Role of MSK1 in the malignant phenotype of Ras-transformed mouse fibroblasts.

Activated by the RAS-MAPK signaling pathway, MSK1 is recruited to immediate-early gene (IEG) regulatory regions, where it phosphorylates histone H3 at Ser-10 or Ser-28. Chromatin remodelers and modifiers are then recruited by 14-3-3 proteins, readers of phosphoserine marks, leading to the occupancy of IEG promoters by the initiation-engaged form of RNA polymerase II and the onset of transcription. In this study, we show that this mechanism of IEG induction, initially elucidated in parental 10T1/2 murine fibroblast cells, applies to metastatic Hras1-transformed Ciras-3 cells. As the RAS-MAPK pathway is constitutively activated in Ciras-3 cells, MSK1 activity and phosphorylated H3 steady-state levels are elevated. We found that steady-state levels of the IEG products AP-1 and COX-2 were also elevated in Ciras-3 cells. When MSK1 activity was inhibited or MSK1 expression was knocked down in Ciras-3 cells, the induction of IEG expression and the steady-state levels of COX-2, FRA-1, and JUN were greatly reduced. Furthermore, MSK1 knockdown Ciras-3 cells lost their malignant phenotype, as reflected by the absence of anchorage-independent growth.

Genomic instability and histone H3 phosphorylation induction by the Ras-mitogen activated protein kinase pathway in pancreatic cancer cells.

Activating mutations in K-Ras occur in most pancreatic cancers. We investigated whether genetic changes (K-Ras mutations) in human pancreatic cancer cell lines altered genomic instability and epigenetic events responding to Ras-mitogen activated protein kinase (MAPK) signaling by characterizing 3 human pancreatic cancer cells lines with and without activating mutations in K-Ras. Activation of the Ras-MAPK pathway results in the stimulation of the histone H3 kinase, mitogen and stress activated kinase (MSK) 1, and increased phosphorylation of histone H3 at serine 10 (H3 S10ph). MSK1 and H3 S10ph have roles in neoplastic transformation. We demonstrate that the presence of a K-Ras mutation did not correlate with elevated chromosomal aberrations or increased genomic instability. Although the levels of the epidermal growth factor receptors and MSK were similar, the Ras-MAPK pathway was differentially induced by phorbol esters (12-O tetradecanoylphorbol-13-acetate) or epidermal growth factor, with the response of this signaling pathway being cell-type specific. This response corresponded downstream at the level of chromatin where stimuli-induced elevation of H3 S10ph typically paralleled the increase in phospho-extracellular signal regulated kinase 1/2. Our results present evidence that nonclonal chromosomal aberrations and epigenetic programming responding to stimulation of the Ras-MAPK pathway may be better markers for cancer progression than the upstream mutated oncogenes.

Chromatin organization and nuclear microenvironments in cancer cells.

Nuclear morphometric descriptors such as nuclear size, shape, DNA content and chromatin organization are used by pathologists as diagnostic markers for cancer. However, our knowledge of events resulting in changes in nuclear shape and chromatin organization in cancer cells is limited. Nuclear matrix proteins, which include lamins, transcription factors (Sp1) and histone modifying enzymes (histone deacetylases), and histone modifications (histone H3 phosphorylation) have roles in organizing chromatin in the interphase nucleus, regulating gene expression programs and determining nuclear shape. Histone H3 phosphorylation, a downstream target of the Ras-mitogen activated protein kinase pathway, is involved in neoplastic transformation. This article will review genetic and epigenetic events that alter chromatin organization in cancer cells and the role of the nuclear matrix in determining nuclear morphology.

Phosphorylated serine 28 of histone H3 is associated with destabilized nucleosomes in transcribed chromatin.

Histone modifications and variants have key roles in the activation and silencing of genes. Phosphorylation of histone H3 at serine 10 and serine 28 is involved in transcriptional activation of genes responding to stress or mitogen-stimulated signaling pathways. The distribution of H3-modified isoforms in G0 phase chicken erythrocyte chromatin was investigated. H3 phosphorylated at serine 28 was found highly enriched in the active/competent gene fractions, as was H3 di- and trimethylated at lysine 4. The H3 variant H3.3 in this chromatin fraction was preferentially phosphorylated at serine 28. Conversely, H3 phosphorylated at serine 10 was present in all chromatin fractions, while H3 dimethylated at lysine 9 was associated with the chromatin-containing repressed genes. H3 phosphorylated at serine 28 was located at the promoter region of the transcriptionally active, but not competent, histone H5 and beta-globin genes. We provide evidence that H3.3 phosphorylated at serine 28 was present in labile nucleosomes. We propose that destabilized nucleosomes containing H3.3 phosphorylated at serine 28 aid in the dynamic disassembly-assembly of nucleosomes in active promoters.

