SHP2 as a primordial epigenetic enzyme expunges histone H3 pTyr-54 to amend androgen receptor homeostasis.

Mutations that decrease or increase the activity of the tyrosine phosphatase, SHP2 (encoded by PTPN11), promotes developmental disorders and several malignancies by varying phosphatase activity. We uncovered that SHP2 is a distinct class of an epigenetic enzyme; upon phosphorylation by the kinase ACK1/TNK2, pSHP2 was escorted by androgen receptor (AR) to chromatin, erasing hitherto unidentified pY54-H3 (phosphorylation of histones H3 at Tyr54) epigenetic marks to trigger a transcriptional program of AR. Noonan Syndrome with Multiple Lentigines (NSML) patients, SHP2 knock-in mice, and ACK1 knockout mice presented dramatic increase in pY54-H3, leading to loss of AR transcriptome. In contrast, prostate tumors with high pSHP2 and pACK1 activity exhibited progressive downregulation of pY54-H3 levels and higher AR expression that correlated with disease severity. Overall, pSHP2/pY54-H3 signaling acts as a sentinel of AR homeostasis, explaining not only growth retardation, genital abnormalities and infertility among NSML patients, but also significant AR upregulation in prostate cancer patients.

Novel therapeutic perspectives in Noonan syndrome and RASopathies.

Noonan syndrome belongs to the family of RASopathies, a group of multiple congenital anomaly disorders caused by pathogenic variants in genes encoding components or regulators of the RAS/mitogen-activated protein kinase (MAPK) signalling pathway. Collectively, all these pathogenic variants lead to increased RAS/MAPK activation. The better understanding of the molecular mechanisms underlying the different manifestations of NS and RASopathies has led to the identification of molecular targets for specific pharmacological interventions. Many specific agents (e.g. SHP2 and MEK inhibitors) have already been developed for the treatment of RAS/MAPK-driven malignancies. In addition, other molecules with the property of modulating RAS/MAPK activation are indicated in non-malignant diseases (e.g. C-type natriuretic peptide analogues in achondroplasia or statins in hypercholesterolemia).  Conclusion: Drug repositioning of these molecules represents a challenging approach to treat or prevent medical complications associated with RASopathies. What is Known: • Noonan syndrome and related disorders are caused by pathogenic variants in genes encoding components or regulators of the RAS/mitogen-activated protein kinase (MAPK) signalling pathway, resulting in increased activation of this pathway. • This group of disorders is now known as RASopathies and represents one of the largest groups of multiple congenital anomaly diseases known. What is New: • The identification of pathophysiological mechanisms provides new insights into the development of specific therapeutic strategies, in particular treatment aimed at reducing RAS/MAPK hyperactivation. • Drug repositioning of specific agents already developed for the treatment of malignant (e.g. SHP2 and MEK inhibitors) or non-malignant diseases (e.g. C-type natriuretic peptide analogues in achondroplasia or statins in hypercholesterolaemia) represents a challenging approach to the treatment of RASopathies.

SHP2 drives inflammation-triggered insulin resistance by reshaping tissue macrophage populations.

Insulin resistance is a key event in type 2 diabetes onset and a major comorbidity of obesity. It results from a combination of fat excess-triggered defects, including lipotoxicity and metaflammation, but the causal mechanisms remain difficult to identify. Here, we report that hyperactivation of the tyrosine phosphatase SHP2 found in Noonan syndrome (NS) led to an unsuspected insulin resistance profile uncoupled from altered lipid management (for example, obesity or ectopic lipid deposits) in both patients and mice. Functional exploration of an NS mouse model revealed this insulin resistance phenotype correlated with constitutive inflammation of tissues involved in the regulation of glucose metabolism. Bone marrow transplantation and macrophage depletion improved glucose homeostasis and decreased metaflammation in the mice, highlighting a key role of macrophages. In-depth analysis of bone marrow-derived macrophages in vitro and liver macrophages showed that hyperactive SHP2 promoted a proinflammatory phenotype, modified resident macrophage homeostasis, and triggered monocyte infiltration. Consistent with a role of SHP2 in promoting inflammation-driven insulin resistance, pharmaceutical SHP2 inhibition in obese diabetic mice improved insulin sensitivity even better than conventional antidiabetic molecules by specifically reducing metaflammation and alleviating macrophage activation. Together, these results reveal that SHP2 hyperactivation leads to inflammation-triggered metabolic impairments and highlight the therapeutical potential of SHP2 inhibition to ameliorate insulin resistance.

