P38 MAPK and Radiotherapy: Foes or Friends?

Over the last 30 years, the study of the cellular response to ionizing radiation (IR) has increased exponentially. Among the various signaling pathways affected by IR, p38 MAPK has been shown to be activated both in vitro and in vivo, with involvement in key processes triggered by IR-mediated genotoxic insult, such as the cell cycle, apoptosis or senescence. However, we do not yet have a definitive clue about the role of p38 MAPK in terms of radioresistance/sensitivity and its potential use to improve current radiotherapy. In this review, we summarize the current knowledge on this family of MAPKs in response to IR as well as in different aspects related to radiotherapy, such as their role in the control of REDOX, fibrosis, and in the radiosensitizing effect of several compounds.

ERK5 Is a Major Determinant of Chemical Sarcomagenesis: Implications in Human Pathology.

Sarcomas are a heterogeneous group of tumors in which the role of ERK5 is poorly studied. To clarify the role of this MAPK in sarcomatous pathology, we used a murine 3-methyl-cholanthrene (3MC)-induced sarcoma model. Our data show that 3MC induces pleomorphic sarcomas with muscle differentiation, showing an increased expression of ERK5. Indeed, this upregulation was also observed in human sarcomas of muscular origin, such as leiomyosarcoma or rhabdomyosarcoma. Moreover, in cell lines derived from these 3MC-induced tumors, abrogation of Mapk7 expression by using specific shRNAs decreased in vitro growth and colony-forming capacity and led to a marked loss of tumor growth in vivo. In fact, transcriptomic profiling in ERK5 abrogated cell lines by RNAseq showed a deregulated gene expression pattern for key biological processes such as angiogenesis, migration, motility, etc., correlating with a better prognostic in human pathology. Finally, among the various differentially expressed genes, Klf2 is a key mediator of the biological effects of ERK5 as indicated by its specific interference, demonstrating that the ERK5-KLF2 axis is an important determinant of sarcoma biology that should be further studied in human pathology.

ERK5 signalling pathway is a novel target of sorafenib: Implication in EGF biology.

Sorafenib is a multikinase inhibitor widely used in cancer therapy with an antitumour effect related to biological processes as proliferation, migration or invasion, among others. Initially designed as a Raf inhibitor, Sorafenib was later shown to also block key molecules in tumour progression such as VEGFR and PDGFR. In addition, sorafenib has been connected with key signalling pathways in cancer such as EGFR/EGF. However, no definitive clue about the molecular mechanism linking sorafenib and EGF signalling pathway has been established so far. Our data in HeLa, U2OS, A549 and HEK293T cells, based on in silico, chemical and genetic approaches demonstrate that the MEK5/ERK5 signalling pathway is a novel target of sorafenib. In addition, our data show how sorafenib is able to block MEK5-dependent phosphorylation of ERK5 in the Ser218/Tyr220, affecting the transcriptional activation associated with ERK5. Moreover, we demonstrate that some of the effects of this kinase inhibitor onto EGF biological responses, such as progression through cell cycle or migration, are mediated through the effect exerted onto ERK5 signalling pathway. Therefore, our observations describe a novel target of sorafenib, the ERK5 signalling pathway, and establish new mechanistic insights for the antitumour effect of this multikinase inhibitor.

Autophagic cell death associated to Sorafenib in renal cell carcinoma is mediated through Akt inhibition in an ERK1/2 independent fashion.

OBJECTIVES: To fully clarify the role of Mitogen Activated Protein Kinase in the therapeutic response to Sorafenib in Renal Cell Carcinoma as well as the cell death mechanism associated to this kinase inhibitor, we have evaluated the implication of several Mitogen Activated Protein Kinases in Renal Cell Carcinoma-derived cell lines. MATERIALS AND METHODS: An experimental model of Renal Cell Carcinoma-derived cell lines (ACHN and 786-O cells) was evaluated in terms of viability by MTT assay, induction of apoptosis by caspase 3/7 activity, autophagy induction by LC3 lipidation, and p62 degradation and kinase activity using phospho-targeted antibodies. Knock down of ATG5 and ERK5 was performed using lentiviral vector coding specific shRNA. RESULTS: Our data discard Extracellular Regulated Kinase 1/2 and 5 as well as p38 Mitogen Activated Protein Kinase pathways as mediators of Sorafenib toxic effect but instead indicate that the inhibitory effect is exerted through the PI3K/Akt signalling pathway. Furthermore, we demonstrate that inhibition of Akt mediates cell death associated to Sorafenib without caspase activation, and this is consistent with the induction of autophagy, as indicated by the use of pharmacological and genetic approaches. CONCLUSION: The present report demonstrates that Sorafenib exerts its toxic effect through the induction of autophagy in an Akt-dependent fashion without the implication of Mitogen Activated Protein Kinase. Therefore, our data discard the use of inhibitors of the RAF-MEK-ERK1/2 signalling pathway in RCC and support the use of pro-autophagic compounds, opening new therapeutic opportunities for Renal Cell Carcinoma.

