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Overview of the HERV-loci differentially expressed in SKOV3WT ovarian carcinoma cells treated with the HDACis romidepsin and vorinostat

ABSTRACT: To check the acetylation levels at the positions harbouring HERV-loci and assess their variation after the treatment with HDACis (Vorinostat, romidepsin), the univocal genomic coordinates of about 3280 HERV-loci have been compared with the position of the H3AcK9 peaks identified by ChIP-seq analysis. The first analysis confirmed that HDACis are able to modulate the acetylation of repetitive elements, including HERV sequences. Subsequently, we considered only the differential peaks (diffpeaks), i.e. the peaks showing significant variation after the treatment with vorinostat and/or romidepsin. Interestingly, even if more than half of HERV-loci co-localised with acetylation peaks after HDACi treatment, only a minority of them (0.9 to 1.9%) co-localised with diffpeaks, showing mostly a reduction in their acetylation levels. Diffpeaks co-localised HERV-loci could be classified into 24 HERV groups and included the ERV-V2 locus. We focused then on the 11 HERV-loci which co-localised with diffpeaks showing an increase in acetylation levels, hence indicating an increased transcriptional activation after HDACi treatment. Remarkably, diffpeaks corresponding to the ERV-V2 locus were identified in all samples and were the most significantly upregulated in both vorinostat and romidepsin treatment. This was in part confirmed by ChIP-qPCR in different OC cells. Beside ERV-V2, only the HML8 sequence at locus 2q11,2 showed increased acetylation levels in all treated samples, independent from the HDACis used, but having lower statistical significance. The analysis revealed that the treatment with HDACis did not only account for the significant increase of ERV-V2 acetylation, but also led to the shift and concentration of acetylation peaks at the 5’ region of the HERV provirus. This observation allows us to hypothesize that HDACi treatment could not only increase ERV-V2 transcriptional activation, but also modify its pattern of expression, and eventually leading to alternative splicing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE164809 | GEO | 2021/01/15

REPOSITORIES: GEO

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Project description:Purpose: In an HIV cure setting, Vorinostat may provide the “shock” capable of flushing HIV out of the persistent reservoir, while antiretroviral therapy is used to prevent new infections. However, this drug may modulate the expression of human endogenous retroviruses (HERVs). This study demonstrates significant modulation of HERVs and suggests that they should be considered as off-target effects of this drug treatment. Methods: Peripheral blood mononuclear cells were collected from 4 healthy donors. Naive CD4 T cells were isolated and utilized to assess HERV dysregulation after treatment with Vorinostat. Cells were collected for study and exposed to a dose of Vorinostat (10uM dose over a 24 hour period). After this, RNA was extracted from cells and their untreated paired counterparts for deep sequencing by Expression Analysis. Sequence reads that passed quality filters were mapped to the HERVd reference database using Bowtie and counted using HTSeq. Any HERV which did not achieve at least 1 count per million mapped and counted reads in at least 4 samples was discarded. Differential expression was performed upon the filtered dataset with edgeR and using TMM normalization. Results: Using an custom built data analysis pipeline, ~100 million reads per sample were mapped to the HERVd reference database and identified 10784 distinct dysregulated elements Bowtie/HTSeq workflow. Differential expression analysis was performed between vorinostat treated and untreated conditions which demonstrated 2102 dysregulated HERV elements with 1007 downregulated and 1095 upregulated (FDR < 0.05) with EdgeR. Results were further subset with a log2FC ± 3 which resulted in 451 upregulated and 363 downregulated HERV elements from 81 and 82 distinct families respectively. HERV elements from the LTR12 family were by far the most dramatically upregulated in family frequency and fold change magnitude. Further confirmation with droplet digital PCR confirmed upregulation of LTR12 elements and demonstrated an exponential dose response curve with HERV expression found at even moderate doses (334nM) of vorinostat treatment. Conclusions: This study represents the first detailed analysis of HERV dysregulation following vorinostat treatment using the untargeted approach of total RNA-Seq. These results demonstrate that vorinostat dysregulates multiple HERV families with a propensity to upregulate members of the LTR12 HERV family. This study also provides a methodology to analyze HERV dysregulation in future treatments with vorinostat or other drugs by providing a mechanism to choose primer and probe sets which will properly represent HERV dysregulation as expression across an HERV is not uniform or continuous. Finally, this work suggests that HERV dysregulation by vorinostat treatment should be considered an off-target effect of this drug and HERV elements, such as LTR12, should be monitored as biomarkers during shock and kill clinical trials with HDAC inhibitors and that trial subjects should be screened to explore further HIV:HERV interactions.

Project description:The identification of recurrent somatic mutations in genes encoding epigenetic enzymes, coupled with biochemical studies demonstrating aberrant recruitment of epigenetic enzymes such as histone deacetylases (HDACs) and histone methyltransferases (HMTs) to promoter regions through association with oncogenic fusion proteins such as PML-RARM-NM-1 and AML1-ETO has provided a strong rationale for the development compounds that target the epigenome for the treatment of cancer. HDAC inhibitors (HDACi) are potent inducers of tumor cell apoptosis but it remains unclear why tumor cells are selectively sensitive to HDACi-induced cell death. Herein we assessed the biological and molecular responses of normal and transformed cells to the FDA-approved HDACi vorinostat. Both HDACi selectively killed cells of diverse tissue origin that had been transformed through the serial introduction of different oncogenes. Time course microarray expression profiling revealed that normal and transformed cells transcriptionally responded to vorinostat treatment. Over 4200 genes responded differently to vorinostat in normal and transformed cells and gene ontology and pathway analyses identified a tumor-cell-selective pro-apoptotic gene-expression signature that consisted of BCL2 family genes. In particular, HDACi induced tumor cell-selective upregulation of the pro-apoptotic gene BMF and downregulation of the pro-survival gene BCL2A1 encoding BFL-1. Maintenance of BFL-1 levels in transformed cells through forced expression conferred vorinostat resistance indicating that specific and selective engagement of the intrinsic apoptosis pathways underlies the tumor cell-selective apoptotic activities of these agents. The ability of HDACi to affect the growth and survival of tumor cells whilst leaving normal cells relatively unharmed is fundamental to their successful clinical application. This study provides new insight into the transcriptional effects of HDACi in human donor-matched normal and transformed cells, and identifies molecules and pathways that could underpin the tumor-selective cytotoxic activity of these compounds. Whole genome expression profiling was performed for vorinostat-treated samples and control samples (DMSO vehicle control). Fourteen samples were included in this study. Each sample has three biological replicates, making forty-two arrays in total.

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Overview of the HERV-loci differentially expressed in SKOV3WT ovarian carcinoma cells treated with the HDACis romidepsin and vorinostat

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