SHP-1 Expression Research Articles

The Tyrosine Phosphatase Shp2 Regulates Yapy357 Phosphorylation, And In Vivo Sensitivity To Cytotoxic Chemotherapy In Cholangiocarcinoma

Presenter: EeeLN Buckarma MD | Mayo Clinic, Rochester Background: Cancer of the biliary tract, cholangiocarcinoma (CCA), is increasing in incidence and has limited treatment options. In an attempt to further understand signaling pathways driving oncogenesis and impacting response to therapy we, and others, have identified altered activation of the transcriptional co-activator, Yes- associated protein (YAP) in CCA. Canonical regulatory pathways impacting YAP consist of serine kinases; however, more recently we have demonstrated a central role for tyrosine phosphorylation in regulating YAP function in CCA. Herein we explore the role of tyrosine phosphatases in regulating YAP tyrosine phosphorylation and sensitivity to current cytotoxic chemotherapy. Methods: Molecular studies utilized the human CCA cell lines HuCCT-1 and KMCH. Baseline tyrosine phosphatase levels were assessed by RT-PCR and immunoblot. YAP-interacting phosphatases were identified by co-immunoprecipitation. The selective tyrosine phosphatse inhibitors NSC87877 and SHP099 were utilized. Tyrosine phosphorylation was assessed by immunoblot. YAP transcriptional activity was evaluated by RT-PCR for YAP target genes. SHP2 levels were modulated utilizing knockout via a doxycycline-inducible CRISPR/Cas9 system. Enforced expression of SHP2 was explored utilizing catalytically active and dead constructs. YAP sub-cellular localization was assessed by immunofluorescence. Proliferation was evaluated by MTS assay and cell viability was assessed with Cell Titer-Glo. Cytotoxic agents’ gemcitabine and cisplatin were utilized for in vitro and in vivo studies. CCA xenografts (via mouse flank model) were generated with non-targeting small guide-bearing KMCH and sgSHP2-KMCH cells. Mice were treated with q3 day cytotoxic treatment x 3 weeks and tumors were evaluated by growth rate, weight and volume. Results: Profiling of tyrosine phosphatase levels by RT-PCR and immunoblot demonstrated higher levels in KMCH cells compared to HuCCT-1; notably the YAP-interacting phosphatase PTPN11 (SHP2) was elevated. Consistent with the anticipated function of the phosphatases, immunoblot demonstrated lower levels of tyrosine phosphorylated YAP (p-YAPY357) in KMCH cells compared to HuCCT-1. The role of SHP2 was further probed by incubation of KMCH and HuCCT-1 cells with NSC87877 which was associated with an increase in p-YAPY357 levels and YAP co-transcriptional activity. In addition, KMCH cells demonstrated increased YAP nuclear localization following incubation with NSC87877. The effects seen with YAP tyrosine phosphorylation were paralleled in doxycycline-inducible CRISPR/Cas9 sgSHP2-KMCH cells. In HuCCT1 cells overexpressing SHP2 (WT-SHP2), pYAPY357 levels and transcriptional activity were decreased, and the localization of YAP was shifted towards the cytoplasmic compartment. Expression of a phosphatase-dead construct (C459S-SHP2) had no demonstrable effect. A deletion-reconstitution approach utilizing induced sgSHP2-KMCH cells followed by reconstitution with expression of either wild-type or phosphatase-dead constructs was associated with a decrease in p-YAP357 levels, YAP transcriptional activity, and YAP baseline nuclear localization in wild-type alone. Increased cell death was observed in KMCH cells as compared the HuCCT-1 and sgSHP2-KMCH cells incubated with gemcitabine and cisplatin. In keeping with these observations, in vivo studies demonstrated that the sgSHP2-KMCH cell lines when compared to wild type, developed tumors with faster growth rates, and subsequently these tumors were less sensitive to the combination of gemcitabine and cisplatin therapy. Conclusion: The tyrosine phosphatase SHP2 regulates p-YAPY357 levels, YAP co-transcriptional activity, and in vivo sensitivity to current standard chemotherapy in cholangiocarcinoma.

B Cell-Intrinsic SHP1 Expression Promotes the Gammaherpesvirus-Driven Germinal Center Response and the Establishment of Chronic Infection.