HEY1 functions are regulated by its phosphorylation at Ser-68.

HEY1 (hairy/enhancer-of-split related with YRPW motif 1) is a member of the basic helix-loop-helix-orange (bHLH-O) family of transcription repressors that mediate Notch signalling. HEY1 acts as a positive regulator of the tumour suppressor p53 via still unknown mechanisms. A MALDI-TOF/TOF MS analysis has uncovered a novel HEY1 regulatory phosphorylation event at Ser-68. Strikingly, this single phosphorylation event controls HEY1 stability and function: simulation of HEY1 Ser-68 phosphorylation increases HEY1 protein stability but inhibits its ability to enhance p53 transcriptional activity. Unlike wild-type HEY1, expression of the phosphomimetic mutant HEY1-S68D failed to induce p53-dependent cell cycle arrest and it did not sensitize U2OS cells to p53-activating chemotherapeutic drugs. We have identified two related kinases, STK38 (serine/threonine kinase 38) and STK38L (serine/threonine kinase 38 like), which interact with and phosphorylate HEY1 at Ser-68. HEY1 is phosphorylated at Ser-68 during mitosis and it accumulates in the centrosomes of mitotic cells, suggesting a possible integration of HEY1-dependent signalling in centrosome function. Moreover, HEY1 interacts with a subset of p53-activating ribosomal proteins. Ribosomal stress causes HEY1 relocalization from the nucleoplasm to perinucleolar structures termed nucleolar caps. HEY1 interacts physically with at least one of the ribosomal proteins, RPL11, and both proteins cooperate in the inhibition of MDM2-mediated p53 degradation resulting in a synergistic positive effect on p53 transcriptional activity. HEY1 itself also interacts directly with MDM2 and it is subjected to MDM2-mediated degradation. Simulation of HEY1 Ser-68 phosphorylation prevents its interaction with p53, RPL11 and MDM2 and abolishes HEY1 migration to nucleolar caps upon ribosomal stress. Our findings uncover a novel mechanism for cross-talk between Notch signalling and nucleolar stress.

eIF2 kinases mediate ß-lapachone toxicity in yeast and human cancer cells.

ß-Lapachone (ß-lap) is a novel anticancer agent that selectively induces cell death in human cancer cells, by activation of the NQO1 NAD(P)H dehydrogenase and radical oxygen species (ROS) generation. We characterized the gene expression profile of budding yeast cells treated with ß-lap using cDNA microarrays. Genes involved in tolerance to oxidative stress were differentially expressed in ß-lap treated cells. ß-lap treatment generated reactive oxygen species (ROS), which were efficiently blocked by dicoumarol, an inhibitor of NADH dehydrogenases. A yeast mutant in the mitochondrial NADH dehydrogenase Nde2p was found to be resistant to ß-lap treatment, despite inducing ROS production in a WT manner. Most interestingly, DNA damage responses triggered by ß-lap were abolished in the nde2Δ mutant. Amino acid biosynthesis genes were also induced in ß-lap treated cells, suggesting that ß-lap exposure somehow triggered the General Control of Nutrients (GCN) pathway. Accordingly, ß-lap treatment increased phosphorylation of eIF2α subunit in a manner dependent on the Gcn2p kinase. eIF2α phosphorylation required Gcn1p, Gcn20p and Nde2p. Gcn2p was also required for cell survival upon exposure to ß-lap and to elicit checkpoint responses. Remarkably, ß-lap treatment increased phosphorylation of eIF2α in breast tumor cells, in a manner dependent on the Nde2p ortholog AIF, and the eIF2 kinase PERK. These findings uncover a new target pathway of ß-lap in yeast and human cells and highlight a previously unknown functional connection between Nde2p, Gcn2p and DNA damage responses.

The transcription factor CREBZF is a novel positive regulator of p53.

CREBZF is a member of the mammalian ATF/CREB family of transcription factors. Here, we describe a novel functional interaction between CREBZF and the tumor suppressor p53. CREBZF was identified in a yeast two-hybrid screen using HEY1, recently characterized as an indirect p53 activator, as bait. CREBZF interacts in vitro with both HEY1 and p53, and CREBZF expression stabilizes and activates p53. Moreover, CREBZF cooperates synergistically with HEY1 to enhance p53 transcriptional activity. On the other hand, partial depletion of endogenous CREBZF diminishes p53 protein levels and inhibits HEY1-mediated activation of p53. CREBZF-positive effects on p53 signaling may reflect, at least in part, an observed induction of posttranslational modifications in p53 known to prevent its degradation. CREBZF expression protects HCT116 cells from UV radiation-induced cell death. In addition, CREBZF expression confers sensitivity to 5-fluorouracil, a p53-activating chemotherapeutic drug. Our study suggests that CREBZF may participate in the modulation of p53 tumor suppressor function.

