Epigenetically maintained SW13+ and SW13- subtypes have different oncogenic potential and convert with HDAC1 inhibition

Abstract

BackgroundThe BRM and BRG1 tumor suppressor genes are mutually exclusive ATPase subunits of the SWI/SNF chromatin remodeling complex. The human adrenal carcinoma SW13 cell line can switch between a subtype which expresses these subunits, SW13+, and one that expresses neither subunit, SW13-. Loss of BRM expression occurs post-transcriptionally and can be restored via histone deacetylase (HDAC) inhibition. However, most previously used HDAC inhibitors are toxic and broad-spectrum, providing little insight into the mechanism of the switch between subtypes. In this work, we explore the mechanisms of HDAC inhibition in promoting subtype switching and further characterize the oncogenic potential of the two epigenetically distinct SW13 subtypes.MethodsSW13 subtype morphology, chemotaxis, growth rates, and gene expression were assessed by standard immunofluorescence, transwell, growth, and qPCR assays. Metastatic potential was measured by anchorage-independent growth and MMP activity. The efficacy of HDAC inhibitors in inducing subtype switching was determined by immunofluorescence and qPCR. Histone modifications were assessed by western blot.ResultsTreatment of SW13- cells with HDAC1 inhibitors most effectively promotes re-expression of BRM and VIM, characteristic of the SW13+ phenotype. During treatment, hyperacetylation of histone residues and hypertrimethylation of H3K4 is pronounced. Furthermore, histone modification enzymes, including HDACs and KDM5C, are differentially expressed during treatment but several features of this differential expression pattern differs from that seen in the SW13- and SW13+ subtypes. As the SW13- subtype is more proliferative while the SW13+ subtype is more metastatic, treatment with HDACi increases the metastatic potential of SW13 cells while restoring expression of the BRM tumor suppressor.ConclusionsWhen compared to the SW13- subtype, SW13+ cells have restored BRM expression, increased metastatic capacity, and significantly different expression of a variety of chromatin remodeling factors including those involved with histone acetylation and methylation. These data are consistent with a multistep mechanism of SW13- to SW13+ conversion and subtype stabilization: histone hypermodification results in the altered expression of chromatin remodeling factors and chromatin epigenetic enzymes and the re-expression of BRM which results in restoration of SWI/SNF complex function and leads to changes in chromatin structure and gene expression that stabilize the SW13+ phenotype.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2353-7) contains supplementary material, which is available to authorized users.

HDAC inhibition induces EMT and alterations in cellular iron homeostasis to augment ferroptosis sensitivity in SW13 cells

“…However, this is consistent with the epigenetically plastic nature of SW13 cells and previous observation that ∼2–3% of the SW13 cell population will spontaneously transition to a mesenchymal phenotype under standard culture conditions [ 8 ]. Notably, not all cells treated with FK228 for 48 h expressed vimentin, but we have previously shown that it takes 72 h to achieve 100% conversion of the entire SW13 cell population to the SW13+ phenotype [ 9 ].

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“…However, ∼2% of the cells in culture exist as a slow growing, highly invasive, mesenchymal-like subtype that expresses SMARCA2 and the mesenchymal markers vimentin and CD44 and are referred to as SW13+ cells [ 8 ]. Intriguingly, HDAC inhibition can induce a phenotype transition from the tumorigenic SW13- to the metastatic SW13+, and the conversion can be confirmed by morphological changes and induction of SMARCA2 expression [ 8 , 9 ].…”

MKL1 mediates TNF-α induced pro-inflammatory transcription by bridging the crosstalk between BRG1 and WDR5

“…For instance, Zager and Johnson have shown that during renal ischemia-reperfusion injury, enhanced binding of BRG1 to gene promoters is paralleled by progressive elevations of H3K4 methylation although it remains undetermined whether there is a cause-effect relationship [28] . Davis et al have reported that treatment of the BRG1 null SW-13 cells with HDAC inhibitors restores BRG1 expression and at the same time stimulates H3K4 hypermethylation [29] .We have previously shown that BRG1 is essential in maintaining H3K4 methylation on the endothelin (ET-1) gene promoter, likely through interacting with WDR5, in response to angiotensin II stimulation [22] . These data clearly establish a role for BRG1 in regulating locus-specific H3K4 methylation.…”

HDAC Inhibitors as Epigenetic Regulators of the Immune System: Impacts on Cancer Therapy and Inflammatory Diseases

“…However, many consequences are downstream of transcription factors and are therefore more difficult to characterize. For instance, treatment with HDAC inhibitors changes histone methylation [ 63 , 64 ], adding to the effect of acetylation on the binding of chromatin regulatory complexes (reviewed in [ 62 ]). Thus, much more characterization of the targets and consequences of acetylation must be completed.…”

“…Similarly, HDAC inhibitors have been proven to be effective in restoring the expression of the mutually exclusive, catalytically active subunits of the SWI/SNF complex, BRM and BRG1, which are silenced in nearly 20% of all cancers [ 92 – 94 ]. However, the therapeutic utility of restoring BRM and BRG1 expression via HDAC inhibition is still in question as in vitro work has demonstrated that while reexpression of these proteins does slow cancer cell growth, it also promotes metastatic traits [ 63 ].…”