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Noonan syndrome with multiple lentigines (NSML) frequently manifests with hypertrophic cardiomyopathy (HCM). Recently, it was demonstrated that mTOR inhibition reverses HCM in NSML mice. We report for the first time on the effects of treatment with a rapamycin analog in an infant with LS and a malignant form of HCM. In the boy, progressive HCM was diagnosed during the first week of life and diagnosis of NSML was established at age 20 weeks by showing a heterozygous Q510E mutation in the PTPN11 gene. Immunoblotting with antibodies against pERK, pAkt, and pS6RP in fibroblasts demonstrated reduced RAS/MAPK and enhanced Akt/mTOR pathway activities. Because of the patient’s critical condition, everolimus therapy was started at age 24 weeks and continued until heart transplantation at age 36 weeks. Prior to surgery, heart failure improved from NYHA stage IV to II and brain natriuretic peptide values decreased from 9600 to

Melanoma tumors have been shown to comprise both invasive and proliferative cell subpopulations. These populations are highly plastic, thus hampering full characterization and therapeutic targeting of dormant and partially dedifferentiated invasive cells. We have reported, previously, that melanoma cells grown in a serum-free neural crest medium, in which they propagate as spheroids, show higher invasiveness and increased immune escape. In addition, in spheroids, we showed the increased expression of several genes which are involved in pluripotency, differentiation, and invasion. We therefore proposed that these culture conditions favor the polarization of proliferative melanoma cells toward an invasive state. As plasticity may suggest a reversible polarization, the aim of this report is to assess the transient phenotype of invasive cells generated through this procedure. We provide evidence that spheroid cells mimic dormant populations, and that this phenotype is fully reversible when cells are reintroduced into culture media that contain serum in which they grow as a monolayer. We also show that most transcriptional deregulations can be reversed. To further explain this plasticity in melanoma cells, we explored the epigenetic status of four gene promoters, assuming changes in acetylation or dimethylation on histone 3. We show reversible modifications on lysine 9 and lysine 4. We propose that spheroids allow the transient polarization of melanoma cells toward enhanced dormancy, loss of differentiation, and invasiveness, thereby reproducing the properties and plasticity of invasive subpopulations in melanoma tumors. This in-vitro model will allow further characterization and targeting of melanoma invasive cell populations.

Hypoxia is a major feature of the solid tumor microenvironment and is known to be associated with tumor progression and poor clinical outcome. Recently, we reported that hypoxia protects human non-small cell lung tumor cells from specific lysis by stabilizing hypoxia-inducible factor-1α and inducing STAT3 phosphorylation. In this study, we show that NANOG, a transcription factor associated with stem cell self renewal, is a new mediator of hypoxia-induced resistance to specific lysis. Our data indicate that under hypoxic conditions, NANOG is induced at both transcriptional and translational levels. Knockdown of the NANOG gene in hypoxic tumor cells is able to significantly attenuate hypoxia-induced tumor resistance to CTL-dependent killing. Such knockdown correlates with an increase of target cell death and an inhibition of hypoxia-induced delay of DNA replication in these cells. Interestingly, NANOG depletion results in inhibition of STAT3 phosphorylation and nuclear translocation. To our knowledge, this study is the first to show that hypoxia-induced NANOG plays a critical role in tumor cell response to hypoxia and promotes tumor cell resistance to Ag-specific lysis.

In this review, we focus on elucidating the cardiac function of germline mutations in the PTPN11 gene, encoding the Src homology-2 (SH2) domain-containing protein tyrosine phosphatase SHP2. PTPN11 mutations cause LEOPARD syndrome (LS) and Noonan syndrome (NS), two disorders that are part of a newly classified family of autosomal dominant syndromes termed "RASopathies," which are caused by germline mutations in components of the RAS/RAF/MEK/ERK mitogen activating protein kinase pathway. LS and NS mutants have opposing biochemical properties, and yet, in patients, these mutations produce similar cardiac abnormalities. Precisely how LS and NS mutations lead to such similar disease etiology remains largely unknown. Recent complementary in vitro, ex vivo, and in vivo analyses reveal new insights into the functions of SHP2 in normal and pathological cardiac development. These findings also reveal the need for individualized therapeutic approaches in the treatment of patients with LS and NS and, more broadly, patients with the other "RASopathy" gene mutations as well.

