Domain-containing Protein Tyrosine Phosphatase Research Articles

Inhibition of RNF6 alleviates traumatic brain injury by suppressing STAT3 signaling in rats.

BackgroundTraumatic brain injury (TBI) has ranked as one of the leading causes of disability and death in the world. The neuroinflammation mediated by signal transducer and activator of transcription 3 (STAT3) signaling during the progression of TBI leads to long‐term neurodegeneration. Ring finger protein 6 (RNF‐6) is an E3 ubiquitin ligase and can regulate the activity of STAT3 signaling pathway by targeting its inhibitors. However, the mechanism underlying this process in TBI remains poorly understood.MethodsIn this research, cortical impact injury was used to construct the TBI rat model. Western blot assay was performed to evaluate the protein levels of RNF6, Src homology 2 domain‐containing protein tyrosine phosphatase 1 (SHP‐1), and STAT3/pSTAT3. QRT‐PCR assay was performed to assess the RNA levels of RNF6 and other cytokines. The neural function of TBI rats was estimated by modified Neurological Severity Scores test.ResultsThe expression of RNF‐6 was up‐regulated in the brain tissues of TBI rats. Down‐regulation of RNF6 alleviated the symptoms and improved the neural recovery postinjury in TBI rats. Inhibition of RNF6 suppressed the cerebral inflammation by up‐regulating the protein level of SHP‐1 and down‐regulating the phosphorylation level of STAT3.ConclusionInhibition of RNF6 alleviated TBI by suppressing the STAT3 signaling in TBI rats.

Abstract 3774: Unraveling mechanisms of resistance to tepotinib and future treatment options

Abstract Background Advanced non-small cell lung cancer (NSCLC) patients with MET exon 14 skipping mutations (METex14) or MET amplification (METamp) derive benefit from MET tyrosine kinase inhibitors (TKIs). Tepotinib is an investigational selective MET TKI. Co-occurring MAPK pathway alterations in MET dependent tumors are frequent and may induce primary or acquired resistance to MET TKIs. Methods METamp (EBC1, NCI-H1993 and MNK-45) and METex14 (Hs746T) cells were obtained from commercial vendors. Tepotinib-resistant (TR) cells (EBC1-TR1, -TR2 and Hs746T-TR) were generated upon continuous exposure of EBC1 and Hs746T cells to tepotinib. The phosphorylation of 49 receptor tyrosine kinases (RTK) and 43 non-RTKs was assessed using Human Phospho-RTK kinase Array Kits (R&D Systems, Minneapolis, MN). Cell viability was evaluated by Resazurin Cell Viability Assay (R&D Systems, Minneapolis, MN) and level of proteins by western blot. Results First, we assessed the short- and long-term effect of tepotinib on MET downstream signaling. Exposure of EBC1 cells to 0.01 µM of tepotinib for 1h abrogated MET, ERK and AKT phosphorylation, an effect that was sustained even 20h after tepotinib withdrawal. Next, we evaluated the phosphorylation status of other than MET RTKs and non-RTKs on the parental EBC1 and EBC1-TR1 cells. We observed activation of several RTKs, including ERBB (EGFR, ErbB2 and ErbB3), FGFR3, AXL, RET, DDR1, and M-CSFR in both cell lines. EBC1-TR1 cells displayed elevated levels of phosphorylated AXL and EGFR compared to EBC1 parental cells. We also observed increased phosphorylation of ERK1/2, AKT, c-Jun and YES on EBC1-TR1 compared to parental EBC1 cells. Similar results were achieved with EBC1-TR2 and Hs746T-TR cells. RTK signaling is mediated by the MAPK pathway which is related among others, with the Src-homology 2 domain-containing protein tyrosine phosphatase (SHP2). Therefore, we tested tepotinib in combination with a MEK inhibitor, pimasertib, or a SHP2 inhibitor, RMC-4550, initially in a panel of treatment-naïve MET dependent cells. A moderate or strong synergism was found when tepotinib was combined with either of the two compounds. A stronger decrease of MET phosphorylation was observed upon treatment with a SHP2 inhibitor combined with tepotinib compared to tepotinib alone. Further molecular characterization of the TR cells as well as combinations in vitro and in vivo of tepotinib with drugs that may prolong its effect and overcome resistance, are ongoing. Conclusions Our results indicate that co-occurring RAS-MAPK alterations may mediate innate or acquired resistance to tepotinib, despite its prolonged on-target activity. The development of therapeutic strategies co-targeting the MAPK pathway may enhance the initial magnitude and duration of response to tepotinib, delay or overcome acquired resistance and ultimately improve clinical outcomes. Citation Format: Linda Pudelko, Frank Jaehrling, Christopher Stroh, Nina Linde, Michael Sanderson, Doreen Musch, Marina Keil, Christina Esdar, Andree Blaukat, Niki Karachaliou. Unraveling mechanisms of resistance to tepotinib and future treatment options [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3774.

