential tumor suppressor in myxoid LPS using DNA methylation and Conflict of interest: The authors have declared that no conflict of interest exists. Submitted: July 15, 2014; Accepted: June 4, 2015. gene expression profiling of 80 sarcomas of mixed histologies (of Reference information: J Clin Invest. 2015;125(8):2965–2978. doi:10.1172/JCI77976. which 12 were DDLPS), Taylor et al. (3) reported epigenetic aber- jci.org Volume 125 Number 8 August 2015 2965 RESEARCH ARTICLE The Journal of Clinical Investigation Table 1. H3K9me3 is differentially expressed in WDLPS and DDLPS tumors Epigenetic mark Score WD (n = 415) DD (n = 157) P value mean (median, range) mean (median, range) H3K9me3 Intensity 0.63 (1, 0–2) 0.78 (1, 0–2) 0.029 Percent 17.34 (5, 0–80) 24.17 (10, 0–80) 0.006 Mult 22.94 (5, 0–140) 32.99 (10, 0–140) 0.009 5mC Intensity 1.88 (2, 0–3) 1.74 (2, 0–3) 0.005 Percent 59.48 (60, 0–90) 63.22 (60, 0–100) <0.001 Mult 114.02 (120, 0–270) 115.07 (120, 0–270) 0.244 5hmC Intensity 1.57 (2, 0–2) 1.31 (1, 1–2) <0.001 Percent 72.48 (70, 0–100) 72.34 (80. 5–100) 0.403 Mult 115.46 (120, 0–200) 96.61 (80, 5–200) <0.001 H3K27Ac Intensity 1.63 (2, 0–3) 1.79 (2, 0–3) 0.074 Percent 50.48 (60, 0–100) 61.63 (60, 0–90) <0.001 Mult 96.45 (120, 0–240) 115.66 (120, 0–210) <0.001 H3K27me3 Intensity 1.07 (1, 0–2) 0.99 (1, 0–2) 0.168 Percent 37.64 (40, 0–90) 35.49 (40, 0–90) 0.618 Mult 55.54 (50, 0–180) 45.49 (40, 0–180) 0.133 H3K4me3 Intensity 1.41 (2, 0–3) 1.47 (2, 0–2) 0.894 Percent 51.31 (60, 0–90) 61.6 (80, 0-90) <0.001 Mult 89.93 (100, 0–240) 98.47 (100, 0–180) 0.201 H3K20me3 Intensity 1.45 (2, 0–2) 1.39 (1, 0–3) 0.173 Percent 56.74 (60, 0–90) 57.73 (60, 0–90) 0.641 Mult 94.31 (100, 0–180) 57.73 (60, 0–90) 0.330 H3K36me2 Intensity 1.14 (1, 0–2) 1.06 (1, 0–2) 0.086 Percent 46 (50, 0–90) 60.25 (55, 0–180) 0.006 Mult 60.25 (55, 0-180) 62.31 (60, 0–180) 0.321 H3K79me3 Intensity 1.63 (2, 0–2) 1.35 (1, 0–2) <0.001 Percent 66.89 (70, 0–90) 64.14 (70, 0–90) 0.409 Mult 111.78 (120, 0–180) 91.04 (80, 0–180) <0.001 Global expression of 9 epigenetic marks in human WDLPS and DDLPS tumors by immunohistochemical analysis of a TMA of WDLPS and DDLPS tumors from 151 unique patients. Cores were assigned a score for intensity of tumor nuclei staining (Intensity), percentage of tumor nuclei stained (Percent), and a third score derived by multiplying the intensity and percentage scores (Mult). Data were analyzed by Student’s t test. Bold text indicates statistical significance (P < 0.05); underlined text highlights H3K9me3 data. rations associated with the promoters of CEBPA and miR-193b tone marks (H3K4me3, H3K27Ac, H3K36me2, H3K79me3, in a subset of DDLPS tumors profiled. However, a comprehen- H3K9me3, H3K20me3, and H3K27me3) and 2 DNA methyla- sive understanding of the different epigenetic modifications that tion marks (5-methylcytosine, 5mC and 5-hydroxymethylcyto- occur has been lacking. An unbiased and systematic approach is sine, 5hmC) known to be important in the regulation of gene needed to examine epigenetic modifications and their contribu- expression (16, 17, 19, 20). Of these, H3K9me3, H3K20me3, tion to liposarcomagenesis, specifically DDLPS. H3K27me3, and 5mC are associated with gene repression, In this study, we explored the epigenomic differences between whereas H3K4me3, H3K27Ac, H3K36me2, and H3K79me3 WDLPS and DDLPS in cell-based systems and human tumor are associated with gene activation. In particular, H3K4me3 samples using unbiased approaches. We identified and validated marks active promoters, H3K27Ac marks active enhancers, and Kruppel-like factor 6 (KLF6) as an H3K9me3-controlled and dif- H3K36me2 and H3K79me3 mark transcribed genes (16, 17, 19, ferentially expressed transcription factor serving a previously 20). Optimal dilutions for immunohistochemical studies using unappreciated tumor-suppressor role in LPS. Mechanistically, Encyclopedia of DNA Elements–validated (ENCODE-validated) KLF6 may drive adipocytic differentiation through increased antibodies against these marks were first determined (data not expression of known regulators of adipogenesis including PPARγ, shown). For each of these 9 antibodies, we performed immuno- thereby leading to the observed phenotypic changes associated histochemical analysis of a tissue microarray (TMA) containing with cellular differentiation and senescence. WDLPS and DDLPS tumors surgically resected from 151 unique patients (Supplemental Figure 1; supplemental material avail- Results able online with this article; doi:10.1172/JCI77976DS1). TMA H3K9me3 is differentially marked in WDLPS and DDLPS tumors. cores were scored by assigning a mean stain intensity score (0 = To gain insight into the role of epigenetic regulation during negative, 1 = low, 2 = intermediate, 3 = high) (Supplemental Fig- liposarcomagenesis, we examined the global levels of 7 his- ure 2) as well as a percentage of tumor cells stained (0%–100%).
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