International audience; The constitutive expression of major histocompatibility complex class II (MHC II) molecules in melanoma is highly unusual and has been associated with unfavorable clinical outcome and higher metastatic dissemination. This association remains poorly understood and therefore, in this study we looked to whether it is caused by intracellular events that promote tumor progression. We previously reported that MHC II expression in melanoma cells requires active mitogen-activated protein kinase/extracellular signal-related kinase. However, our comparative and molecular analyses of a panel of melanoma cell lines herein provide clear evidence that mitogen-activated protein kinase/extracellular signal-related kinase is not sufficient for HLA-DR expression. We found that the expression of HLA-DR in these tumors rather coincides with the expression of CXCL-1 and CXCL-8 chemokines, both known to be expressed in tumors that invade early and are related to invasive stages of melanoma. The expression of HLA-DR also nicely paralleled that of the nuclear NFkappaB p50 subunit, regulating the expression of these chemokines in melanoma and previously correlated with poor prognosis of melanoma patients, although we provide evidence that NFkappaB is not directly regulating MHC II expression level. The molecular basis for class II transactivator and HLA-DR expression in melanoma therefore remains unsolved, but our findings linking together the expression of HLA-DR, of chemokines involved in invasiveness, and of nuclear NFkappaB p50 strongly support the content that MHC II may be a marker of invasive primary melanoma, and could explain the long-standing association of MHC II expression with overall poor prognosis and unfavorable clinical outcome.

Using a series of immunoprecipitation (IP) – tandem mass spectrometry (LC-MS/MS) experiments and reciprocal BLAST, we conducted a fly-human cross-species comparison of the phosphoinositide-3-kinase (PI3K) interactome in a drosophila S2R+ cell line and several NSCLC and human multiple myeloma cell lines to identify conserved interacting proteins to PI3K, a critical signaling regulator of the AKT pathway. Using H929 human cancer cells and drosophila S2R+ cells, our data revealed an unexpected direct binding of Corkscrew, the drosophila ortholog of the non-receptor protein tyrosine phosphatase type II (SHP2) to the Pi3k21B (p60) regulatory subunit of PI3K (p50/p85 human ortholog) but no association with Pi3k92e, the human ortholog of the p110 catalytic subunit. The p85-SHP2 association was validated in human cell lines and formed a ternary regulatory complex with GRB2-associated-binding protein 2 (GAB2). Validation experiments with knockdown of GAB2 and Far-Western blots proved the direct interaction of SHP2 with p85, independent of adaptor proteins and transfected FLAG-p85 provided evidence that SHP2 binding on p85 occurred on the SH2 domains. A disruption of the SHP2-p85 complex took place after insulin/IGF1 stimulation or imatinib treatment, suggesting that the direct SHP2-p85 interaction was both independent of AKT activation and positively regulates the ERK signaling pathway.

Congenital heart disease (CHD) is the most common birth defect worldwide; however, underlying mechanisms remain unknown. Loss-of-function mutations in PTPN11, the gene encoding the protein tyrosine phosphatase SHP2, are implicated in CHD and cause Noonan Syndrome with Multiple Lentigines (NSML). NSML presents with multiple cardiac defects, including hypertrophy. Here, we found that the NSML-associated adult-onset cardiac hypertrophy stems from aberrant signaling originating from developing endocardium. Embryonic NSML hearts showed diminished trabeculation and valvular hyperplasia, defects recapitulated in endocardial-, but not myocardial- or neural crest-, specific NSML mice. NSML hearts also developed ventricular septal defects, a phenotype reproduced only in myocardial-specific NSML hearts, suggesting NSML mutations have both cell autonomous and non-autonomous functions in cardiac development. Importantly, endocardial-specific expression of NSML was sufficient to induce adult-onset cardiac hypertrophy. Mechanistically, we observed aberrant AKT activity in NSML embryos, with decreased downstream FOXP1/FGF and NOTCH1/EPHB2 signaling, two pathways necessary for reciprocal crosstalk between developing endocardium and myocardium. Taken together, our data provide the first functional and disease-based evidence to suggest that critical mechanisms exist to control endocardial-myocardial crosstalk, the aberrant regulation of which may lead to CHD and adult-onset cardiac disease.