The Noonan syndrome-associated D61G variant of the protein tyrosine phosphatase SHP2 prevents synaptic down-scaling

Homeostatic scaling of the synapse, such as synaptic down-scaling, has been proposed to offset deleterious effects induced by sustained synaptic strength enhancement. Proper function and subcellular distribution of Src homology 2 domain-containing nonreceptor protein tyrosine phosphatase (SHP2) are required for synaptic plasticity. However, the role of SHP2 in synaptic down-scaling remains largely unknown. Here, using biochemical assays and cell-imaging techniques, we found that synaptic SHP2 levels are temporally regulated during synaptic down-scaling in cultured hippocampal neurons. Furthermore, we observed that a Noonan syndrome-associated mutation of SHP2, resulting in a D61G substitution, prevents synaptic down-scaling. We further show that this effect is due to an inability of the SHP2-D61G variant to properly disassociate from postsynaptic density protein 95, leading to impaired SHP2 dispersion from synaptic sites after synaptic down-scaling. Our findings reveal a molecular mechanism of the Noonan syndrome-associated genetic variant SHP2-D61G that contributes to deficient synaptic down-scaling.

Correlation between SHP-1 and carotid plaque vulnerability in humans

BackgroundAdvanced atherosclerotic plaques tend to indicate an increased risk of cerebral ischemic events. SH2 domain-containing protein tyrosine phosphatase 1 (SHP-1) is a class I classical nonreceptor protein tyrosine phosphatase associated with plaque stability, as shown by analysis of a Gene Expression Omnibus (GEO) dataset showing differences in mRNA levels. However, the correlation between SHP-1 and human carotid plaque stability at the protein level remains unclear. Methods and resultsThirty-nine carotid plaque tissue samples were acquired from 39 carotid artery stenosis patients after carotid endarterectomy. Hematoxylin and eosin, Masson trichrome, and CD68 staining was performed for pathological characterization, and immunohistochemical staining for SHP-1 was carried out. Within stable and unstable plaques, SHP-1 mainly accumulated in the necrotic area, plaque shoulder, and fibrous cap, similar to the distribution of CD68. A quantitative analysis of SHP-1 was carried out. The relative SHP-1-positive cell area was higher in the vulnerable group than in the stable group (P < .001). The number of symptomatic patients in the vulnerable group was no greater than that in the stable group (P = .098). Moreover, the integrated optical density (IOD)/area of SHP-1 was significantly higher in the vulnerable group than in the stable group (P < .001). Besides, SHP-1 colocalized with CD68 and vascular cell adhesion protein 1(VCAM-1). ConclusionsWe demonstrate that SHP-1 expression increases during carotid atherosclerotic plaque progression. The protein expression of SHP-1 was related to an increase in plaque instability in not only symptomatic but also asymptomatic patients with carotid artery stenosis. SHP-1 may play a role in atherosclerosis progression by macrophage polarization-mediated efferocytosis. Furthermore, SHP-1 may become a promising biomarker for plaque vulnerability in the future.