The RASopathy Family: Consequences of Germline Activation of the RAS/MAPK Pathway.

Noonan syndrome [NS; Mendelian Inheritance in Men (MIM) #163950] and related syndromes [Noonan syndrome with multiple lentigines (formerly called LEOPARD syndrome; MIM #151100), Noonan-like syndrome with loose anagen hair (MIM #607721), Costello syndrome (MIM #218040), cardio-facio-cutaneous syndrome (MIM #115150), type I neurofibromatosis (MIM #162200), and Legius syndrome (MIM #611431)] are a group of related genetic disorders associated with distinctive facial features, cardiopathies, growth and skeletal abnormalities, developmental delay/mental retardation, and tumor predisposition. NS was clinically described more than 50 years ago, and disease genes have been identified throughout the last 3 decades, providing a molecular basis to better understand their physiopathology and identify targets for therapeutic strategies. Most of these genes encode proteins belonging to or regulating the so-called RAS/MAPK signaling pathway, so these syndromes have been gathered under the name RASopathies. In this review, we provide a clinical overview of RASopathies and an update on their genetics. We then focus on the functional and pathophysiological effects of RASopathy-causing mutations and discuss therapeutic perspectives and future directions.

SHP2 sails from physiology to pathology.

Over the two past decades, mutations of the PTPN11 gene, encoding the ubiquitous protein tyrosine phosphatase SHP2 (SH2 domain-containing tyrosine phosphatase 2), have been identified as the causal factor of several developmental diseases (Noonan syndrome (NS), Noonan syndrome with multiple lentigines (NS-ML), and metachondromatosis), and malignancies (juvenile myelomonocytic leukemia). SHP2 plays essential physiological functions in organism development and homeostasis maintenance by regulating fundamental intracellular signaling pathways in response to a wide range of growth factors and hormones, notably the pleiotropic Ras/Mitogen-Activated Protein Kinase (MAPK) and the Phosphoinositide-3 Kinase (PI3K)/AKT cascades. Analysis of the biochemical impacts of PTPN11 mutations first identified both loss-of-function and gain-of-function mutations, as well as more subtle defects, highlighting the major pathophysiological consequences of SHP2 dysregulation. Then, functional genetic studies provided insights into the molecular dysregulations that link SHP2 mutants to the development of specific traits of the diseases, paving the way for the design of specific therapies for affected patients. In this review, we first provide an overview of SHP2's structure and regulation, then describe its molecular roles, notably its functions in modulating the Ras/MAPK and PI3K/AKT signaling pathways, and its physiological roles in organism development and homeostasis. In the second part, we describe the different PTPN11 mutation-associated pathologies and their clinical manifestations, with particular focus on the biochemical and signaling outcomes of NS and NS-ML-associated mutations, and on the recent advances regarding the pathophysiology of these diseases.

Noonan syndrome-causing SHP2 mutants inhibit insulin-like growth factor 1 release via growth hormone-induced ERK hyperactivation, which contributes to short stature.

