The most important problems and needs of rasopathy patients with a noonan syndrome spectrum disorder.

BACKGROUND: Noonan syndrome spectrum disorders (NSSDs) constitute a group within the Rasopathies, and are one of the largest groups of syndromes with impact on multi-organ involvement known. The extreme variability of the clinical phenotype is, among others, due to the numerous different genes that are involved, and the differences in clinical presentation over the life span. We have studied the needs of patients and their relatives aiming to develop, evaluate and choose focus in research, medical care and policy to better meet their perspectives. METHODS: Using the participatory and interactive Dialogue method, 80 patients and relatives mentioned 53 different problems or needs (topics) that were categorized into eight themes. These themes and the topics within each theme, were subsequently prioritized by putting them in order of importance methodologically. RESULTS: The four highest prioritized themes were: (1) Physical problems (non-musculoskeletal related); (2) Social, emotional and behavioral problems; (3) Cognitive functioning and information processing; and (4) Problems related to the musculoskeletal system. Nineteen out of the 53 topics were physical problems. According to the total group of respondents, the top 3 prioritized topics within theme 1 were coagulation problems, heart problems, and feeding problems. Also data stratified by age groups, phenotype (NS and other NSSDs) and gender showed some remarkable results. For instance, feeding problems were prioritized as the most important topic of the highest prioritized theme, according to patients aged 0-12 years. Also feeding problems show a significant difference in its prioritization according to female patients (2) compared to male patients (7). On the other hand, heart problems were not mentioned in the top three prioritized topics in the youngest age groups, although heart problems are generally considered most important for patients with NSSD. CONCLUSIONS: With our results we underline the importance of methodologically inventorying the needs of NSSD patients, not only at the group level, but to also focus on specific needs according to e.g. age, phenotype and gender. For instance, it is remarkable that both the current Clinical Guidelines and the Noonan Syndrome diagnostic criteria give little to no attention to feeding problems, though our results indicate that, to the youngest patients, these problems have top priority. A similar situation appears to apply to the clinical management of e.g. coagulation, neuropsychological and musculoskeletal problems (like physiotherapy or occupational therapy) and to a need for (educational) tools to support patients at school or at work. Our study may help to shape targeted (clinical) management, research and policy inside and outside medical (research) institutes and shed light on the complex phenotypes of NSSDs, the families' and patients' perspectives on the everyday consequences of the many different problems, as well as their needs.

Glucocorticoids affect metabolic but not muscle microvascular insulin sensitivity following high versus low salt intake.

BACKGROUNDSalt-sensitive hypertension is often accompanied by insulin resistance in obese individuals, but the underlying mechanisms are obscure. Microvascular function is known to affect both salt sensitivity of blood pressure and metabolic insulin sensitivity. We hypothesized that excessive salt intake increases blood pressure and decreases insulin-mediated glucose disposal, at least in part by impairing insulin-mediated muscle microvascular recruitment (IMMR).METHODSIn 20 lean and 20 abdominally obese individuals, we assessed mean arterial pressure (MAP; 24-hour ambulatory blood pressure measurements), insulin-mediated whole-body glucose disposal (M/I value; hyperinsulinemic-euglycemic clamp technique), IMMR (contrast-enhanced ultrasound), osmolyte and water balance, and excretion of mineralocorticoids, glucocorticoids, and amino and organic acids after a low- and high-salt diet during 7 days in a randomized, double-blind, crossover design.RESULTSOn a low-, as compared with a high-salt, intake, MAP was lower, M/I value was lower, and IMMR was greater in both lean and abdominally obese individuals. In addition, natural logarithm IMMR was inversely associated with MAP in lean participants on a low-salt diet only. On a high-salt diet, free water clearance decreased, and excretion of glucocorticoids and of amino acids involved in the urea cycle increased.CONCLUSIONOur findings imply that hemodynamic and metabolic changes resulting from alterations in salt intake are not necessarily associated. Moreover, they are consistent with the concept that a high-salt intake increases muscle glucose uptake as a response to high salt-induced, glucocorticoid-driven muscle catabolism to stimulate urea production and thereby renal water conservation.TRIAL REGISTRATIONClinicalTrials.gov, NCT02068781.