SHP2 mutations induce precocious gliogenesis of Noonan syndrome-derived iPSCs during neural development in vitro

BackgroundNoonan syndrome (NS) is a developmental disorder caused by mutations of Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2). Although NS patients have diverse neurological manifestations, the mechanisms underlying the involvement of SHP2 mutations in neurological dysfunction remain elusive.MethodsInduced pluripotent stem cells generated from dermal fibroblasts of three NS-patients (NS-iPSCs) differentiated to the neural cells by using two different culture systems, 2D- and 3D-cultured systems in vitro.ResultsHere we represent that SHP2 mutations cause aberrant neural development. The NS-iPSCs exhibited impaired development of EBs in which BMP and TGF-β signalings were activated. Defective early neuroectodermal development of NS-iPSCs recovered by inhibition of both signalings and further differentiated into NPCs. Intriguingly, neural cells developed from NS-NPCs exhibited abundancy of the glial cells, neurites of neuronal cells, and low electrophysiological property. Those aberrant phenotypes were also detected in NS-cerebral organoids. SHP2 inhibition in the NS-NPCs and NS-cerebral organoids ameliorated those anomalies such as biased glial differentiation and low neural activity.ConclusionOur findings demonstrate that SHP2 mutations contribute to precocious gliogenesis in NS-iPSCs during neural development in vitro.

Recent Advances of SHP2 Inhibitors in Cancer Therapy: Current Development and Clinical Application

SHP2 (Src homology-2 domain-containing protein tyrosine phosphatase-2) is a non-receptor protein tyrosine phosphatase that removes tyrosine phosphorylation. Functionally, SHP2 serves as an important hub to connect several intracellular oncogenic signaling pathways, such as Jak/STAT, PI3K/AKT, RAS/Raf/MAPK, and PD-1/PD-L1 pathways. Mutations and/or overexpression of SHP2 has been associated with genetic developmental diseases and cancers. Because of the role of SHP2 plays in many diseases, the development of inhibitors targeting the catalytic site in SHP2 has been pursued for more than a decade, but none has advanced to clinical development. Recent discovery of allosteric inhibitors has inspired a novel approach to selectively target SHP2 via the noncatalytic site. To date, four SHP2 allosteric inhibitors have entered clinical trials for the treatment of solid tumors. This review will provide a summary of the physiological and biological functions of SHP2 and discuss the development of nonallosteric/allosteric SHP2 inhibitors in recent years.

Protein tyrosine phosphatase SHP-1 modulates microtubule organization in later stages of mast cell activation

Abstract Antigen-mediated activation of mast cells initiates signaling events leading to degranulation and release of inflammatory mediators. Although rapid and transient microtubule reorganization during activation has been described, the molecular mechanisms that control their rearrangement are largely unknown. Important role in microtubule nucleation from centrosomes play γ-tubulin complexes. Here we report on the regulation of microtubule organization in bone marrow-derived mast cells (BMMCs) by Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 1 (SHP-1). Reciprocal immunoprecipitation experiments revealed that SHP-1 is present in complexes containing γ-tubulin complex proteins and protein tyrosine kinase Syk. Pull-down experiments with truncated forms of γ-tubulin and SHP-1 unveiled corresponding interaction domains. When compared to controls, activated BMMCs without SHP-1 had more protrusions containing microtubules, higher level of tyrosine phosphorylation, enhanced activity of Syk kinase, as well as increased expression of cytokines and prostaglandins. Intracellular calcium mobilization and degranulation also increased. Microtubule regrowth experiments in cells lacking SHP-1 revealed stimulation of microtubule nucleation, and phenotypic rescue experiments confirmed that SHP-1 represents a negative regulator of microtubule nucleation in BMMCs. Moreover, inhibition of the SHP-1 activity by inhibitors TPI-1 and NSC87877 also augmented microtubule nucleation. The regulation was due to changes of γ-tubulin accumulation in centrosomes. Our data suggest a novel SHP-1 dependent mechanism for the suppression of microtubule formation in later stages of mast cell activation.

P0-Related Protein Accelerates Human Mesenchymal Stromal Cell Migration by Modulating VLA-5 Interactions with Fibronectin.