Hypertrophic cardiomyopathy is a common cause of mortality in congenital heart disease (CHD). Many gene abnormalities are associated with cardiac hypertrophy, but their function in cardiac development is not well understood. Loss-of-function mutations in PTPN11, which encodes the protein tyrosine phosphatase (PTP) SHP2, are implicated in CHD and cause Noonan syndrome with multiple lentigines (NSML), a condition that often presents with cardiac hypertrophic defects. Here, we found that NSML-associated hypertrophy stems from aberrant signaling mechanisms originating in developing endocardium. Trabeculation and valvular hyperplasia were diminished in hearts of embryonic mice expressing a human NSML-associated variant of SHP2, and these defects were recapitulated in mice expressing NSML-associated SHP2 specifically in endothelial, but not myocardial or neural crest, cells. In contrast, mice with myocardial- but not endothelial-specific NSML SHP2 expression developed ventricular septal defects, suggesting that NSML-associated mutations have both cell-autonomous and nonautonomous functions in cardiac development. However, only endothelial-specific expression of NSML-associated SHP2 induced adult-onset cardiac hypertrophy. Further, embryos expressing the NSML-associated SHP2 mutation exhibited aberrant AKT activity and decreased downstream forkhead box P1 (FOXP1)/FGF and NOTCH1/EPHB2 signaling, indicating that SHP2 is required for regulating reciprocal crosstalk between developing endocardium and myocardium. Together, our data provide functional and disease-based evidence that aberrant SHP2 signaling during cardiac development leads to CHD and adult-onset heart hypertrophy.

The Ras-related guanosine triphosphatase RhoA mediates pathological cardiac hypertrophy, but also promotes cell survival and is cardioprotective after ischemia/reperfusion injury. To understand how RhoA mediates these opposing roles in the myocardium, we generated mice with a cardiomyocyte-specific deletion of RhoA. Under normal conditions, the hearts from these mice showed functional, structural, and growth parameters similar to control mice. Additionally, the hearts of the cardiomyocyte-specific, RhoA-deficient mice subjected to transverse aortic constriction (TAC)-a procedure that induces pressure overload and, if prolonged, heart failure-exhibited a similar amount of hypertrophy as those of the wild-type mice subjected to TAC. Thus, neither normal cardiac homeostasis nor the initiation of compensatory hypertrophy required RhoA in cardiomyocytes. However, in response to chronic TAC, hearts from mice with cardiomyocyte-specific deletion of RhoA showed greater dilation, with thinner ventricular walls and larger chamber dimensions, and more impaired contractile function than those from control mice subjected to chronic TAC. These effects were associated with aberrant calcium signaling, as well as decreased activity of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and AKT. In addition, hearts from mice with cardiomyocyte-specific RhoA deficiency also showed less fibrosis in response to chronic TAC, with decreased transcriptional activation of genes involved in fibrosis, including myocardin response transcription factor (MRTF) and serum response factor (SRF), suggesting that the fibrotic response to stress in the heart depends on cardiomyocyte-specific RhoA signaling. Our data indicated that RhoA regulates multiple pathways in cardiomyocytes, mediating both cardioprotective (hypertrophy without dilation) and cardio-deleterious effects (fibrosis).

Congenital heart disease is the most common human developmental disorder, affecting ∼1:100 newborns, and is the primary cause of birth-defect related deaths worldwide. As a major regulator of receptor tyrosine kinase (RTK), cytokine and G-protein coupled receptor signaling, the non-receptor protein tyrosine phosphatase SHP2 plays a critical role in normal cardiac development and function. Indeed, SHP2 participates in a wide variety of cellular functions, including proliferation, survival, differentiation, migration, and cell–cell communication. Moreover, human activating and inactivating mutations of SHP2 are responsible for two related developmental disorders called Noonan and LEOPARD Syndromes, respectively, which are both characterized, in part, by congenital heart defects. Structural, enzymologic, biochemical, and SHP2 mouse model studies have together greatly enriched our knowledge of SHP2 and, as such, have also uncovered the diverse roles for SHP2 in cardiac development, including its contribution to progenitor cell specification, cardiac morphogenesis, and maturation of cardiac valves and myocardial chambers. By delineating the precise mechanisms by which SHP2 is involved in regulating these processes, we can begin to better understand the pathogenesis of cardiac disease and find more strategic and effective therapies for treatment of patients with congenital heart disorders.