The emerging role of GATA transcription factors in development and disease.

The GATA family of transcription factors consists of six proteins (GATA1-6) which are involved in a variety of physiological and pathological processes. GATA1/2/3 are required for differentiation of mesoderm and ectoderm-derived tissues, including the haematopoietic and central nervous system. GATA4/5/6 are implicated in development and differentiation of endoderm- and mesoderm-derived tissues such as induction of differentiation of embryonic stem cells, cardiovascular embryogenesis and guidance of epithelial cell differentiation in the adult.

MK3 modulation affects BMI1-dependent and independent cell cycle check-points.

Although the MK3 gene was originally found deleted in some cancers, it is highly expressed in others. The relevance of MK3 for oncogenesis is currently not clear. We recently reported that MK3 controls ERK activity via a negative feedback mechanism. This prompted us to investigate a potential role for MK3 in cell proliferation. We here show that overexpression of MK3 induces a proliferative arrest in normal diploid human fibroblasts, characterized by enhanced expression of replication stress- and senescence-associated markers. Surprisingly, MK3 depletion evokes similar senescence characteristics in the fibroblast model. We previously identified MK3 as a binding partner of Polycomb Repressive Complex 1 (PRC1) proteins. In the current study we show that MK3 overexpression results in reduced cellular EZH2 levels and concomitant loss of epigenetic H3K27me3-marking and PRC1/chromatin-occupation at the CDKN2A/INK4A locus. In agreement with this, the PRC1 oncoprotein BMI1, but not the PCR2 protein EZH2, bypasses MK3-induced senescence in fibroblasts and suppresses P16INK4A expression. In contrast, BMI1 does not rescue the MK3 loss-of-function phenotype, suggesting the involvement of multiple different checkpoints in gain and loss of MK3 function. Notably, MK3 ablation enhances proliferation in two different cancer cells. Finally, the fibroblast model was used to evaluate the effect of potential tumorigenic MK3 driver-mutations on cell proliferation and M/SAPK signaling imbalance. Taken together, our findings support a role for MK3 in control of proliferation and replicative life-span, in part through concerted action with BMI1, and suggest that the effect of MK3 modulation or mutation on M/SAPK signaling and, ultimately, proliferation, is cell context-dependent.

Spectrin repeat containing nuclear envelope 1 and forkhead box protein E1 are promising markers for the detection of colorectal cancer in blood.

Identifying biomarkers in body fluids may improve the noninvasive detection of colorectal cancer. Previously, we identified N-Myc downstream-regulated gene 4 (NDRG4) and GATA binding protein 5 (GATA5) methylation as promising biomarkers for colorectal cancer in stool DNA. Here, we examined the utility of NDRG4, GATA5, and two additional markers [Forkhead box protein E1 (FOXE1) and spectrin repeat containing nuclear envelope 1 (SYNE1)] promoter methylation as biomarkers in plasma DNA. Quantitative methylation-specific PCR was performed on plasma DNA from 220 patients with colorectal cancer and 684 noncancer controls, divided in a training set and a test set. Receiver operating characteristic analysis was performed to measure the area under the curve of GATA5, NDRG4, SYNE1, and FOXE1 methylation. Functional assays were performed in SYNE1 and FOXE1 stably transfected cell lines. The sensitivity of NDRG4, GATA5, FOXE1, and SYNE1 methylation in all stages of colorectal cancer (154 cases, 444 controls) was 27% [95% confidence interval (CI), 20%-34%), 18% (95% CI, 12%-24%), 46% (95% CI, 38%-54%), and 47% (95% CI, 39%-55%), with a specificity of 95% (95% CI, 93%-97%), 99% (95% CI, 98%-100%), 93% (95% CI, 91%-95%), and 96% (95% CI, 94%-98%), respectively. Combining SYNE1 and FOXE1, increased the sensitivity to 56% (95% CI, 48%-64%), while the specificity decreased to 90% (95% CI, 87%-93%) in the training set and to 58% sensitivity (95% CI, 46%-70%) and 91% specificity (95% CI, 80%-100%) in a test set (66 cases, 240 controls). SYNE1 overexpression showed no major differences in cell proliferation, migration, and invasion compared with controls. Overexpression of FOXE1 significantly decreased the number of colonies in SW480 and HCT116 cell lines. Overall, our data suggest that SYNE1 and FOXE1 are promising markers for colorectal cancer detection.