Noonan syndrome (NS), a genetic disease caused in half of cases by activating mutations of the tyrosine phosphatase SHP2 (PTPN11), is characterized by congenital cardiopathies, facial dysmorphic features, and short stature. How mutated SHP2 induces growth retardation remains poorly understood. We report here that early postnatal growth delay is associated with low levels of insulin-like growth factor 1 (IGF-1) in a mouse model of NS expressing the D61G mutant of SHP2. Conversely, inhibition of SHP2 expression in growth hormone (GH)-responsive cell lines results in increased IGF-1 release upon GH stimulation. SHP2-deficient cells display decreased ERK1/2 phosphorylation and rat sarcoma (RAS) activation in response to GH, whereas expression of NS-associated SHP2 mutants results in ERK1/2 hyperactivation in vitro and in vivo. RAS/ERK1/2 inhibition in SHP2-deficient cells correlates with impaired dephosphorylation of the adaptor Grb2-associated binder-1 (GAB1) on its RAS GTPase-activating protein (RASGAP) binding sites and is rescued by interfering with RASGAP recruitment or function. We demonstrate that inhibition of ERK1/2 activation results in an increase of IGF-1 levels in vitro and in vivo, which is associated with significant growth improvement in NS mice. In conclusion, NS-causing SHP2 mutants inhibit GH-induced IGF-1 release through RAS/ERK1/2 hyperactivation, a mechanism that could contribute to growth retardation. This finding suggests that, in addition to its previously shown beneficial effect on NS-linked cardiac and craniofacial defects, RAS/ERK1/2 modulation could also alleviate the short stature phenotype in NS caused by PTPN11 mutations.

Functional effects of PTPN11 (SHP2) mutations causing LEOPARD syndrome on epidermal growth factor-induced phosphoinositide 3-kinase/AKT/glycogen synthase kinase 3beta signaling.

LEOPARD syndrome (LS), a disorder with multiple developmental abnormalities, is mainly due to mutations that impair the activity of the tyrosine phosphatase SHP2 (PTPN11). How these alterations cause the disease remains unknown. We report here that fibroblasts isolated from LS patients displayed stronger epidermal growth factor (EGF)-induced phosphorylation of both AKT and glycogen synthase kinase 3beta (GSK-3beta) than fibroblasts from control patients. Similar results were obtained in HEK293 cells expressing LS mutants of SHP2. We found that the GAB1/phosphoinositide 3-kinase (PI3K) complex was more abundant in fibroblasts from LS than control subjects and that both AKT and GSK-3beta hyperphosphorylation were prevented by reducing GAB1 expression or by overexpressing a GAB1 mutant unable to bind to PI3K. Consistently, purified recombinant LS mutants failed to dephosphorylate GAB1 PI3K-binding sites. These mutants induced PI3K-dependent increase in cell size in a model of chicken embryo cardiac explants and in transcriptional activity of the atrial natriuretic factor (ANF) gene in neonate rat cardiomyocytes. In conclusion, SHP2 mutations causing LS facilitate EGF-induced PI3K/AKT/GSK-3beta stimulation through impaired GAB1 dephosphorylation, resulting in deregulation of a novel signaling pathway that could be involved in LS pathology.

Signal strength dictates phosphoinositide 3-kinase contribution to Ras/extracellular signal-regulated kinase 1 and 2 activation via differential Gab1/Shp2 recruitment: consequences for resistance to epidermal growth factor receptor inhibition.

Phosphoinositide 3-kinase (PI3K) participates in extracellular signal-regulated kinase 1 and 2 (ERK1-2) activation according to signal strength, through unknown mechanisms. We report herein that Gab1/Shp2 constitutes a PI3K-dependent checkpoint of ERK1-2 activation regulated according to signal intensity. Indeed, by up- and down-regulation of signal strength in different cell lines and through different methods, we observed that Gab1/Shp2 and Ras/ERK1-2 in concert become independent of PI3K upon strong epidermal growth factor receptor (EGFR) stimulation and dependent on PI3K upon limited EGFR activation. Using Gab1 mutants, we observed that this conditional role of PI3K is dictated by the EGFR capability of recruiting Gab1 through Grb2 or through the PI3K lipid product PIP(3), according to a high or weak level of receptor stimulation, respectively. In agreement, Grb2 siRNA generates, in cells with maximal EGFR stimulation, a strong dependence on PI3K for both Gab1/Shp2 and ERK1-2 activation. Therefore, Ras/ERK1-2 depends on PI3K only when PIP(3) is required to recruit Gab1/Shp2, which occurs only under weak EGFR mobilization. Finally, we show that, in glioblastoma cells displaying residual EGFR activation, this compensatory mechanism becomes necessary to efficiently activate ERK1-2, which could probably contribute to tumor resistance to EGFR inhibitors.