P0-related protein (PZR), a Noonan and LEOPARD syndrome target, is a member of the transmembrane Immunoglobulin superfamily. Its cytoplasmic tail contains two immune-receptor tyrosine-based inhibitory motifs (ITIMs), implicated in adhesion-dependent signaling and regulating cell adhesion and motility. PZR promotes cell migration on the extracellular matrix (ECM) molecule, fibronectin, by interacting with SHP-2 (Src homology-2 domain-containing protein tyrosine phosphatase-2), a molecule essential for skeletal development and often mutated in Noonan and LEOPARD syndrome patients sharing overlapping musculoskeletal abnormalities and cardiac defects. To further explore the role of PZR, we assessed the expression of PZR and its ITIM-less isoform, PZRb, in human bone marrow mesenchymal stromal cells (hBM MSC), and its ability to facilitate adhesion to and spreading and migration on various ECM molecules. Furthermore, using siRNA knockdown, confocal microscopy, and immunoprecipitation assays, we assessed PZR and PZRb interactions with β1 integrins. PZR was the predominant isoform in hBM MSC. Migrating hBM MSCs interacted most effectively with fibronectin and required the association of PZR, but not PZRb, with the integrin, VLA-5(α5β1), leading to modulation of focal adhesion kinase phosphorylation and vinculin levels. This raises the possibility that dysregulation of PZR function may modify hBM MSC migratory behavior, potentially contributing to skeletal abnormalities.

Ethacrynic acid inhibits STAT3 activity through the modulation of SHP2 and PTP1B tyrosine phosphatases in DU145 prostate carcinoma cells

To identify signal transducer and activator of transcription factor 3 (STAT3) inhibitors, we generated STAT3-dependent gene expression signature by analyzing gene expression profiles of DU145 cancer cells treated with STAT3 inhibitor, piperlongumine and 2-hydroxycinnamaldehyde. Then we explored gene expression signature-based strategies using a connectivity map database and identified several STAT3 inhibitors, including ethacrynic acid (EA). EA is currently used as a diuretic drug. EA inhibited STAT3 activation in DU145 prostate cancer cells and consequently decreased the levels of STAT3 target genes such as cyclin A and MCL-1. Furthermore, EA treatment inhibited tumor growth in mice xenografted with DU145 cells and decreased p-STAT3 expression in tumor tissues. Knockdown of Src homology region 2 domain-containing phosphatase-2 (SHP2) or Protein tyrosine phosphatase 1B (PTP1B) gene expression by siRNA suppressed the ability of EA to inhibit STAT3 activation. When EA was combined with an activator of SHP2 or PTP1B, p-STAT3 expression was synergistically decreased; when EA was combined with an inhibitor of SHP2 or PTP1B, p-STAT3 expression was rescued. By using an affinity pulldown assay with biotinyl-EA, EA was shown to associate with SHP2 and PTP1B in vitro. Additionally, the drug affinity responsive target stability (DARTS) assay confirmed the direct binding of EA to SHP2 and PTP1B. SHP2 is activated by EA through active phosphorylation at Y580 and direct binding to SHP2. Collectively, our results suggest that EA inhibits STAT3 activity through the modulation of phosphatases such as SHP2 and PTP1B and may be a potential anticancer drug to target STAT3 in cancer progression.

SHP1 tyrosine phosphatase gets involved in host defense against Streptococcus agalactiae infection and BCR signaling pathway in Nile tilapia (Oreochromis niloticus)

Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1), a kind of protein tyrosine phosphatases (PTPs), is a critical regulator of antigen receptor signal transduction. Signal transduction of BCR is regulated by phosphatases in teleost as in mammals. In this study, SHP1 from Nile tilapia (Oreochromis niloticus) (OnSHP1) was identified and characterized, including the expression pattern against bacterial infection and regulation function in BCR signaling pathway. The open reading frame of OnSHP1 contains 1749 bp of nucleotide sequence, encoding a protein of 582 amino acids. The OnSHP1 protein was highly conversed compared to that of other species, including two amino-terminal SH2 domains at the N terminus and a PTP catalytic domain. Transcriptional expression analysis revealed that OnSHP1 was detected in all examined tissues and highly expressed in spleen. The up-regulated OnSHP1 expression was observed in peripheral blood, spleen and anterior kidney following challenge with Streptococcus agalactiae or lipopolysaccharide (LPS) in vivo, as well as that displayed in leukocytes stimulated with S. agalactiae or LPS in vitro. Further, after induction with mouse anti-tilapia IgM monoclonal antibody in vitro, OnSHP1 was significantly up-regulated in leukocytes. When spleen leukocytes treated with PTP Inhibitor II in vitro, the phosphorylation level of OnSHP1 at the phosphorylation sites (Y535 and Y557) and the cytoplasmic free Ca2+ concentration were up-regulated significantly. Overall, the findings of this study indicate that SHP1 gets involved in host defense against bacterial infection and BCR signaling pathway in Nile tilapia.

