Transformation by different oncogenes relies on specific metabolic adaptations

Metabolic adaptations are emerging as common traits of cancer cells and tumor progression. In vitro transformation of NIH 3T3 cells allows the analysis of the metabolic changes triggered by a single oncogene. In this work, we have compared the metabolic changes induced by H-RAS and by the nuclear resident mutant of histone deacetylase 4 (HDAC4). RAS-transformed cells exhibit a dominant aerobic glycolytic phenotype characterized by up-regulation of glycolytic enzymes, reduced oxygen consumption and a defect in complex I activity. In this model of transformation, glycolysis is strictly required for sustaining the ATP levels and the robust cellular proliferation. By contrast, in HDAC4/TM transformed cells, glycolysis is only modestly up-regulated, lactate secretion is not augmented and, instead, mitochondrial oxygen consumption is increased. Our results demonstrate that cellular transformation can be accomplished through different metabolic adaptations and HDAC4/TM cells can represent a useful model to investigate oncogene-driven metabolic changes besides the Warburg effect

The co-existence of transcriptional activator and transcriptional repressor MEF2 complexes influences tumor aggressiveness

The contribution of MEF2 TFs to the tumorigenic process is still mysterious. Here we clarify that MEF2 can support both pro-oncogenic or tumor suppressive activities depending on the interaction with co-activators or co-repressors partners. Through these interactions MEF2 supervise histone modifications associated with gene activation/repression, such as H3K4 methylation and H3K27 acetylation. Critical switches for the generation of a MEF2 repressive environment are class IIa HDACs. In leiomyosarcomas (LMS), this two-faced trait of MEF2 is relevant for tumor aggressiveness. Class IIa HDACs are overexpressed in 22% of LMS, where high levels of MEF2, HDAC4 and HDAC9 inversely correlate with overall survival. The knock out of HDAC9 suppresses the transformed phenotype of LMS cells, by restoring the transcriptional proficiency of some MEF2-target loci. HDAC9 coordinates also the demethylation of H3K4me3 at the promoters of MEF2-target genes. Moreover, we show that class IIa HDACs do not bind all the regulative elements bound by MEF2. Hence, in a cell MEF2-target genes actively transcribed and strongly repressed can coexist. However, these repressed MEF2-targets are poised in terms of chromatin signature. Overall our results candidate class IIa HDACs and HDAC9 in particular, as druggable targets for a therapeutic intervention in LMS

Transcriptomic and genomic studies classify NKL54 as a histone deacetylase inhibitor with indirect influence on MEF2-dependent transcription

In leiomyosarcoma class IIa HDACs (histone deacetylases) bind MEF2 and convert these transcription factors into repressors to sustain proliferation. Disruption of this complex with small molecules should antagonize cancer growth. NKL54, a PAOA (pimeloylanilide o-aminoanilide) derivative, binds a hydrophobic groove of MEF2, which is used as a docking site by class IIa HDACs. However, NKL54 could also act as HDAC inhibitor (HDACI). Therefore, it is unclear which activity is predominant. Here, we show that NKL54 and similar derivatives are unable to release MEF2 from binding to class IIa HDACs. Comparative transcriptomic analysis classifies these molecules as HDACIs strongly related to SAHA/vorinostat. Low expressed genes are upregulated by HDACIs, while abundant genes are repressed. This transcriptional resetting correlates with a reorganization of H3K27 acetylation around the transcription start site (TSS). Among the upregulated genes there are several BH3-only family members, thus explaining the induction of apoptosis. Moreover, NKL54 triggers the upregulation of MEF2 and the downregulation of class IIa HDACs. NKL54 also increases the binding of MEF2D to promoters of genes that are upregulated after treatment. In summary, although NKL54 cannot outcompete MEF2 from binding to class IIa HDACs, it supports MEF2-dependent transcription through several actions, including potentiation of chromatin binding

MEF2D silencing causes opposite effect in SK-LMS-1 and SK-UT-1 cells.