Epigenetics in radiotherapy: where are we heading?

Radiotherapy is an important component of anti-cancer treatment. However, not all cancer patients respond to radiotherapy, and with current knowledge clinicians are unable to predict which patients are at high risk of recurrence after radiotherapy. There is therefore an urgent need for biomarkers to guide clinical decision-making. Although the importance of epigenetic alterations is widely accepted, their application as biomarkers in radiotherapy has not been studied extensively. In addition, it has been suggested that radiotherapy itself introduces epigenetic alterations. As epigenetic alterations can potentially be reversed by drug treatment, they are interesting candidate targets for anticancer therapy or radiotherapy sensitizers. The application of demethylating drugs or histone deacetylase inhibitors to sensitize patients for radiotherapy has been studied in vitro, in vivo as well as in clinical trials with promising results. This review describes the current knowledge on epigenetics in radiotherapy.

MK3 controls Polycomb target gene expression via negative feedback on ERK.

BACKGROUND: Gene-environment interactions are mediated by epigenetic mechanisms. Polycomb Group proteins constitute part of an epigenetic cellular transcriptional memory system that is subject to dynamic modulation during differentiation. Molecular insight in processes that control dynamic chromatin association and dissociation of Polycomb repressive complexes during and beyond development is limited. We recently showed that MK3 interacts with Polycomb repressive complex 1 (PRC1). The functional relevance of this interaction, however, remained poorly understood. MK3 is activated downstream of mitogen- and stress-activated protein kinases (M/SAPKs), all of which fulfill crucial roles during development. We here use activation of the immediate-early response gene ATF3, a bona fide PRC1 target gene, as a model to study how MK3 and its effector kinases MAPK/ERK and SAPK/P38 are involved in regulation of PRC1-dependent ATF3 transcription. RESULTS: Our current data show that mitogenic signaling through ERK, P38 and MK3 regulates ATF3 expression by PRC1/chromatin dissociation and epigenetic modulation. Mitogenic stimulation results in transient P38-dependent H3S28 phosphorylation and ERK-driven PRC1/chromatin dissociation at PRC1 targets. H3S28 phosphorylation by itself appears not sufficient to induce PRC1/chromatin dissociation, nor ATF3 transcription, as inhibition of MEK/ERK signaling blocks BMI1/chromatin dissociation and ATF3 expression, despite induced H3S28 phosphorylation. In addition, we establish that concomitant loss of local H3K27me3 promoter marking is not required for ATF3 activation. We identify pERK as a novel signaling-induced binding partner of PRC1, and provide evidence that MK3 controls ATF3 expression in cultured cells via negative regulatory feedback on M/SAPKs. Dramatically increased ectopic wing vein formation in the absence of Drosophila MK in a Drosophila ERK gain-of-function wing vein patterning model, supports the existence of MK-mediated negative feedback regulation on pERK. CONCLUSION: We here identify and characterize important actors in a PRC1-dependent epigenetic signal/response mechanism, some of which appear to be nonspecific global responses, whereas others provide modular specificity. Our findings provide novel insight into a Polycomb-mediated epigenetic mechanism that dynamically controls gene transcription and support a direct link between PRC1 and cellular responses to changes in the microenvironment.

Genetics and epigenetics of cutaneous malignant melanoma: a concert out of tune.

Cutaneous malignant melanoma (CMM) is the most life-threatening neoplasm of the skin and is considered a major health problem as both incidence and mortality rates continue to rise. Once CMM has metastasized it becomes therapy-resistant and is an inevitably deadly disease. Understanding the molecular mechanisms that are involved in the initiation and progression of CMM is crucial for overcoming the commonly observed drug resistance as well as developing novel targeted treatment strategies. This molecular knowledge may further lead to the identification of clinically relevant biomarkers for early CMM detection, risk stratification, or prediction of response to therapy, altogether improving the clinical management of this disease. In this review we summarize the currently identified genetic and epigenetic alterations in CMM development. Although the genetic components underlying CMM are clearly emerging, a complete picture of the epigenetic alterations on DNA (DNA methylation), RNA (non-coding RNAs), and protein level (histone modifications, Polycomb group proteins, and chromatin remodeling) and the combinatorial interactions between these events is lacking. More detailed knowledge, however, is accumulating for genetic and epigenetic interactions in the aberrant regulation of the INK4b-ARF-INK4a and microphthalmia-associated transcription factor (MITF) loci. Importantly, we point out that it is this interplay of genetics and epigenetics that effectively leads to distorted gene expression patterns in CMM.

