Increased H3K9me3 drives dedifferentiated phenotype via KLF6 repression in liposarcoma
Emily Z. Keung, Kadir C. Akdemir, Ghadah A. Al Sannaa, Jeannine Garnett, Dina Lev, Keila E. Torres, Alexander J. Lazar, Kunal Rai, Lynda Chin
Emily Z. Keung, Kadir C. Akdemir, Ghadah A. Al Sannaa, Jeannine Garnett, Dina Lev, Keila E. Torres, Alexander J. Lazar, Kunal Rai, Lynda Chin
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Research Article Oncology

Increased H3K9me3 drives dedifferentiated phenotype via KLF6 repression in liposarcoma

Abstract

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.

Authors

Emily Z. Keung, Kadir C. Akdemir, Ghadah A. Al Sannaa, Jeannine Garnett, Dina Lev, Keila E. Torres, Alexander J. Lazar, Kunal Rai, Lynda Chin

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Figure 6

Chaetocin, an inhibitor of SUV39H1, inhibits DDLPS proliferation, inhibits H3K9 trimethylation globally and at the KLF6 locus, and induces upregulation of KLF6 and the proadipogenesis factors PPARG, CEBPA, and CEBPB.

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Chaetocin, an inhibitor of SUV39H1, inhibits DDLPS proliferation, inhibi...
(A) Proliferation of DDLPS cell lines 246 and 224a upon chaetocin treatment (mean ± SEM; n = 6). Data were analyzed by 2-way ANOVA. (B) KLF6 expression by qRT-PCR analysis in DDLPS cell lines 246 and 224a following chaetocin treatment (30 nM), normalized to GAPDH (mean ± SD, n = 3). Data were analyzed by Student’s t test. (C) Global levels of H3K9me3 by Western blot analysis in DDLPS cell lines 246 and 224a following chaetocin treatment (30 nM). (D) ChIP-qPCR analysis of H3K9me3 enrichment at the KLF6 locus in DDLPS 246 and 224a cells treated with vehicle versus chaetocin (30 nM) for 24 hours (mean ± SD, n = 3). Data were analyzed by Student’s t test. (E) Relative expression of PPARG, CEBPA, and CEBPB by qRT-PCR in DDLPS cell lines 246 and 224a following chaetocin treatment (30 nM) for 24 hours, normalized to GAPDH (mean ± SD, n = 3). Data were analyzed by Student’s t test. (F) Global levels of H3K9me3 by Western blot analysis in DDLPS 224a stably expressing a control shRNA (shCntl) or KLF6 shRNA (shKLF6) following chaetocin treatment (30 nM). (G) Proliferation of DDLPS 224a stably expressing a control shRNA (shCntl) or KLF6 shRNA (shKLF6) upon chaetocin treatment (mean ± SEM; n = 6). (H) Relative expression of PPARG, CEBPA, and CEBPB by qRT-PCR in DDLPS 224a stably expressing a control shRNA (shCntl) or KLF6 shRNA (shKLF6) following chaetocin treatment (30 nM) for 24 hours, normalized to GAPDH (mean ± SD, n = 3). (I) Proposed model of KLF6 as a differentially expressed and epigenetically (H3K9me3) regulated transcription factor with a tumor-suppressive role in LPS. In DDLPS relative to WDLPS, higher H3K9me3 levels at the KLF6 locus suppresses KLF6 expression, resulting in decreased expression of the proadipocytic regulators PPARγ, CEBPα, and CEBPβ. Data are representative of 2 experiments. *P < 0.05; **P < 0.001.