<p>A) MEF2D expression was silenced by lentiviral infection using two different shRNAs. Immunoblot analysis of MEF2D and CDKN1A levels in SK-LMS-1 cells expressing the control shRNA or two different shRNAs against MEF2D. RACK1 was used as loading control. B) qRT-PCR analysis of mRNA expression levels of MEF2D and of some MEF2-target genes (<i>KLF2</i>, <i>RHOB</i>, <i>CDKN1A</i>, <i>JUN</i>, <i>CNN1</i>, <i>IRS1</i>) in SK-LMS-1 cells expressing the different shRNAs. mRNA levels are relative to control shRNA. Data are presented as mean ± SD; n = 4. C) Analysis of the cells synthetizing DNA as scored after BrdU staining. Mean ± SD; n = 3. D) SK-LMS-1 cells expressing the indicated shRNAs were seeded at 2x10⁴/ml in plates coated with 10μg/ml fibronectin, or BSA; after 16h they were subjected to time-lapse analysis for 6 hours. Results represent the individual migration rate and the average (bar) from at least 140 cells from three independent experiments. Mean and SEM are indicated. E) Invasion properties of the SK-LMS-1 cells expressing the shRNA2 against MEF2D or the control. Data are presented as mean ± SD; n = 4. Invasion of the Matrigel was scored after 16 hours and was expressed as ratio between cells invading the matrix in presence (oriented motility) and absence (random invasion) of the chemoattractant. Cells were evidenced with Hoechst 33342 staining. At least 5 fields for each condition were acquired and the invading cells were counted by using ImageJ. F) Growth in soft agar of SK-LMS-1 cells expressing the indicated shRNAs, foci were stained with MTT and counted. Data are presented as mean ± SD; n = 4. G) MEF2D expression was silenced by lentiviral infection using two different shRNAs. Immunoblot analysis of MEF2D and CDKN1A levels in SK-UT-1 cells expressing the control shRNA or two different shRNAs against MEF2D. RACK1 was used as loading control. H) qRT-PCR analysis of mRNA expression levels of MEF2D and of some MEF2-target genes (<i>KLF2</i>, <i>RHOB</i>, <i>CDKN1A</i>, <i>JUN</i>, <i>CNN1</i>, <i>IRS1</i>) in SK-UT-1 cells expressing the different shRNAs. mRNA levels are relative to control shRNA. Data are presented as mean ± SD; n = 4. I) Analysis of the cells synthetizing DNA as scored after BrdU staining. Mean ± SD; n = 3. J) SK-UT-1 cells expressing the indicated shRNAs were subjected to time-lapse analysis for 6 hours as in Fig 3D. Results represent the individual migration rate and the average (bar) from at least 140 cells from three independent experiments. Mean and SEM are indicated. K) Invasion properties of the SK-UT-1 cells expressing the shRNA2 against MEF2D or the control. Data are presented as mean ± SD; n = 4. Invasion of the Matrigel was scored after 16 hours and was expressed as ratio between cells invading the matrix in presence (oriented motility) and absence (random invasion) of the chemoattractant. Cells were evidenced with Hoechst 33342 staining. At least 5 fields for each condition were acquired and the invading cells were counted by using ImageJ. L) Growth in soft agar of SK-UT-1 cells expressing the indicated shRNAs, foci were stained with MTT and counted. Data are presented as mean ± SD; n = 4. * p < 0.05, ** p < 0.01, *** p < 0.001</p

Epigenetic changes monitored by HDAC9.