Identification, recombinant expression and immunological study of Lja-SHP2 in Lampetra japonica.

The protein tyrosine phosphatase SHP2 of higher vertebrates, encoded by ptpn11 gene, catalyzes the dephosphorylation of tyrosine phosphorylation site, and plays regulatory roles in various signaling pathways by cooperating with other protein tyrosine kinase. Previous studies have shown that SHP2 plays an important role in the activation and signal transduction of T and B cells in higher vertebrates. To study the role of a SHP2 homologous molecule of lampreys (Lja-SHP2) in immune response, we cloned and expressed the open reading frame sequence of Lja-SHP2 gene in prokaryotic expression vector pET-32a. The recombinant protein was successfully expressed in E. coli and the rabbit-derived polyclonal antibody was prepared. Lampetra japonica were immunized with mixed bacteria, and the mRNA and protein of Lja-SHP2 in immune-related cells and tissues were detected by real-time quantitative PCR and Western blotting after immunization. The Lja-SHP2 mRNA and protein were not significantly affected in leukocytes and supraneural myeloid bodies, but up-regulated significantly in gill tissues (P<0.05) after challenged by mixed bacteria, which indicated that Lja-SHP2 mainly participates in the immune response of gill tissues after mixed bacteria stimulation. To further investigate whether Lja-SHP2 level was affected in three lymphocyte subsets, the B-cell mitogen lipopolysaccharide (LPS) and T-cell mitogen phytohaemagglutinin (PHA) were employed to boost the immune response in L. japonica. LPS immune stimulation increased Lja-SHP2 in leucocytes significantly compared with the control group, and but had a marginal effect on Lja-SHP2 expression in gills and supraneural myeloid bodies. PHA immune stimulation could up-regulate Lja-SHP2 level in leukocytes, gill tissues and supraneural myeloid bodies. The change of Lja-SHP2 was especially dramatical in leukocytes, which was about 2.5 times higher than that in the control group, suggesting that Lja-SHP2 is involved in the lamprey immune response mediated by PHA. Consistent with the previous finding that PHA could induce the activation of VLRA+ lymphocytes, our results showed that Lja-SHP2 might be included in the immune response of VLRA+ lymphocytes mediated by PHA in gills. This research will benefit exploring the functions of Lja-SHP2 in the immune response of lamprey and will provide clues for understanding the phylogenesis of SHP2 molecular family, and its roles in the early occurrence and evolution of adaptive immune system in higher vertebrates.

Trans-Fatty acids facilitate DNA damage-induced apoptosis through the mitochondrial JNK-Sab-ROS positive feedback loop

trans-Fatty acids (TFAs) are unsaturated fatty acids that contain one or more carbon-carbon double bonds in trans configuration. Epidemiological evidence has linked TFA consumption with various disorders, including cardiovascular diseases. However, the underlying pathological mechanisms are largely unknown. Here, we show a novel toxic mechanism of TFAs triggered by DNA damage. We found that elaidic acid (EA) and linoelaidic acid, major TFAs produced during industrial food manufacturing (so-called as industrial TFAs), but not their corresponding cis isomers, facilitated apoptosis induced by doxorubicin. Consistently, EA enhanced UV-induced embryonic lethality in C. elegans worms. The pro-apoptotic action of EA was blocked by knocking down Sab, a c-Jun N-terminal kinase (JNK)-interacting protein localizing at mitochondrial outer membrane, which mediates mutual amplification of mitochondrial reactive oxygen species (ROS) generation and JNK activation. EA enhanced doxorubicin-induced mitochondrial ROS generation and JNK activation, both of which were suppressed by Sab knockdown and pharmacological inhibition of either mitochondrial ROS generation, JNK, or Src-homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1) as a Sab-associated protein. These results demonstrate that in response to DNA damage, TFAs drive the mitochondrial JNK-Sab-ROS positive feedback loop and ultimately apoptosis, which may provide insight into the common pathogenetic mechanisms of diverse TFA-related disorders.

3'-sialyllactose targets cell surface protein, SIGLEC-3, and induces megakaryocyte differentiation and apoptosis by lipid raft-dependent endocytosis.