Identification and characterization of novel potentially oncogenic mutations in the human BAF57 gene in a breast cancer patient.

BAF57 is a core subunit present in all mammalian SWI/SNF ATP-dependent chromatin remodeling complexes, which regulates important biological processes including gene transcription, DNA recombination, DNA repair, and DNA replication. Among other functions, BAF57 mediates the recruitment of SWI/SNF to sequence-specific transcription factors. Thus, BAF57 plays a crucial role in regulating estrogen-dependent gene expression and proliferation in human cell lines derived from breast tumors. Increasing genetic and biochemical evidences suggest that mutations in BAF57 or alterations in its expression could play an oncogenic role in the mammary gland. Here, we describe two novel mutations in the BAF57 gene found in a breast cancer patient. Both mutations originate premature stop codons, leading to truncated proteins, structurally similar to another BAF57 mutant previously found in a human cell line derived from a breast tumor (BT-549). The expression of these novel BAF57 mutants has abnormally high estrogen receptor alpha (ERα) coactivating potential, suggesting that they might be involved in the aberrant estrogen-dependent proliferation that occur in the majority of breast tumors that retain ERα expression. In addition, the mutations in BAF57 affect its functional interaction with the androgen receptor and ETS2, two transcription factors that play an important role in breast cell biology. Therefore, mutations in BAF57 could impinge on several oncogenic signaling pathways contributing to the origin and/or development of breast cancer.

Repression of androgen receptor activity by HEYL, a third member of the Hairy/Enhancer-of-split-related family of Notch effectors.

The Hairy/Enhancer-of-split-related with YRPW-like motif (HEY) family of proteins are transcriptional repressors and downstream effectors of Notch signaling. We previously reported that HEY1 and HEY2 selectively repress androgen receptor (AR) signaling in mammalian cell lines and have shown that in human tissue HEY1 is excluded from the nuclei in prostate cancer but not benign prostatic hyperplasia. We have now characterized a third member of this family, HEYL, which is a more potent repressor of AR activity. HEYL interacted with and repressed AR activation function-1 domain and competitively inhibited SRC1e activation of AR transcriptional activity. Using a cell line inducibly expressing exogenous HEYL, we showed that HEYL represses endogenous AR-regulated genes and reduces androgen-dependent prostate cancer cell growth. Using a trans-repression assay, we identified both trichostatin-sensitive and -insensitive domains within HEYL; however, analysis of endogenous AR target genes suggested that HEYL represses AR activity through histone deacetylase I/II-independent mechanisms. Immunohistochemical analyses of tissue indicated that, in a fashion similar to that previously reported for HEY1, HEYL is excluded from the nuclei in prostate cancer but not adjacent benign tissue. This suggests that nuclear exclusion of HEY proteins may be an important step in the progression of prostate cancer.

The SWI/SNF chromatin remodeling subunit BAF57 is a critical regulator of estrogen receptor function in breast cancer cells.

Estrogen receptors (ERs) play critical roles in both normal mammary gland development and in the formation and progression of breast tumors, constituting a major therapeutic target for breast cancer treatment. We have previously described that ER transcriptional activity is potentiated by BAF57, a core subunit of the mammalian SWI/SNF chromatin remodeling complex. Here we provide evidence demonstrating an important role for BAF57 as regulator of ER functions in breast cancer cells. Different experimental manipulations leading to the abrogation of BAF57 expression and/or function severely reduced the expression of various endogenous ER target genes and blocked estrogen-stimulated proliferation in ZR-75-1 breast cancer cells. Moreover, using a structure-function analysis, we have defined the protein domains required for the functional interaction between ERalpha and BAF57, including a key region within the hinge of ERalpha that is essential for BAF57 recruitment and its function on ER-mediated transcription. Interestingly, we found that BAF57 is an ER subtype-selective modulator that specifically regulates ERalpha-mediated transcription. Taken together, our results suggest that targeting BAF57 could represent a new way to effectively inhibit the action of ERalpha.

Nuclear receptor regulation gears up another Notch.

In this perspective we describe examples of crosstalk between nuclear receptors (NRs) and Notch signaling by means of direct functional interactions between components of both pathways. This crosstalk may provide eukaryotic organisms with molecular mechanisms for the coordination of llong-distance endocrine signals with cell-to-cell juxtacrine communication.

Identification of BAF57 mutations in human breast cancer cell lines.