<p>A) ChIP in SK-UT-1 cells WT or KO for HDAC9. Chromatin was immunoprecipitated using the anti-HDAC9 antibody. Normal rabbit IgGs were used as control. <i>TK</i> promoter was used as negative control. Atypical MEF2-targets are indicated in orange and the classical ones in blue. The genomic regions amplified by qPCR are the same as reported in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006752#pgen.1006752.s009" target="_blank">S5E Fig</a>. Data are presented as mean ± SD; n = 4. B) ChIP in SK-UT-1 cells WT or KO for HDAC9. Chromatin was immunoprecipitated using the anti-H3K27ac and the H3K4me3 antibodies as indicated. Normal rabbit IgGs were used as control. <i>TK</i> promoter was used as negative control. Atypical MEF2-targets are indicated in orange and the classical ones in blue. The genomic regions amplified by qPCR are the same as reported in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006752#pgen.1006752.s010" target="_blank">S6A and S6B Fig</a> (for H3H27ac) and in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006752#pgen.1006752.s010" target="_blank">S6A and S6B Fig</a> (for H3K4me3). Data are presented as mean ± SD; n ≥ 4. C) Scatter dot plot representing the coding mRNA TPM levels of the four class IIa HDACs in a cohort of 26 LMS samples. The horizontal lines indicate the median. Dunn's Multiple Comparison Test was applied to test the significance of HDAC9 up-regulation. D) Scatter dot plot representing the IsoPCT of the two main isoforms of HDAC9: HDAC9 WT (red) and MITR (blue). Individual tumors (n = 26) were aligned along the x axis. E) Kaplan-Meier survival analysis related to the expression levels of HDAC9, performed on the same samples and according to the same criteria as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006752#pgen.1006752.g001" target="_blank">Fig 1H</a>. F) Summary of the shift in MEF2 transcriptional activities in relation to tumor progression in LMS. Possible co-activators and co-repressor are illustrated. The scheme describes the condition for the atypical MEF2-targets only. * p < 0.05, ** p < 0.01, *** p < 0.001.</p

Analysis of MEF2D-HDAC4 repressive complexes in LMS cells.

<p>A) Quantitative analysis of the immunofluorescence studies. LMS cells were treated or not for 2 hours with 5ng/ml leptomycin B (LC Laboratories). After fixation of the cells, immunofluorescence analysis was performed to visualize HDAC4. Nuclei were stained with Hoechst 33342. Data are presented as mean ± SD (n = 3). B) MEF2D-HDAC4 complexes were immunoprecipitated using 1μg of anti-HDAC4, or anti-USP33, as a control, antibodies. Immunoblotting using an anti-MEF2D antibody was next used for the detection. The same amounts of cellular lysates were immunoprecipitated and the immunoblot were developed under the same circumstances. C) Chromatin was immunoprecipitated from SK-LMS-1 or SK-UT-1 cells using the anti-MEF2D and the anti-HDAC4 antibodies. Anti-FLAG antibody was used as control. <i>TK</i> promoter was used as negative control. The MEF2 binding site, the amplified region and the TSS are indicated for each tested gene, respectively with a vertical arrow, two arrowheads and a horizontal arrow. The TK promoter was used as negative control.</p

The MEF2 transcriptome.

<p>A) Venn diagrams showing the number of transcripts differing significantly in response to MEF2A and MEF2D silencing in SK-LMS-1 cells (green) or in SK-UT-1 cells (violet). Differentially expressed genes (DEGs) were selected based on fold change >1.5 and <-1.5 fold and p values <0.05. B) Gene ontology (GO) analysis was performed to interpret the principal biological processes under the regulation of MEF2 in SK-LMS-1 and SK-UT-1 cells. The NES (normalized enrichment score), the FDR (false discovery rate) and the p-value are provided. C) GSEA was performed by using SK-UT-1 DNA microarray data and genes repressed or induced by MEF2D/A KD in SK-LMS-1 cells, as indicated. Two groups were created in SK-UT-1 samples: A = shControl; B = shMEF2D/A. D) GSEA was performed by using SK-UT-1 DNA microarray data and genes repressed both by MEF2D/A KD in SK-LMS-1 and significantly modulated also in SK-UT-1 cells. Two groups were created in SK-UT-1 samples: A = shControl; B = shMEF2D/A. E) Scatter plot representing genes commonly down-regulated after MEF2A and MEF2D silencing in SK-LMS-1 cells (blue dots) and significantly modulated, after the same silencing, also in SK-UT-1 cells (red squares). F) Scatter plot representing genes commonly up-regulated after MEF2A and MEF2D silencing in SK-LMS-1 cells (blue dots) and significantly modulated, after the same silencing, also in SK-UT-1 cells (red squares). G) Turkey box-plots illustrating the mRNA expression levels of classical and atypical MEF2-target genes in SK-LMS-1 and SK-UT-1 cells in our DNA microarray and in another public available DNA microarray study (GSE39262). * p < 0.05, ** p < 0.01, *** p < 0.001</p