Heart failure is a progressive disease characterized by loss of cardiac contractility, interstitial fibrosis, and chamber remodeling. Though several signaling pathways have been identified, the precise mechanisms involved in regulating pathological cardiac responses remain unclear. The Ras-related small G protein RhoA is an identified mediator of in vivo pathological hypertrophy. However, it also promotes cell survival and is cardioprotective after ischemia/repurfusion injury. Therefore, to determine the molecular mechanisms that underlie these opposing roles for RhoA in the myocardium, we generated mice with cardiomyocyte-specific deletion of RhoA (RhoAfl/fl-αMHC-Cre). As compared to wildtype mice, hearts from RhoAfl/fl-αMHC-Cre mice showed similar functional, structural, and growth parameters, both at baseline and in response to acute transverse aortic constriction (TAC) (2wks), suggesting RhoA plays no major role in either homeostatic or acute stress-induced cardiac conditions. However, after exposure to chronic TAC for 8wks, hearts from RhoAfl/fl-αMHC-Cre mice developed an accelerated dilation, with significant loss of contractile function. Mechanistically, cardiomyocyte-specific loss of RhoA reduced ERK1/2 activation and perturbed agonist-evoked calcium cycling, through aberrant IP3 and DAG signaling, indicating that RhoA mediates its cardioprotective effects through regulation of these pathways. Despite this, and surprisingly, hearts from RhoAfl/fl-αMHC mice also showed significantly decreased TAC-induced cardiac fibrosis, with a demonstrated decrease in transcriptional activation of genes involved in the fibrotic response, including the serum response factor (SRF), as well as the secreted factor BMP7. Taken together, our data reveal an interesting, yet highly complex signaling and regulatory mechanism for RhoA, whereby RhoA signaling is both cardioprotective against development of heart failure in response to chronic stress, but also cardio-deleterious through its capacity to activate pro-fibrotic genes.

Loss of function mutations in PTPN11 , the gene encoding the protein tyrosine phosphatase SHP2, cause LEOPARD Syndrome (LS), an autosomal dominant disorder with multiple cardiac defects, including hypertrophy. However, the mechanisms by which the LS mutants cause cardiac disease and regulate development remain unclear. Here, we utilized our inducible LS mouse model to determine whether LS mutations affect onset of cardiac hypertrophy via aberrant regulation of developmental processes. We examined hearts from wildtype (WT) or ubiquitously-expressing heterozygous LS (LS/+) and homozygous LS (LS/LS) embryos. As compared to WT, both LS/+ and LS/LS developing hearts showed diminished trabeculation, valvular hyperplasia and ventricular septal defects. In addition, LS/LS embryos had dextraposition of the aorta, leading to embryonic lethality. Gene expression analysis revealed that LS mutant hearts had decreased Foxp1/Fgf and Notch1/EphB2 signaling, suggesting perturbation of the reciprocal cross-talk between the developing endocardium and myocardium To assess this directly and to determine the in-vivo effects of lineage-specific LS expression during development, we generated, endothelial (Tie2)-, myocardial (Nkx2.5)- and neural crest (Wnt1)- specific LS/+ expressing mice. Surprisingly, the majority of LS cardiac defects, including the adult-onset cardiac hypertrophy, were recapitulated by expression of endocardial-specific LS alone, suggesting a functional necessity for the endocardium in hypertrophy of the heart. Moreover, though myocardial-specific expression of LS showed only minor developmental effects, these mice still recapitulated the adult-onset cardiac hypertrophy. Interestingly, no cardiac abnormalities were observed in the neural crest-specific expressing LS mice. Taken together, our data indicate that the LS mutations affect both cell autonomous and non-autonomous pathways necessary for the reciprocal cross-talk between endocardium and myocardium, and, importantly, that the adult-onset hypertrophy in LS is caused by aberrant signaling effects that occur during cardiac development.