Promoter CpG island methylation markers in colorectal cancer: the road ahead.

Despite increasing knowledge on the biology, detection and treatment of colorectal cancer (CRC), the disease is still a major health problem. Hypermethylation of promoter regions of genes has been studied extensively as a contributor in CRC carcinogenesis. In addition, it is the topic of many studies focusing on biomarkers for the early detection, prediction of prognosis and treatment outcome. Methylation markers may be preferred over current screening and test methods as they are stable and easy to detect. However, almost no methylation marker is currently being used in clinical practice, often due to a lack of sensitivity, specificity, or validation of the results. This review summarizes the current knowledge of hypermethylation biomarkers for CRC detection, progression and treatment outcome.

Analysis of promoter CpG island hypermethylation in cancer: location, location, location!

The genetic and epigenetic alterations that underlie cancer pathogenesis are rapidly being identified. This provides novel insights in tumor biology as well as in potential cancer biomarkers. The somatic mutations in cancer genes that have been implemented in clinical practice are well defined and very specific. For epigenetic alterations, and more specifically aberrant methylation of promoter CpG islands, evidence is emerging that these markers could be used for the early detection of cancer as well as prediction of prognosis and response to therapy. However, the exact location of biologically and clinically relevant hypermethylation has not been identified for the majority of methylation markers. The most widely used approaches to analyze DNA methylation are based on primer- and probe-based assays that provide information for a limited number of CpG dinucleotides and thus for only part of the information available in a given CpG island. Validation of the current data and implementation of hypermethylation markers in clinical practice require a more comprehensive and critical evaluation of DNA methylation and limitations of the techniques currently used in methylation marker research. Here, we discuss the emerging evidence on the importance of the location of CpG dinucleotide hypermethylation in relation to gene expression and associations with clinicopathologic characteristics in cancer.

The unfolded protein response protects human tumor cells during hypoxia through regulation of the autophagy genes MAP1LC3B and ATG5.

Tumor hypoxia is a common microenvironmental factor that adversely influences tumor phenotype and treatment response. Cellular adaptation to hypoxia occurs through multiple mechanisms, including activation of the unfolded protein response (UPR). Recent reports have indicated that hypoxia activates a lysosomal degradation pathway known as autophagy, and here we show that the UPR enhances the capacity of hypoxic tumor cells to carry out autophagy, and that this promotes their survival. In several human cancer cell lines, hypoxia increased transcription of the essential autophagy genes microtubule-associated protein 1 light chain 3beta (MAP1LC3B) and autophagy-related gene 5 (ATG5) through the transcription factors ATF4 and CHOP, respectively, which are regulated by PKR-like ER kinase (PERK, also known as EIF2AK3). MAP1LC3B and ATG5 are not required for initiation of autophagy but mediate phagophore expansion and autophagosome formation. We observed that transcriptional induction of MAP1LC3B replenished MAP1LC3B protein that was turned over during extensive hypoxia-induced autophagy. Correspondingly, cells deficient in PERK signaling failed to transcriptionally induce MAP1LC3B and became rapidly depleted of MAP1LC3B protein during hypoxia. Consistent with these data, autophagy and MAP1LC3B induction occurred preferentially in hypoxic regions of human tumor xenografts. Furthermore, pharmacological inhibition of autophagy sensitized human tumor cells to hypoxia, reduced the fraction of viable hypoxic tumor cells, and sensitized xenografted human tumors to irradiation. Our data therefore demonstrate that the UPR is an important mediator of the hypoxic tumor microenvironment and that it contributes to resistance to treatment through its ability to facilitate autophagy.

