| Abstract |
To fit within the confines of the cell nucleus, DNA wraps around histones and folds to assume a higher order condensed structure termed chromatin. However, this condensation hinders important processes, such as transcription, replication, repair or recombination. Various covalent modifications on the histone proteins or the DNA itself, together with the active re-positioning of nucleosomes, regulate accessibility to the DNA. These modifications are functionally linked and cross-regulated, and their combination forms a code for the interpretation of the genetic information. Some of these modifications are transferred from mother to daughter cells or even from one generation of a multicellular organism to the next. These heritable alterations in chromatin that are not accompanied by changes in the DNA sequence are termed epigenetic, and are necessary for the temporal and spatial interpretation of the genome. Alterations in chromatin structure impact fundamental cellular processes and play a central role in human disease, and, in particular, cancer.
In cancer, many physiological cellular activities are altered, resulting in uncontrolled cell proliferation, unrestricted survival, and pronounced defects in cellular morphogenesis. The latter plays a central role in conferring cancer cells with the inappropriate migratory and invasive characteristics that are important for the progression of the disease. In the metastatic process, cancer cells leave the primary tumor, and are disseminated throughout the body to seed secondary tumors at distant sites. In this process, epithelial cells lose their polarity and cell-cell adhesion, and gain migratory and invasive properties, in a process termed epithelial-mesenchymal transition (EMT), which involves a dramatic reorganization of the cytoskeleton accompanied by the formation of membrane protrusions required for invasive growth.
Several lines of evidence suggest that the cytoskeleton is regulated by epigenetic mechanisms and alterations in chromatin regulators have now been linked to deregulated cytoskeletal functions. However, the detailed mechanisms behind these processes are only now beginning to emerge. The long term goal of this proposal will be the understanding of how chromatin regulation and epigenetic mechanisms influence the onset and progression of cancer. For this we will focus on the epigenetic regulation of one of the main cellular machineries involved in the metastatic process, the cytoskeleton. Our central hypothesis is that, in cancer, epigenetic alterations frequently result in the deregulation of the mechanisms maintaining normal cytoskeleton organization and function, which in turn promotes motility and invasiveness. To address this hypothesis, it is important to elucidate the chromatin factors that are involved in the regulation of the cytoskeleton, to decipher the epigenetic mechanisms by which these critical cellular factors regulate the expression of cytoskeleton genes, and to establish how these mechanisms contribute to tumor survival, invasiveness and migration.
Actin cytoskeleton reorganization and signaling play an essential role in promoting cell movement, changing cell shape and establishing intracellular adhesion, and are thus implicated in malignant cell growth, transformation and invasion. Actin remodelling is accomplished by assembling multi-molecular complexes of structural and regulatory proteins at specific cell contact sites. Subsequently, actin filaments undergo elongation, branching, bundling, and depolymerization, leading to assembled superstructures. Actin-binding proteins are the mediators for these processes. The regulation of actin assembly and disassembly is under the control of complex signaling systems that link external signals to remodelling events, which result in altered cellular activities that adapt cell shape and/or behavior to suit new environmental conditions. During cancer progression key actin regulators, such as the members of the Rho GTPase family, become deregulated.
To elucidate our hypothesis, we examined the effect of the chromatin regulator NDY1/ KDM2B. The histone demethylase NDY1/KDM2B was isolated in a screen for novel oncogenes using retroviral insertion mutagenesis in rodents in 2008. KDM2B is downregulated during senescence in mouse embryonic fibroblasts (MEFs), while ectopic expression of KDM2B enables MEFs to bypass replicative senescence by co-operating with PRC complexes in the repression the lnk4o-Arf-Ink4b locus. KDM2B represses the Ink4a-Arf-Ink4b locus by an elaborate mechanism. On the one hand, KDM2B expression counteracts the senescence-associated downregulation of EZH2, resulting in elevated global H3K27me3 levels. Increased trimethylation of H3K27 is also observed specifically in the Ink4a-Arf-Ink4b locus and results in the recruitment of PRC1 to the locus. In addition, KDM2B is specifically recruited to the Ink4a-Arf-Ink4b locus via a PRC 1-type complex and it removes local H3K36me2 and H3K4me3 marks, marks that lead to transcriptional repression. KDM2B accelerates cell cycle progression and suppresses cell senescence during vitamin C-induced reprogramming by repressing the Ink4a-Arf~Ink4b locus and has also the capacity to promote iPSC generation in a vitamin C-independent manner, by co-operating with reprogramming factors Oct4, Sox2, and Klf4. This capacity depends on its demethylase and DNA- binding activities, but is largely independent of its role in antagonizing senescence. The function of KDM2B depends on its CxxC-ZF domain, which mediates its genome-wide binding to CpG islands. The histone demethylase KDM2B is an epigenetic factor with oncogenic properties that is regulated by the basic fibroblasts growth factor (FGF-2 or bFGF). FGF-2 is one of the 23 members of the FGF growth factor family. FGF-2 is produced by a variety of cell types and binds to the FGF receptors (primarily FGFR-1 and -2). In human cancer, FGF-2 is produced by the tumor cells or by the surrounding stroma and promotes tumor cell proliferation, migration, and invasiveness, as well as angiogenesis and the cycling of cancer stem cells. It has recently been shown that KDM2B co¬operates with Polycomb Group proteins to promote cell migration and angiogenesis in tumors. The Polycomb group (PcG) proteins mediate epigenetic inheritance of silent and active chromatin states, are essential for normal development, and are implicated in cell proliferation, stem cell identity and cancer, genomic imprinting and X-inactivation. Polycomb Repressive Complex 2 (PRC2) contains the PcG proteins EZH2, which catalyzes the tri- and di- methylation of lysine 27 of histone H3 (H3K27). The H3K27me3 mark is specifically recognized by Poly comb Repressive Complex 1 (PRC1). The core of PRC1, of which many variants are thought to exist, contains BMI1 and KDM2B, polyhomeotic (Ph) and RING/RNF-type proteins. The RING/RNF proteins have ubiquitin E3 ligase activity that targets K119 of histone H2A. Ubiquitination of H2A appears to be a critical event in gene silencing.