The adaptor protein Gab1 couples the stimulation of vascular endothelial growth factor receptor-2 to the activation of phosphoinositide 3-kinase.

Phosphoinositide 3-kinase (PI3K) mediates essential functions of vascular endothelial growth factor (VEGF), including the stimulation of endothelial cell proliferation and migration. Nevertheless, the mechanisms coupling the receptor VEGFR-2 to PI3K remain obscure. We observed that the Grb2-bound adapter Gab1 is tyrosine-phosphorylated and relocated to membrane fractions upon VEGF stimulation of endothelial cells. We could detect the PI3K regulatory subunit p85 in immunoprecipitates of endogenous Gab1, and vice versa, and measure a Gab1-associated lipid kinase activity upon VEGF stimulation. Furthermore, transfection of the Gab1-YF3 mutant lacking all p85-binding sites strongly repressed PI3K activation measured in vitro. Moreover, Gab1-YF3 severely decreased the cellular amount of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) generated in response to VEGF. Furthermore, adenoviral expression of Gab1-YF3 suppressed both Akt phosphorylation and recovery of wounded human umbilical vein endothelial cell monolayers, a VEGF-dependent process involving cell migration and proliferation under PI3K control. Transfection of other Gab1 mutants, lacking Grb2-binding sites or the pleckstrin homology (PH) domain, also prevented Akt activation, further demonstrating Gab1 involvement in PI3K activation. These mutants were also used to show that interactions with both Grb2 and PtdIns(3,4,5)P3 mediate Gab1 recruitment by VEGFR-2. Importantly, Gab1 mobilization was impaired by (i) PI3K inhibitors, (ii) deletion of Gab1 PH domain, (iii) PTEN (phosphatase and tensin homolog deleted on chromosome 10) overexpression to repress PtdIns(3,4,5)P3 production, and (iv) overexpression of a competitor PH domain for PtdIns(3,4,5)P3 binding, which altogether demonstrated that PI3K is also an upstream regulator of Gab1. Gab1 thus appears as a primary actor in coupling VEGFR-2 to PI3K/Akt, recruited through an amplification loop involving PtdIns(3,4,5)P3 and its PH domain.

The HRPT2 tumor suppressor gene product parafibromin associates with human PAF1 and RNA polymerase II.

Inactivation of the HRPT2 tumor suppressor gene is associated with the pathogenesis of the hereditary hyperparathyroidism-jaw tumor syndrome and malignancy in sporadic parathyroid tumors. The cellular function of the HPRT2 gene product, parafibromin, has not been defined yet. Here we show that parafibromin physically interacts with human orthologs of yeast Paf1 complex components, including PAF1, LEO1, and CTR9, that are involved in transcription elongation and 3' end processing. It also associates with modified forms of the large subunit of RNA polymerase II, in particular those phosphorylated on serine 5 or 2 within the carboxy-terminal domain, that are important for the coordinate recruitment of transcription elongation and RNA processing machineries during the transcription cycle. These interactions depend on a C-terminal domain of parafibromin, which is deleted in ca. 80% of clinically relevant mutations. Finally, RNAi-induced downregulation of parafibromin promotes entry into S phase, implying a role for parafibromin as an inhibitor of cell cycle progression. Taken together, these findings link the tumor suppressor parafibromin to the transcription elongation and RNA processing pathway as a PAF1 complex- and RNA polymerase II-bound protein. Dysfunction of this pathway may be a general phenomenon in the majority of cases of hereditary parathyroid cancer.

Gab1, SHP-2 and other novel regulators of Ras: targets for anticancer drug discovery?