Chromatin modification of the trefoil factor 1 gene in human breast cancer cells by the Ras/mitogen-activated protein kinase pathway.

Histone H3 phosphorylation is a downstream response to activation of the Ras/mitogen-activated protein kinase (MAPK) pathway. This modification is thought to have a role in chromatin remodeling and in the initiation of gene transcription. In MCF-7 breast cancer cells, we observed that phosphorylated histone H3 (phospho-H3) at Ser(10) but not Ser(28) increased with phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) treatment. Although phosphorylated extracellular signal-regulated kinase 1/2 levels in these cells cultured under estradiol deplete and replete conditions displayed no change, a significant induction was observed after TPA treatment. Furthermore, whereas both estradiol and TPA increased trefoil factor 1 (TFF1) mRNA levels in these cells, only TPA-induced and not estradiol-induced TFF1 expression was inhibited by the H3 kinase mitogen and stress activated protein kinase (MSK) inhibitor H89 and MAPK kinase inhibitor UO126, showing the involvement of the Ras/MAPK following TPA induction. Mutation of the activator protein 1 (AP-1) binding site abrogated the TPA-induced transcriptional response of the luciferase reporter gene under the control of the TFF1 promoter, showing the requirement for the AP-1 site. In chromatin immunoprecipitation assays, estradiol treatment resulted in the association of the estrogen receptor-alpha (ERalpha) and acetylated H3 with the TFF1 promoter. The levels of phospho-H3 and MSK1 associated with the TFF1 promoter were moderately increased. In the presence of TPA, whereas ERalpha was not bound to the promoter, a strong association of acetylated and/or phospho-H3, MSK1, and c-Jun was observed. These results show that although both stimuli lead to TFF1 gene activation, estradiol and TPA exert their effects on TFF1 gene expression by different mechanisms.

Phosphorylation of histones by tissue transglutaminase.

Tissue transglutaminase 2 (TG2) has recently been shown to have intrinsic serine/threonine kinase activity. Since histones are known to be cross-linked by TG2, we investigated whether histones are also substrates for TG2 kinase activity. TG2 was able to phosphorylate H1, H2A, H2B, H3, and H4 histones in vitro. Using peptide substrates and phosphospecific antibodies we demonstrated that TG2 phosphorylated Ser(10) in H3 and that this phosphorylation was reduced by acetylation, whereas phosphorylation of Ser(10) by TG2 enhanced acetylation. Furthermore we demonstrated that exogenous TG2 phosphorylated H1 and H3 in nucleosome preparations. We examined the abundance of TG2 in DNA-associated proteins from MCF-7 cells treated with phorbol ester (TPA) and 17beta-estradiol (E2). TG2 abundance was significantly reduced in E2-treated cells and enhanced in TPA-treated cells. In summary we have demonstrated that TG2 is able to phosphorylate purified histone proteins, and H3 and H1 in chromatin preparations, and it is associated with chromatin in breast cancer cells. These studies suggest a novel role for TG2 in the regulation of chromatin structure and function.

Abnormalities of chromatin in tumor cells.

Nuclear morphometric descriptors such as nuclear size, shape, DNA content and chromatin organization are used by pathologists as diagnostic markers for cancer. Tumorigenesis involves a series of poorly understood morphological changes that lead to the development of hyperplasia, dysplasia, in situ carcinoma, invasive carcinoma, and in many instances finally metastatic carcinoma. Nuclei from different stages of disease progression exhibit changes in shape and the reorganization of chromatin, which appears to correlate with malignancy. Multistep tumorigenesis is a process that results from alterations in the function of DNA. These alterations result from stable genetic changes, including those of tumor suppressor genes, oncogenes and DNA stability genes, and potentially reversible epigenetic changes, which are modifications in gene function without a change in the DNA sequence. DNA methylation and histone modifications are two epigenetic mechanisms that are altered in cancer cells. The impact of genetic (e.g., mutations in Rb and ras family) and epigenetic alterations with a focus on histone modifications on chromatin structure and function in cancer cells are reviewed here.