In our study, focusing on the role of KDM2B, we addressed its role in the epigenetic regulation of actin cytoskeleton signaling, cell-cell adhesion, cell growth, motility and migration of prostate and colon tumor cells and we elucidated the underlying mechanism. We have found that this histone demethylase play an important role in cell migration and result in major alterations of several proteins involved in cytoskeleton regulation, cell-cell adhesion and Ca2+ homeostasis. We report here that KDM2B is functionally expressed in DU-145 prostate and HCT-116 colon cancer cells and it is activated by FGF-2. KDM2B regulates the PRC proteins EZH2 and BMI1, resulting in the up-regulation or down-regulation of gene transcription and protein expression of the protein upon overexpression or knockdown of KDM2B, respectively. Also, KDM2B knockdown induced potent up-regulation of gene transcription and protein expression of the epithelial markers E- Cadherin and ZO-1, while KDM2B overexpression down-regulated the levels of both markers, suggesting control of cell adhesion by KDM2B. The opposite was obvious for the mesenchymal marker N-Cadherin in colon cancer cells. Knockdown of this epigenetic factor induced potent down-regulation of the protein levels of N-Cadherin and on the other hand, KDM2B overexpression upregulated the mesenchymal marker, suggesting control of EMT by KDM2B. These results establish a clear functional role of the epigenetic factor KDM2B in the regulation of EMT in colon tumor cells and further support the recently postulated oncogenic role of this histone demethylase in various tumors. In addition, RhoA, RhoB and RhoC protein levels diminished upon KDM2B- knockdown, while all three small GTPases became upregulated in KDM2B-overexpressing DU-145 and HCT-116 cell clones. While the transcription levels of the three small GTPases remained unchanged, RhoA and RhoB activity were diminished upon KDM2B-knockdown and upregulated in KDM2B-overexpressing cell clones. Interestingly, Racl GTPase levels increased upon KDM2B- knockdown and diminished in KDM2B-overexpressing HCT-116 colon tumor- and DU-145 prostate cancer cells. In accordance, actin reorganization with formation of stress fibers became evident in KDM2B-overexpressing cells and abolished in the presence of the Rho inhibitor C3 transferase. Furthermore, DU-145 cell migration was significantly enhanced in KDM2B overexpressing cells and abolished in C3-pretreated cells. Conversely, the retardation of cell migration observed in KDM2B knockdown cells was enhanced in C3-pretreated cells. Besides Rho- GTPases signaling, we have further analyzed whether KDM2B may as well control additional signaling effectors that regulate actin reorganization, cell growth and motility. For this reason, we have focused on the FAK/PI3K metabolic pathway and we analyzed the underlying mechanism upon silencing and/or overexpression of KDM2B. KDM2B overexpression or silencing controls the activity of FAK in DU-145 prostate- and HCT-116 colon-tumor cells without affecting gene transcription and protein expression of this kinase. Upon KDM2B overexpression in DU-145 cells, significantly enhanced migration was observed, which was abolished in cells pretreated by the specific phosphoinositide-3 kinase (PI3K) inhibitor LY294002, implying involvement of FAK/PI3K signaling in the migration process. In line with this, the p8 5-PI3K-subunit was downregulated upon knockdown of KDM2B in DU-145 cells, while the opposite effect became evident in KDM2B- overexpressing cells. On the other hand, expression and activation of the FAK/PI3K-downstream acting pro-survival kinases AKT and SGK1 were not changed. Short-term proliferation of DU-145 cells remained unaffected upon overexpression or knockdown of KDM2B, an observation being in line with the observed, unaltered expression and activation profiles of the pro-survival kinases AKT and SGK1. These results uncover novel molecular targets namely FAK and PI3K and revealed a novel functional role of KDM2B in regulating the activation of the FAK/PI3K signaling in prostate and colon cancer cells that participates in the control of cell motility without affecting cell proliferation. Last, we examined the effect of KDM2B in Ca2+ homeostasis. KDM2B overexpression in DU-145 cells, induced potent up-regulation of gene transcription of Orail and Stiml and regulated Store Operated Calcium Entry (SOCE). These results revealed a novel functional role of KDM2B in regulating FAK/PI3K signaling and Ca2+ homeostasis in prostate cancer cells.
The results of this study, establish for the first time a clear functional link between the epigenetic factor KDM2B and the regulation of cell-cell adhesion, Rho-GTPases signaling, FAK/ PI3K signaling and Ca2+ homeostasis that in turn controls actin cytoskeleton reorganization and cell migration. The chromatin factor KDM2B regulates cell motility via four at least different metabolic pathways; though actin cytoskeleton organization and RhoGTPases signaling, FAK/PI3K pathway and Ca2+ entry. Our findings imply that in tumor cells, chromatin regulators may contribute to tumor growth by controlling expression and function of cytoskeletal and cell-cell adhesion genes, invasiveness and migration and establish a clear mechanistic link, how alterations in epigenetic factors may lead to deregulated cytoskeletal functions.
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