3'-sialyllactose is one of the abundant components in human milk oligosaccharides (HMOs) that protect infants from various viral infections in early stages of immune system development. 3SL is a combination of lactose and sialic acid. Most sialic acids are widely expressed in animal cells and they bind to siglec proteins. In this study, we demonstrate that 3SL specifically binds to CD33. It induces megakaryocyte differentiation and subsequent apoptosis by targeting cell surface protein siglec-3 (CD33) in human chronic myeloid leukemia K562 cells. The 3SL-bound CD33 was internalized to the cytosol via caveolae-dependent endocytosis. At the molecular level, 3SL-bound CD33 recruits the suppressor of cytokine signaling 3 (SOCS3) and SH2 domain-containing protein tyrosine phosphatase 1 (SHP1). SOCS3 is degraded with CD33 by proteasome degradation, while SHP-1 activates extracellular signal-regulated kinase (ERK) to induce megakaryocytic differentiation and subsequent apoptosis. The present study, therefore, suggests that 3SL is a potential anti-leukemia agent affecting differentiation and apoptosis.

Hyperglycemia-induced ubiquitination and degradation of β-catenin with the loss of platelet endothelial cell adhesion molecule-1 in retinal endothelial cells.

Increased retinal vascular permeability is one of the earliest manifestations of diabetic retinopathy. The aim of this study was to investigate the role of hyperglycemia-induced platelet endothelial cell adhesion molecule-1 loss on retinal vascular permeability via the β-catenin pathway. Type I diabetes was induced in male Wistar rats using streptozotocin injections, with age-matched non-diabetic rats as controls. Rat retinal microvascular endothelial cells were grown under normal or high glucose conditions for 6days. Small interfering Ribonucleic Acid was used to knock down platelet endothelial cell adhesion molecule-1 in rat retinal microvascular endothelial cells for loss-of-function studies. Retinas and rat retinal microvascular endothelial cells were subjected to Western blot, immunofluorescence labeling, and co-immunoprecipitation analyses to assess protein levels and interactions. A biotinylated gelatin and fluorescein isothiocyanate-avidin assay was used for retinal endothelial cell permeability studies. β-catenin, β-catenin/platelet endothelial cell adhesion molecule-1 interaction, active Src homology 2 domain-containing protein tyrosine phosphatasewere significantly decreased, while β-catenin ubiquitination levels and endothelial permeability were significantly increased, in hyperglycemic retinal endothelial cells. Similar results were observed with platelet endothelial cell adhesion molecule-1 partial knockdown, where β-catenin and active Src homology 2 domain-containing protein tyrosine phosphatase levels were decreased, while phospho-β-catenin and retinal endothelial cell permeability were increased. Platelet endothelial cell adhesion molecule-1 loss may contribute to increased retinal endothelial cell permeability by attenuating β-catenin levels under hyperglycemic conditions.

SHP-2 in Lymphocytes' Cytokine and Inhibitory Receptor Signaling

Somewhat counterintuitively, the tyrosine phosphatase SHP-2 (SH2 domain-containing protein tyrosine phosphatase-2) is crucial for the activation of extracellular signal-regulated kinase (ERK) downstream of various growth factor receptors, thereby exerting essential developmental functions. This phosphatase also deploys proto-oncogenic functions and specific inhibitors have recently been developed. With respect to the immune system, the role of SHP-2 in the signaling of cytokines relevant for myelopoiesis and myeloid malignancies has been intensively studied. The function of this phosphatase downstream of cytokines important for lymphocytes is less understood, though multiple lines of evidence suggest its importance. In addition, SHP-2 has been proposed to mediate the suppressive effects of inhibitory receptors (IRs) that sustain a dysfunctional state in anticancer T cells. Molecules involved in IR signaling are of potential pharmaceutical interest as blockade of these inhibitory circuits leads to remarkable clinical benefit. Here, we discuss the dichotomy in the functions ascribed to SHP-2 downstream of cytokine receptors and IRs, with a focus on T and NK lymphocytes. Further, we highlight the importance of broadening our understanding of SHP-2′s relevance in lymphocytes, an essential step to inform on side effects and unanticipated benefits of its therapeutic blockade.

