Abstract
Novel therapies are required for the treatment of metastatic renal cell carcinoma (RCC), which is associated with inoperable disease and patient death. Histone deacetylases (HDACs) are epigenetic modifiers and potential drug targets. Additional information on molecular pathways that are altered by histone deacetylase inhibitors (HDACi) in RCC cells is warranted. It should equally be delineated further which individual members of the 18 mammalian HDACs determine the survival and tumor-associated gene expression programs of such cells. Most importantly, an ongoing dispute whether HDACi promote or suppress metastasis-associated epithelial-to-mesenchymal transition (EMT) has to be resolved before HDACi are considered further as clinically relevant drugs. Here we show how HDACi affect murine and primary human RCC cells. We find that these agents induce morphological alterations resembling the metastasis-associated EMT. However, individual and proteomics-based analyses of epithelial and mesenchymal marker proteins and of EMT-associated transcription factors (EMT-TFs) reveal that HDACi do not trigger EMT. Pathway deconvolution analysis identifies reduced proliferation and apoptosis induction as key effects of HDACi. Furthermore, these drugs lead to a reduction of the cell adhesion molecule E-cadherin and of the platelet-derived growth factor receptor-β (PDGFRβ), which is a key driver of RCC metastasis formation. Accordingly, HDACi reduce the pulmonary spread of syngeneic transplanted renal carcinoma cells in mice. Specific genetic elimination of the histone deacetylases HDAC1/HDAC2 reflects the effects of pharmacological HDAC inhibition regarding growth suppression, apoptosis, and the downregulation of E-cadherin and PDGFRβ. Thus, these epigenetic modifiers are non-redundant gatekeepers of cell fate and precise pharmacological targets.
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Acknowledgements
The Wilhelm Sander-Stiftung (2010.078 to O.H.K) supported the major part of this work. The laboratory of O.H.K is additionally supported by the Deutsche Forschungsgemeinschaft (KR2291/4-1, KR2291/5-1 and KR2291/7-1 to O.H.K), the Deutsche Krebshilfe (110909 to O.H.K; German Cancer Aid) and intramural funding (University Medical Center Mainz and Naturwissenschaftlich-medizinisches Forschungszentrum Mainz, NMFZ). We thank Franziska Müller, Dagmar Faust and Tina Brachetti for excellent technical assistance.
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204_2018_2229_MOESM1_ESM.tif
Supplementary material 1 Morphological changes in Renca cells upon HDACi treatment. Renca cells were treated with the indicated concentrations of VPA (mM) and MS-275 (µM) for 24–48 h and analyzed for morphological changes with phase-change light microscopy. Images are representative for four independent experiments. Shown are the same, complete pictures of Fig. 1B. (TIF 25509 KB)
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Supplementary material 2 Regulation of EMT associated factors. (A) Renca cells were treated with the indicated concentrations of MS-275 (µM) for 24–48 h. Four independent replicates were analyzed for global protein expression by label-free quantitation (LFQ) via mass spectrometry. Heatmap lists changes in LFQ expression levels of the indicated proteins. (B) Renca cells were treated with the indicated concentrations of MS-275 (µM) for 24–48 h. Four independent replicates were analyzed for global protein expression by label-free quantitation (LFQ) via mass spectrometry. Heatmap lists changes in LFQ expression levels of the indicated proteins. (C) Renca cells were treated with 1.5 and 5 µM MS-275 for 24–48 h and independent triplicates were analyzed for quantitative mRNA expression of Cdh1 encoding E-cadherin by qPCR. Graph shows mean ± SD (n = 3); one-way ANOVA; Dunnett multiple comparisons test; **P<0.01) (TIF 25509 KB)
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Supplementary material 3 HDACi reduce proliferation of Renca cells in vitro. (A) Graph depicts percentage of cells in S phase and G2 phase of Fig. 4c. (n = 4; one-way ANOVA; Dunnett multiple comparisons test; *P <0.05, ***P <0.001, ****P <0.0001). (B) Representative pictures for quantification of Giemsa staining in Fig. 4 F (TIF 25509 KB)
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Supplementary material 4 Quantification of HDAC1 and HDAC2 expression and morphological changes in Renca cells transfected with siRNAs against HDAC1 and HDAC2. (A) Densitometric analysis of HDAC1 levels detected by Western blot in Fig. 7a. Data were normalized to the respective loading controls. Results display relative amount of HDAC1 as mean ± SD (n = 3). (B) Densitometric analysis of HDAC2 levels detected by Western blot in Fig. 7a. Data were normalized to the respective loading controls. Results display relative amount of HDAC2 as mean ± SD (n = 3). (C) Cells were transfected with siRNAs against HDAC1 and HDAC2 and analyzed for morphological changes with phase-change light microscopy. Images are representative for three independent experiments. Shown are the same, complete pictures of Fig. 7b (TIF 25509 KB)
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Supplementary material 5 Table S1 Primer sequences for qPCR analyses shown in Fig. 3a, b and Supplemental Figure S2C (PDF 19 KB)
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Supplementary material 6 Table S2 Complete raw data set acquired by mass-spectrometry showing global protein expression (as log2 LFQ values) of Renca cells in response to 24–48 h treatment with 1.5 and 5 µM MS-275. (XLSX 107 KB)
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Kiweler, N., Brill, B., Wirth, M. et al. The histone deacetylases HDAC1 and HDAC2 are required for the growth and survival of renal carcinoma cells. Arch Toxicol 92, 2227–2243 (2018). https://doi.org/10.1007/s00204-018-2229-5
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DOI: https://doi.org/10.1007/s00204-018-2229-5