Impact of class I HDACs and their inhibitors on renal and colorectal tumor cell fate decisions
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Abstract
Advanced, metastasized renal and colorectal carcinomas are difficult to treat and frequently incurable diseases. Therefore, a key focus of modern cancer research is the identification of improved chemotherapeutic treatment options. Overexpression of histone deacetylases (HDACs) is a frequent characteristic of malignant neoplasms and often correlates with disadvantageous disease progression and therapy outcome. In recent years, inhibitors of histone deacetylases (HDACi) were introduced as a new class of small-molecule anticancer therapeutics. Since HDACs deacetylate numerous histones and non-histone proteins, the inhibition of these enzymes has implications for a large number of cellular processes. As a result of the pleiotropic actions of HDACs, the precise impact of HDACi on cellular signaling pathways remains poorly understood. This work investigated the effects of HDACs and HDACi on key cellular signaling pathways in kidney and colon tumor cell lines on a global and functional scale.
Having found that HDACi induce morphological alterations in renal cell carcinoma (RCC) cells that resemble the characteristics of a mesenchymal phenotype, this work investigated individual and proteomics-based analyses of epithelial-mesenchymal transition (EMT) signaling pathways. Proteomics and GO-term enrichment analyses revealed disturbed EMT signaling and an associated loss of cell cycle control. Detailed analysis of EMT transcription factor expression by qPCR, epithelial and mesenchymal marker protein expression by Western blot, migratory potential, and β-catenin localization by immunofluorescence confirmed these findings. Furthermore, HDACi significantly induced cell death that due to caspase-mediated apoptosis. These findings were confirmed in primary renal cells. RNAi-based knockdown experiments unveiled HDAC1 and HDAC2 as mechanistic targets of HDACi.
In colorectal cancer (CRC) cells, the specific modification of HDAC2 by sumoylation affects p53- and nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB)-dependent gene expression. The work presented demonstrates that a loss of HDAC2 sumoylation enhances CRC sensitivity towards the antimetabolite 5-fluorouracil (5-FU) as well as the DNA-crosslinker nimustine (ACNU). HDAC2 sumoylation did thereby not affect p53 activation and ATM- and ATR-dependent DNA damage signaling. In contrast, DNA damage-induced accumulation of γH2AX and γH2AX foci formation depended on HDAC2 sumoylation. Mass spectrometric analysis of global protein expression, knockdown experiments, and analyses of DNA damage repair by immunofluorescence identified the specific loss of the chromatin remodeling complex-associated DNA-helicases Brahma-related gene 1 (BRG1) and SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A containing DEAD/H box 1 (SMARCAD1) as potential cause for the observed sensitization effects. Curiously, the main cytotoxic effects of 5-FU were rather based on its RNA damage than on its DNA damage. Thus, this work reveals a multitude of novel effects of commonly used chemotherapeutics.