Evaluation of SHP1-P2 methylation as a biomarker of lymph node metastasis in patients with squamous cell carcinoma of the head and neck

Abstract Background Hypermethylation of Src homology region 2 domain-containing protein-tyrosine phosphatase 1 promoter 2 (SHP1-P2) has been proven as an epithelial-specific marker. This marker has been used for the detection of lymph node metastasis in patients with lung cancer or colon cancer. Objectives To investigate SHP1-P2 methylation in patients with squamous cell carcinoma of the head and neck (HNSCC) and determine its potential for micrometastasis detection in the lymph nodes of patients with HNSCC. Methods SHP1-P2 methylation levels were analyzed by combined methylation-specific primer TaqMan real-time PCR in 5 sample groups: normal tonsils (n = 10), microdissected squamous cell carcinoma epithelia (n = 9), nonmetastatic head and neck cancer lymph nodes (LN N0, n = 15), metastatic HNSCC histologically negative for tumor cells (LN–, n = 18), and matched cases histologically positive for tumor cells (LN+, n = 18). Results SHP1-P2 methylation of 10.27 ± 4.05% was found in normal tonsils as a lymphoid tissue baseline, whereas it was 61.31 ± 17.00% in microdissected cancer cell controls. In the 3 lymph node groups, the SHP1-P2 methylation levels were 9.99 ± 6.61% for LN N0, 14.49 ± 10.03% for LN- Nx, and 41.01 ± 24.51% for LN+ Nx. The methylation levels for LN- Nx and LN+ Nx were significantly different (P = 0.0002). Receiver operating characteristic curve analysis of SHP1-P2 methylation demonstrated an area under the curve of 0.637 in distinguishing LN N0 from LN– Nx. Conclusions SHP1-P2 methylation was high in HNSCC, and low in lymphoid tissues. This methylation difference is concordant with lymph node metastasis.

PTPN3 suppresses lung cancer cell invasiveness by counteracting Src-mediated DAAM1 activation and actin polymerization

Cancer cell migration plays a crucial role during the metastatic process. Reversible tyrosine phosphorylation by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) have been implicated in the regulation of cancer cell migration and invasion. However, the underlying mechanisms have not been fully elucidated. Here, we show that depletion of the FERM and PDZ domain-containing protein tyrosine phosphatase PTPN3 enhances lung cancer cell migration/invasion and metastasis by promoting actin filament assembly and focal adhesion dynamics. We further identified Src and DAAM1 (dishevelled associated activator of morphogenesis 1) as interactors of PTPN3. DAAM1 is a formin-like protein involved in the regulation of actin cytoskeletal remodeling. PTPN3 inhibits Src activity and Src-mediated phosphorylation of Tyr652 on DAAM1. The tyrosine phosphorylation of DAAM1 is essential for DAAM1 homodimer formation and actin polymerization. Ectopic expression of a DAAM1 phosphodeficient mutant inhibited F-actin assembly and suppressed lung cancer cell migration and invasion. Our findings reveal a novel mechanism by which reversible tyrosine phosphorylation of DAAM1 by Src and PTPN3 regulates actin dynamics and lung cancer invasiveness.

Investigating the reason for loss-of-function of Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2) caused by Y279C mutation through molecular dynamics simulation

Noonan syndrome with multiple lentigines (NSML), formerly known as LEOPARD syndrome (LS), is an autosomal dominant inherited multisystemic disorder. Most patients involve mutation in SHP2 encoded by tyrosine-protein phosphatase non-receptor type 11 (PTPN11) gene. Studies have shown that NSML-associated Y279C mutation exhibited the reduced phosphatase activity, leading to loss-of-function (LOF) of SHP2. However, the effect of the Y279C mutation on the SHP2 at the molecular level is unclear. In this study, molecular dynamics simulations of SHP2 wild-type (SHP2WT) and Y279C mutant (SHP2Y279C) were performed to investigate the structural differences in proteins after Y279C mutation and to find out the reason for loss-of-function of SHP2. Through a series of post-dynamic analyses, it was found that the protein occupied a smaller phase space after Y279C mutation, showing reduced flexibility. Specifically, due to the mutation of Y279C, the secondary structures of these two regions (residues Lys70-Ala72 and Gly462-Arg465) were significantly transformed from Turn to α-helix and β-strand. Furthermore, by calculating the residue interaction network, hydrogen bond occupancy and binding free energy, it was further revealed that the conformational differences between SHP2WT and SHP2Y279C systems were mainly caused by the differences in the interaction between Arg465–Phe469, Ile463–Gly467, Cys279–Lys70, Cys459–Ala72, Gly464–Phe71, Phe71–Ile463, Ile463–Ala505 and Arg465–Glu361. Consequently, this finding is expected to provide a new insight into the reason for loss-of-function of SHP2 caused by Y279C mutation.Communicated by Ramaswamy H. Sarma