Talking to chromatin: post-translational modulation of polycomb group function.

Polycomb Group proteins are important epigenetic regulators of gene expression. Epigenetic control by polycomb Group proteins involves intrinsic as well as associated enzymatic activities. Polycomb target genes change with cellular context, lineage commitment and differentiation status, revealing dynamic regulation of polycomb function. It is currently unclear how this dynamic modulation is controlled and how signaling affects polycomb-mediated epigenetic processes at the molecular level. Experimental evidence on regulation of polycomb function by post-translational mechanisms is steadily emerging: Polycomb Group proteins are targeted for ubiquitylation, sumoylation and phosphorylation. In addition, specific Polycomb Group proteins modify other (chromatin) associated proteins via similar post-translational modifications. Such modifications affect protein function by affecting protein stability, protein-protein interactions and enzymatic activities. Here, we review current insights in covalent modification of Polycomb Group proteins in the context of protein function and present a tentative view of integrated signaling to chromatin in the context of phosphorylation. Clearly, the available literature reveals just the tip of the iceberg, and exact molecular mechanisms in, and the biological relevance of post-translational regulation of polycomb function await further elucidation. Our understanding of causes and consequences of post-translational modification of polycomb proteins will gain significantly from in vivo validation experiments. Impaired polycomb function has important repercussions for stem cell function, development and disease. Ultimately, increased understanding of signaling to chromatin and the mechanisms involved in epigenetic remodeling will contribute to the development of therapeutic interventions in cell fate decisions in development and disease.

Hypoxia-induced expression of carbonic anhydrase 9 is dependent on the unfolded protein response.

Adaptation to tumor hypoxia is mediated in large part by changes in protein expression. These are driven by multiple pathways, including activation of the hypoxia inducible factor-1 (HIF-1) transcription factor and the PKR-like endoplasmic reticulum kinase PERK, a component of the unfolded protein response. Through gene expression profiling we discovered that induction of the HIF-1 target gene CA9 was defective in mouse embryo fibroblasts derived from mice harboring an eIF2alpha S51A knock-in mutation. This finding was confirmed in two isogenic human cell lines with an engineered defect in eIF2alpha phosphorylation. We show that impaired CA9 expression was not due to changes in HIF activity or CA9 mRNA stability. Using chromatin immunoprecipitation we show that the eIF2alpha-dependent translationally regulated gene ATF4 binds directly to the CA9 promoter and is associated with loss of the transcriptional repressive histone 3 lysine 27 tri-methylation mark. Loss or overexpression of ATF4 confirmed its role in CA9 induction during hypoxia. Our data indicate that expression of CA9 is regulated through both the HIF-1 and unfolded protein response hypoxia response pathways in vitro and in vivo.

Promoter CpG island hypermethylation- and H3K9me3 and H3K27me3-mediated epigenetic silencing targets the deleted in colon cancer (DCC) gene in colorectal carcinogenesis without affecting neighboring genes on chromosomal region 18q21.

Chromosomal loss of 18q21 is a frequent event in colorectal cancer (CRC) development, suggesting that this region harbors tumor suppressor genes (TSGs). Several candidate TSGs, among which methyl-CpG-binding domain protein 1 (MBD1), CpG-binding protein CXXC1, Sma- and Mad-related protein 4 (SMAD4), deleted in colon cancer (DCC) and methyl-CpG-binding domain protein 2 (MBD2) are closely linked on a 4-Mb DNA region on chromosome18q21. As TSGs can be epigenetically silenced, this study investigates whether MBD1, CXXC1, SMAD4, DCC and MBD2 are subject to epigenetic silencing in CRC. Methylation-specific polymerase chain reaction and sodium bisulfite sequencing of these genes show that DCC, but not MBD1, CXXC1, SMAD4 and MBD2, has promoter CpG island methylation in CRC cell lines and tissues {normal mucosa [29.5% (18/61)], adenomas [81.0% (47/58)] and carcinomas [82.7% (62/75)] (P = 8.6 x 10(-9))} that is associated with reduced DCC expression, independent of 18q21 loss analyzed by multiplex ligation-dependent probe amplification. Reduced gene expression of CXXC1, SMAD4 and MBD2 correlates with 18q21 loss in CRC cell lines (P = 0.04, 0.02 and 0.02, respectively). Treatment with the demethylating agent 5-aza-2'-deoxycytidine, but not with the histone deacetylase inhibitor trichostatin A exclusively restored DCC expression in CRC cell lines. Chromatin immunoprecipitation studies reveal that the DCC promoter is marked with repressive histone-tail marks H3K9me3 and H3K27me3, whereas activity related H3K4me3 was absent. Only active epigenetic marks were detected for MBD1, CXXC1, SMAD4 and MBD2. This study demonstrates specific epigenetic silencing of DCC in CRC as a focal process not affecting neighboring genes on chromosomal region 18q21.