HDAC4 and HDAC9 KO in SK-UT-1 cells.

<p>A) Immunoblot analysis of HDAC4, HDAC9 and MEF2D in the indicated SK-UT-1 clones. Two different HDAC9 KO clones were selected. Actin was used as loading control. B) mRNA expression levels of the indicated atypical and classical MEF2-target genes in SK-UT-1 cells WT or KO for HDAC9. Data are presented as mean ± SD; n = 3. C) Turkey box-plots illustrating the mRNA expression levels of classical and atypical MEF2-target genes in SK-UT-1 cells WT or KO for HDAC9. Dunn's Multiple Comparison Test was applied to test the significance. D) Immunoblot analysis of HDAC4 and HDAC9 in the indicated SK-UT-1 clones. Two different HDAC4 KO clones generated by different sgRNAs were selected. Actin was used as loading control. E) mRNA expression levels of the indicated atypical and classical MEF2-target genes in SK-UT-1 cells WT or KO for HDAC4. Data are presented as mean ± SD; n = 3. F) Turkey box-plots illustrating the mRNA expression levels of classical and atypical MEF2-target genes in SK-UT-1 cells WT or KO for HDAC4. Dunn's Multiple Comparison Test was applied to test the significance. G) Invasion properties of the SK-UT-1 cells WT, KO for HDAC4 or KO for HDAC9, as indicated. Data are presented as mean ± SD; n = 4. H) Example of growth in soft agar of SK-UT-1 cells WT or KO for HDAC9. Foci were stained with MTT. I) Quantitative results of colony formation assay for SK-UT-1 cells WT, KO for HDAC4 or KO for HDAC9, as indicated. Data are presented as mean ± SD; n ≥ 3. * p < 0.05, ** p < 0.01, *** p < 0.001</p

Defect in UPS-mediated MEF2-degradation in the most aggressive LMS cell line.

<p>A) Immunoblot analysis of HDAC4, SKP2 and MEF2 family members in LMS cells. Cells were treated for 8 hours with 2.5μM of the UPS inhibitor MG132. RACK1 was used as loading control. B) Immunoblot analysis of MEF2D isoforms in LMS cells engineered to express the dominant negative version of SKP2 (DN). RACK1 was used as loading control. C) Immunoblot analysis of MEF2D in LMS cells engineered to express an inducible version of SKP2 fused to ER as indicated. SKP2 was induced for 30 hours with 0.5μM 4-OHT. RACK1 was used as loading control and the nuclear relocalization of SKP2 after 4-OHT treatment was scored by immunofluorescence. D) Cellular lysates obtained in SK-LMS-1 cells expressing the DN mutant of SKP2 were immunoprecipitated using anti-MEF2Dα1 antibody and immunoblotted with the indicated antibodies. Immunoblots with total lysates (input) are also included. E) Cellular lysates obtained in SK-UT-1 cells expressing the DN mutant of SKP2 were immunoprecipitated using anti-MEF2Dα1 isoform and immunoblotted with the indicated antibodies. Immunoblots with total lysates (input) using the indicated antibodies are also included.</p