Sauchinone suppresses FcεRI-mediated mast cell signaling and anaphylaxis through regulation of LKB1/AMPK axis and SHP-1-Syk signaling module

Sauchinone, the biologically active lignan of Saururus chinensis, has been reported to have anti-inflammatory, antitumor, antioxidant, and hepatoprotective properties. However, little is known about the effect of sauchinone on FcεRI-mediated mast cell activation. The aim of this study was to evaluate the anti-allergic activity of sauchinone and the underlying mechanism using mouse bone marrow-derived mast cells (BMMCs) and the mast cell-mediated passive cutaneous anaphylaxis (PCA) model. Sauchinone markedly suppressed FcεRI-mediated activation of positive signaling mediators, including Syk, linker for activation of T cells (LAT), phospholipase C (PLC)γ, mitogen-activated protein (MAP) kinases, Akt, IκB kinase (IKK), and intracellular Ca2+, and increased the activation of negative signaling mediators, including liver kinase B (LKB)1/AMP-activated protein kinase (AMPK) and Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1. Interestingly, sauchinone increased the interaction between SHP-1 and Syk. Consequently, sauchinone significantly suppressed FcεRI-mediated BMMC degranulation and synthesis of eicosanoids and cytokines. These inhibitory effects of sauchinone were diminished in BMMCs treated with siRNAs targeting LKB1, AMPKα2, or SHP-1, and in BMMCs isolated from AMPKα2-deficient mice. In addition, administration of sauchinone markedly suppressed the IgE-mediated PCA reaction in wild-type mice, and this inhibitory effect was significantly reduced in AMPKα2−/− mice. Taken together, these data suggest that sauchinone suppresses FcεRI-mediated mast cell activation and anaphylaxis through modulation of the LKB1/AMPK and SHP-1/Syk pathways. Therefore, sauchinone might be a potential therapeutic agent for the treatment of allergic inflammatory diseases.

Probing the acting mode and advantages of RMC-4550 as an Src-homology 2 domain-containing protein tyrosine phosphatase (SHP2) inhibitor at molecular level through molecular docking and molecular dynamics

The over-activation of Ras/mitogen-activated protein kinase (MAPK) signaling pathway associated with a variety of cancers is usually related with abnormal activation of Src-homology 2 domain-containing protein tyrosine phosphatase (SHP2). For this purpose, SHP2 has attracted extensive interest as a potential target for cancer treatment. RMC-4550, as a newly developed selective inhibitor of SHP2, possesses an overwhelming advantage over the previous generation inhibitor SHP099 in terms of in vitro activity. However, the binding mode of SHP2 with RMC-4550 and the reason for the high efficiency of RMC-4550 as SHP2 inhibitor at molecular level are still unclear. Therefore, in this study, the binding mode of RMC-4550 with SHP2 and the superiorities of RMC-4550 as inhibitor at binding affinity and dynamic interactive behavior with SHP2 were probed by molecular docking and molecular dynamics (MD) simulations. By comparing the results of molecular docking, it was found that SHP2 formed more tight interaction with RMC-4550 than that with SHP099. Subsequently, a series of post-dynamic analyses on three simulation trajectories (SHP2WT, SHP2SHP099 and SHP2RMC-4550) were performed and found that the SHP2 protein bound with RMC-4550 maintained a firmer interaction between N-Src-homology 2 (N-SH2) and PTP domain throughout the MD simulation, leading to a more stable protein conformation. The finding here provides new clues for the design of SHP2 inhibitor against the over-activation of Ras/MAPK pathway.Communicated by Ramaswamy H. Sarma