Anti-tumor chemotherapy utilizing peptide-based approaches--apoptotic pathways, kinases, and proteasome as targets.

The pharmacological sciences are taking advantage of recent discoveries that have defined the molecular pathways governing apoptosis. These signaling cascades are frequently inactivated or distorted by mutations in cancer cells. Peptides derived from critical interaction, phosphorylation, or cleavage sites are the preferred leads (starting points) for the development of new drugs. In this review we summarize recent peptide-based approaches that target MDM2, p53, NF-kappaB, ErbB2, MAPK, as well as Smac/DIABLO, IAP BIR domains, and Bcl-2 interaction domains, with a specific focus on the BH3 domain. Separate parts of the review deal with proteasome inhibitors, integrin-derived peptides, and molecules that are being tested for tumor-selective delivery of anticancer drugs ("magic bullet" approach). The proteasome inhibitors and integrin-derived peptides show a variety of effects, targeting not only tumor growth, but also angiogenesis, metastasizing potential, and other cancer cell functions. The last part of this review describes approaches that use specific properties (surface receptors, increased enzymatic activities) of cancer cells in order to target them specifically. These new generations of anticancer drugs provide the foundations for therapies with fewer side effects and higher efficacy.

The Ras-MAPK signal transduction pathway, cancer and chromatin remodeling.

Stimulation of the Ras-mitogen-activated protein kinase (MAPK) signal transduction pathway results in a multitude of events including expression of the immediate-early genes, c-fos and c-myc. Downstream targets of this stimulated pathway are the mitogen- and stress-activated protein kinases (MSK) 1 and 2, which are histone H3 kinases. In chromatin immunoprecipitation assays, it has been shown that the mitogen-induced phosphorylated H3 is associated with the immediate-early genes and that MSK1/2 activity and H3 phosphorylation have roles in chromatin remodeling and transcription of these genes. In oncogene-transformed fibroblasts in which the Ras-MAPK pathway is constitutively active, histone H1 and H3 phosphorylation is increased and the chromatin of these cells has a more relaxed structure than the parental cells. In this review we explore the deregulation of the Ras-MAPK pathway in cancer, with an emphasis on breast cancer. We discuss the features of MSK1 and 2 and the impact of a constitutively activated Ras-MAPK pathway on chromatin remodeling and gene expression.

Histone modifications as a platform for cancer therapy.

Tumorigenesis and metastasis are a progression of events resulting from alterations in the processing of the genetic information. These alterations result from stable genetic changes (mutations) involving tumor suppressor genes and oncogenes (e.g., ras, BRAF) and potentially reversible epigenetic changes, which are modifications in gene function without a change in the DNA sequence. Mutations of genes coding for proteins that directly or indirectly influence epigenetic processes will alter the cell's gene expression program. Epigenetic mechanisms often altered in cancer cells are DNA methylation and histone modifications (acetylation, methylation, phosphorylation). This article will review the potential of these reversible epigenetic processes as targets for cancer therapies.

Mitogen- and stress-activated protein kinase 1 activity and histone h3 phosphorylation in oncogene-transformed mouse fibroblasts.

Activation of the Ras-Raf-mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase-ERK signal transduction pathway or the SAPK2/p38 pathway results in the activation of mitogen- and stress-activated protein kinase 1 (MSK1). This activation of MSK1 leads to a rapid phosphorylation of histone H3 at Ser(10). Previously, we had demonstrated that Ser(10) phosphorylated H3 was elevated in Ciras-3 (c-Ha-ras-transformed 10T12) mouse fibroblasts and that H3 phosphatase activity was similar in Ciras-3 and 10T12 cells. Here, we demonstrate that the activities of ERK and MSK1, but not p38, are elevated in Ciras-3 cells relative to these activities in the parental 10T12 cells. Analyses of the subcellular distribution of MSK1 showed that the H3 kinase was similarly distributed in Ciras-3 and 10T12 cells, with most MSK1 being present in the nucleus. In contrast to many other chromatin modifying enzymes, MSK1 was loosely bound in the nucleus and was not a component of the nuclear matrix. Our results provide evidence that oncogene-mediated activation of the Ras-mitogen-activated protein kinase signal transduction pathway elevates the activity of MSK1, resulting in the increased steady-state levels of phosphorylated H3, which may contribute to the chromatin decondensation and aberrant gene expression observed in these cells.