Ras proteins function as molecular switches that cycle between an inactive GDP-bound state, and an active GTP-bound form that triggers different signaling pathways. Because Ras can integrate both proliferative and anti-apoptotic stimuli, GTP-locked Ras mutants play a critical role in the development of human tumors. Moreover, wild-type Ras relays the transforming potential of a number of molecules involved in tumor development, including protein tyrosine kinases. Consequently, the molecular intermediates that control Ras activation are potential targets of anti-tumoral pharmacology. Besides the canonical Shc/Grb2/Sos module classically involved in Ras activation, novel effectors have recently been shown to participate in this pathway, including the multivalent Grb2-associated docking protein Gab1, the protein tyrosine phosphatase SHP-2, and the phosphoinositide 3-kinase. Recent genetic advances have shown that these proteins are critically involved in cell proliferation and survival, further suggesting that they could be interesting targets for selective tumor therapy. Here we review recent progress in our understanding of the role of Gab1 and its partners in Ras activation, and other survival/proliferation pathways. Implications for the pharmacological manipulation of this pathway in the treatment of cancer will also be discussed.

Modulation of phosphoinositide 3-kinase activation by cholesterol level suggests a novel positive role for lipid rafts in lysophosphatidic acid signalling.

Methyl-beta-cyclodextrin (MbetaCD) was used to explore a role for cholesterol-enriched plasma membrane microdomains in coupling lysophosphatidic acid (LPA) stimulation to phosphoinositide 3-kinase (PI3K) activation. Cholesterol depletion strongly inhibited the production of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate in Vero cells stimulated with LPA. In agreement, the phosphorylation of Akt/protein kinase B, but not of Erk kinases, was suppressed by MbetaCD. MbetaCD did not interfere with the overall phospholipid metabolism, and its effects were reversed in cholesterol add-back experiments. Finally, PI3K was detected in lipid rafts prepared from control but not MbetaCD-treated cells, suggesting that these microdomains contribute to LPA signalling by compartmentalising component(s) of the PI3K pathway.

A function for phosphoinositide 3-kinase beta lipid products in coupling beta gamma to Ras activation in response to lysophosphatidic acid.

Although Gbetagamma is thought to mediate mitogen-activated protein kinase (MAPK) activation in response to G protein-coupled receptor stimulation, the mechanisms involved in this pathway have not been clearly defined. Phosphoinositide 3-kinase (PI3K) has been proposed as an early intermediate in this process, but its role has remained elusive. We have observed that dominant negative mutants of p110beta, but not of p110gamma, inhibited MAPK stimulation in response to lysophosphatidic acid (LPA). The role of p110beta was located upstream from Ras. To determine which of the lipid or protein kinase activities of p110beta were important for Ras activation, we produced a mutant p110beta lacking the lipid but not the protein kinase activity. This protein displayed a dominant negative activity similar to a kinase-dead mutant, indicating that p110beta lipid kinase activity was essentially involved in Ras activation. In agreement, overexpression of the lipid phosphatase PTEN was found to specifically inhibit Ras stimulation induced by LPA. In addition, we have observed that the PH domain-containing adapter protein Gab1, which is involved in p110beta activation during LPA stimulation, is also implicated in this pathway downstream of p110beta. Indeed, both membrane redistribution and phosphorylation of Gab1 were reduced in the presence of PI3K inhibitors or dominant negative p110beta. Downstream of Gab1, the tyrosine phosphatase SHP2 was found to mediate Ras activation in response to LPA and to be recruited through PI3K and Gab1, because transfection of Gab1 mutant deficient for SHP2 binding inhibited Ras activation without interfering with PI3K activation. We conclude that LPA-induced Ras activation is mediated by a p110beta/Gab1/SHP2 pathway. Moreover, we present data indicating that p110beta is effectively the target of betagamma in this pathway, suggesting that the p110beta/Gab1/SHP2 pathway provides a novel link between betagamma and Ras by integrating two early events of LPA signaling, i.e. Gbetagamma release and tyrosine kinase receptor transactivation.