Accumulating genetic and biochemical evidences support a role for the SWI/SNF chromatin-remodeling complex in cancer development and multiple core subunits of these complexes have been found to function as tumor suppressor genes. The core SWI/SNF subunit BAF57 mediates direct interactions with estrogen and androgen receptors (ER and AR) regulating their transcriptional activity. BAF57 gene maps to chromosome band 17 q21 in close proximity to the BRCA1 gene. This locus has been associated with frequent loss of heterozygosity (LOH) and allelic imbalance in breast cancers; however, BRCA1 mutations are rare events in sporadic breast cancer with LOH in the region, suggesting that another tumor suppressor gene resides in this area. All these reasons prompted us to screen for mutations in the BAF57 gene using a panel of the most commonly used human breast cancer cell lines. All cell lines analysed contain wild-type copies of BAF57 gene with the only exception of the breast ductal carcinoma cell line BT549. Sequencing of genomic DNA and cDNA generated from BT549 mRNA demonstrated the presence of a CA dinucleotide insertion in exon 5 of BAF57. The absence of wild-type BAF57 alleles indicates that this is a biallelic inactivating mutation that causes a frameshift and as a consequence a premature stop codon leading to a truncated BAF57 protein. A functional characterisation of the truncated BAF57 showed that it has lost the ability to bind to ER but still binds to the nuclear receptor coactivator SRC1e. Furthermore, we observed that the expression of the truncated BAF57 increased the ability of SRC1e to potentiate transcriptional activation by ERalpha, suggesting that mutations in BAF57 could contribute to the oncogenic transformation in breast cancer cells.

Hey1, a mediator of notch signaling, is an androgen receptor corepressor.

Hey1 is a member of the basic helix-loop-helix-Orange family of transcriptional repressors that mediate Notch signaling. Here we show that transcription from androgen-dependent target genes is inhibited by Hey1 and that expression of a constitutively active form of Notch is capable of repressing transactivation by the endogenous androgen receptor (AR). Our results indicate that Hey1 functions as a corepressor for AF1 in the AR, providing a mechanism for cross talk between Notch and androgen-signaling pathways. Hey1 colocalizes with AR in the epithelia of patients with benign prostatic hyperplasia, where it is found in both the cytoplasm and the nucleus. In marked contrast, we demonstrate that Hey1 is excluded from the nucleus in most human prostate cancers, raising the possibility that an abnormal Hey1 subcellular distribution may have a role in the aberrant hormonal responses observed in prostate cancer.

A response unit in the first exon of the beta-amyloid precursor protein gene containing thyroid hormone receptor and Sp1 binding sites mediates negative regulation by 3,5,3'-triiodothyronine.

Thyroid hormones repress expression of APP (beta-amyloid precursor protein) in cultured cells of neuronal origin. The effect involves binding to the nuclear thyroid hormone receptor (TR) and is mediated by DNA sequences located within the first exon of the gene. These sequences contain a thyroid hormone response element that is necessary, but not sufficient, to mediate the inhibitory effect of the thyroid hormone T(3). In this report, we show that repression by T(3) is mediated by a response unit composed by the thyroid hormone response element and 5'-flanking sequences that bind Sp1 and mediate stimulation by this transcription factor. In that unit, binding sites for TR and Sp1 overlap and a complex mechanism appears to account for the TR-mediated regulation of APP. Unliganded TR does not bind to DNA and allows Sp1 to bind to DNA and stimulate APP basal expression. Binding of ligand T(3), which increases affinity of TR by DNA, precludes binding of Sp1 to DNA and decreases the Sp1-dependent expression of APP.

Nuclear receptors: a rendezvous for chromatin remodeling factors.

Nuclear receptors (NRs) are a large family of ligand-induced transcription factors that include the vitamin D receptor. The recent discovery of WINAC, a novel ATP-dependent chromatin remodeling complex, has shed new light on the molecular mechanisms by which the vitamin D receptor controls gene expression with unexpected clinical implications.

Targeting of SWI/SNF chromatin remodelling complexes to estrogen-responsive genes.

SWI/SNF complexes are ATP-dependent chromatin remodelling enzymes that have been implicated in the regulation of gene expression in yeast and higher eukaryotes. BRG1, a catalytic subunit in the mammalian SWI/SNF complex, is required for transcriptional activation by the estrogen receptor, but the mechanisms by which the complex is recruited to estrogen target genes are unknown. Here, we have identified an interaction between the estrogen receptor and BAF57, a subunit present only in mammalian SWI/SNF complexes, which is stimulated by estrogen and requires both a functional hormone-binding domain and the DNA-binding region of the receptor. We also found an additional interaction between the p160 family of coactivators and BAF57 and demonstrate that the ability of p160 coactivators to potentiate transcription by the estrogen receptor is dependent on BAF57 in transfected cells. Moreover, chromatin immunoprecipitation assays demonstrated that BAF57 is recruited to the estrogen-responsive promoter, pS2, in a ligand-dependent manner. These results suggest that one of the mechanisms for recruiting SWI/SNF complexes to estrogen target genes is by means of BAF57.