Etomoxir-induced partial carnitine palmitoyltransferase-I (CPT-I) inhibition in vivo does not alter cardiac long-chain fatty acid uptake and oxidation rates.

Although CPT-I (carnitine palmitoyltransferase-I) is generally regarded to present a major rate-controlling site in mitochondrial beta-oxidation, it is incompletely understood whether CPT-I is rate-limiting in the overall LCFA (long-chain fatty acid) flux in the heart. Another important site of regulation of the LCFA flux in the heart is trans-sarcolemmal LCFA transport facilitated by CD36 and FABPpm (plasma membrane fatty acid-binding protein). Therefore, we explored to what extent a chronic pharmacological blockade of the LCFA flux at the level of mitochondrial entry of LCFA-CoA would affect sarcolemmal LCFA uptake. Rats were injected daily with saline or etomoxir, a specific CPT-I inhibitor, for 8 days at 20 mg/kg of body mass. Etomoxir-treated rats displayed a 44% reduced cardiac CPT-I activity. Sarcolemmal contents of CD36 and FABPpm, as well as the LCFA transport capacity, were not altered in the hearts of etomoxir-treated versus control rats. Furthermore, rates of LCFA uptake and oxidation, and glucose uptake by cardiac myocytes from etomoxir-treated rats were not different from control rats, neither under basal nor under acutely induced maximal metabolic demands. Finally, hearts from etomoxir-treated rats did not display triacylglycerol accumulation. Therefore CPT-I appears not to present a major rate-controlling site in total cardiac LCFA flux. It is likely that sarcolemmal LCFA entry rather than mitochondrial LCFA-CoA entry is a promising target for normalizing LCFA flux in cardiac metabolic diseases.

MAPKAP kinase 3pK phosphorylates and regulates chromatin association of the polycomb group protein Bmi1.

Polycomb group (PcG) proteins form chromatin-associated, transcriptionally repressive complexes, which are critically involved in the control of cell proliferation and differentiation. Although the mechanisms involved in PcG-mediated repression are beginning to unravel, little is known about the regulation of PcG function. We showed previously that PcG complexes are phosphorylated in vivo, which regulates their association with chromatin. The nature of the responsible PcG kinases remained unknown. Here we present the novel finding that the PcG protein Bmi1 is phosphorylated by 3pK (MAPKAP kinase 3), a convergence point downstream of activated ERK and p38 signaling pathways and implicated in differentiation and developmental processes. We identified 3pK as an interaction partner of PcG proteins, in vitro and in vivo, by yeast two-hybrid interaction and co-immunoprecipitation, respectively. Activation or overexpression of 3pK resulted in phosphorylation of Bmi1 and other PcG members and their dissociation from chromatin. Phosphorylation and subsequent chromatin dissociation of PcG complexes were expected to result in de-repression of targets. One such reported Bmi1 target is the Cdkn2a/INK4A locus. Cells overexpressing 3pK showed PcG complex/chromatin dissociation and concomitant de-repression of p14(ARF), which was encoded by the Cdkn2a/INK4A locus. Thus, 3pK is a candidate regulator of phosphorylation-dependent PcG/chromatin interaction. We speculate that phosphorylation may not only affect chromatin association but, in addition, the function of individual complex members. Our findings linked for the first time MAPK signaling pathways to the Polycomb transcriptional memory system. This suggests a novel mechanism by which a silenced gene status can be modulated and implicates PcG-mediated repression as a dynamically controlled process.