International audience; PURPOSE: Melanoma tumors are highly heterogeneous and can undergo phenotypic modifications depending on their plasticity and the microenvironment, with shifts between proliferative and invasive states. We have shown that melanoma cells, grown as spheroids in a neural crest cell medium, polarize toward an invasive and motile phenotype, in agreement with transcriptomic modulations, including the up-regulation of Nanog and Oct4. Overexpression of these genes was shown to be associated with poor prognosis and metastatic forms of some cancers. We thus investigated implication of Nanog and Oct4, two embryonic transcription factors, in melanoma motility. METHODS: Our team used stable transfection of Nanog or Oct4 in A375 melanoma cell line to investigate motility in a wound healing assay and a transendothelial migration assay. Using semiquantitative RT-PCR, expression of two gene panels involved either in mesenchymal motility or in amoeboid migration was studied. RESULTS: Strongly enhanced capacities of motility and extravasation were observed with cells overexpressing Oct4 and Nanog. The A375 cell line has been described as having a mesenchymal migration type. However, in the Oct4 and Nanog transfectants, several amoeboid migration markers are strongly induced. Accordingly, amoeboid migration inhibitors decrease significantly the transmigration of Oct4- and Nanog-expressing cells through endothelial cells. CONCLUSIONS: We propose here that Nanog and Oct4 pluripotency marker expression in melanoma cells increases the transmigration capacity of these cells through the gain of amoeboid motility, leading to higher invasiveness and aggressiveness.

International audience; BACKGROUND: The aggressiveness of melanoma tumors is likely to rely on their well-recognized heterogeneity and plasticity. Melanoma comprises multi-subpopulations of cancer cells some of which may possess stem cell-like properties. Although useful, the sphere-formation assay to identify stem cell-like or tumor initiating cell subpopulations in melanoma has been challenged, and it is unclear if this model can predict a functional phenotype associated with aggressive tumor cells. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the molecular and functional phenotypes of melanoma spheroids formed in neural crest cell medium. Whether from metastatic or advanced primary tumors, spheroid cells expressed melanoma-associated markers. They displayed higher capacity to differentiate along mesenchymal lineages and enhanced expression of SOX2, NANOG, KLF4, and/or OCT4 transcription factors, but not enhanced self-renewal or tumorigenicity when compared to their adherent counterparts. Gene expression profiling attributed a neural crest cell signature to these spheroids and indicated that a migratory/invasive and immune-function modulating program could be associated with these cells. In vitro assays confirmed that spheroids display enhanced migratory/invasive capacities. In immune activation assays, spheroid cells elicited a poorer allogenic response from immune cells and inhibited mitogen-dependent T cells activation and proliferation more efficiently than their adherent counterparts. Our findings reveal a novel immune-modulator function of melanoma spheroids and suggest specific roles for spheroids in invasion and in evasion of antitumor immunity. CONCLUSION/SIGNIFICANCE: The association of a more plastic, invasive and evasive, thus a more aggressive tumor phenotype with melanoma spheroids reveals a previously unrecognized aspect of tumor cells expanded as spheroid cultures. While of limited efficiency for melanoma initiating cell identification, our melanoma spheroid model predicted aggressive phenotype and suggested that aggressiveness and heterogeneity of melanoma tumors can be supported by subpopulations other than cancer stem cells. Therefore, it could be constructive to investigate melanoma aggressiveness, relevant to patients and clinical transferability.

LEOPARD syndrome (LS) is an autosomal dominant "RASopathy" that manifests with congenital heart disease. Nearly all cases of LS are caused by catalytically inactivating mutations in the protein tyrosine phosphatase (PTP), non-receptor type 11 (PTPN11) gene that encodes the SH2 domain-containing PTP-2 (SHP2). RASopathies typically affect components of the RAS/MAPK pathway, yet it remains unclear how PTPN11 mutations alter cellular signaling to produce LS phenotypes. We therefore generated knockin mice harboring the Ptpn11 mutation Y279C, one of the most common LS alleles. Ptpn11(Y279C/+) (LS/+) mice recapitulated the human disorder, with short stature, craniofacial dysmorphia, and morphologic, histologic, echocardiographic, and molecular evidence of hypertrophic cardiomyopathy (HCM). Heart and/or cardiomyocyte lysates from LS/+ mice showed enhanced binding of Shp2 to Irs1, decreased Shp2 catalytic activity, and abrogated agonist-evoked Erk/Mapk signaling. LS/+ mice also exhibited increased basal and agonist-induced Akt and mTor activity. The cardiac defects in LS/+ mice were completely reversed by treatment with rapamycin, an inhibitor of mTOR. Our results demonstrate that LS mutations have dominant-negative effects in vivo, identify enhanced mTOR activity as critical for causing LS-associated HCM, and suggest that TOR inhibitors be considered for treatment of HCM in LS patients.