Gammaherpesviruses are ubiquitous pathogens that establish lifelong infections in the majority of adults worldwide. Chronic gammaherpesvirus infection has been implicated in both lymphomagenesis and, somewhat controversially, autoimmune disease development. Pathogenesis is largely associated with the unique ability of gammaherpesviruses to usurp B cell differentiation, specifically, the germinal center response, to establish long-term latency in memory B cells. The host tyrosine phosphatase SHP1 is known as a brake on immune cell activation and is downregulated in several gammaherpesvirus-driven malignancies. However, here we demonstrate that B cell- but not T cell-intrinsic SHP1 expression supports the gammaherpesvirus-driven germinal center response and the establishment of viral latency. Furthermore, B cell-intrinsic SHP1 deficiency cooperated with gammaherpesvirus infection to increase the levels of double-stranded DNA-reactive antibodies at the peak of viral latency. Thus, in spite of decreased SHP1 levels in gammaherpesvirus-driven B cell lymphomas, B cell-intrinsic SHP1 expression plays a proviral role during the establishment of chronic infection, suggesting that the gammaherpesvirus-SHP1 interaction is more nuanced and is modified by the stage of infection and pathogenesis.IMPORTANCE Gammaherpesviruses establish lifelong infection in a majority of adults worldwide and are associated with a number of malignancies, including B cell lymphomas. These viruses infect naive B cells and manipulate B cell differentiation to achieve a lifelong infection of memory B cells. The germinal center stage of B cell differentiation is important as both an amplifier of the viral latent reservoir and the target of malignant transformation. In this study, we demonstrate that expression of tyrosine phosphatase SHP1, a negative regulator that normally limits the activation and proliferation of hematopoietic cells, enhances the gammaherpesvirus-driven germinal center response and the establishment of chronic infection. The results of this study uncover an intriguing beneficial interaction between gammaherpesviruses that are presumed to profit from B cell activation and a cellular phosphatase that is traditionally perceived to be a negative regulator of the same processes.

SHP2 deficiency promotes Staphylococcus aureus pneumonia following influenza infection.

ObjectivesSecondary bacterial pneumonia is common following influenza infection. However, it remains unclear about the underlying molecular mechanisms.Materials and methodsWe established a mouse model of post‐influenza S aureus pneumonia using conditional Shp2 knockout mice (LysMCre/+:Shp2flox/flox). The survival, bacterial clearance, pulmonary histology, phenotype of macrophages, and expression of type I interferons and chemokines were assessed between SHP2 deletion and control mice (Shp2flox/flox). We infused additional KC and MIP‐2 to examine the reconstitution of antibacterial immune response in LysMCre/+:Shp2flox/flox mice. The effect of SHP2 on signal molecules including MAPKs (JNK, p38 and Erk1/2), NF‐κB p65 and IRF3 was further detected.Results LysMCre/+:Shp2flox/flox mice displayed impaired antibacterial immunity and high mortality compared with control mice in post‐influenza S aureus pneumonia. The attenuated antibacterial ability was associated with the induction of type I interferon and suppression of chemo‐attractants KC and MIP‐2, which reduced the infiltration of neutrophils into the lung upon secondary bacterial invasion. In additional, Shp2 knockout mice displayed enhanced polarization to alternatively activated macrophages (M2 phenotype). Further in vitro analyses consistently demonstrated that SHP2‐deficient macrophages were skewed towards an M2 phenotype and had a decreased antibacterial capacity. Moreover, SHP2 modulated the inflammatory response to secondary bacterial infection via interfering with NF‐κB and IRF3 signalling in macrophages.ConclusionsOur findings reveal that the SHP2 expression enhances the host immune response and prompts bacterial clearance in post‐influenza S aureus pneumonia.

Overexpression and Targeted Activation of the Protein Phosphatases SHP-1 Abrogates Survival Pathways in Large Granular Lymphocyte Leukemia (LGLL)

Introduction: Aberrant phosphorylation-mediated signaling is a well-established condition involved in the onset and progression of virtually all types of cancer. That not only depends on the over-activation of protein kinases, but also on the lack of the proper counterbalance of protein phosphatases, because of their decreased expression or activity. In turn, this is likely to be due to genetic and epigenetic alterations, or interactions with cellular inhibitors and inhibitory post-translational modifications, respectively. In Large Granular Lymphocyte Leukemia (LGLL), a rare lymphoproliferative disorder characterized by clonal expansion of either cytotoxic T lymphocytes (T-LGLL) or natural killer cells (chronic lymphoproliferative disorder of NK cells, CLPD-NK), constitutive activation of phosphorylation-regulated survival pathways, especially the JAK2/STAT3 axis, but also PI3K/Akt and NF-κB pathways among others, has been found to account for the molecular mechanisms sustaining monoclonal cell proliferation and resistance to apoptosis. Interestingly, since such pathways are modulated by the tyrosine phosphatase SHP-1 under normal conditions, it is conceivable that putative alterations of this phosphatase, and consequent inability to disrupt hyperactive pathways, play a role in LGLL pathogenesis. In this study, we assessed the ability of small molecules inducing the expression and stimulating the activity of SHP-1 to abrogate the survival pathways in LGL by counteracting aberrant signals generated by constitutively activated protein kinases. Methods: Peripheral blood specimens were collected from untreated patients with LGLL (both T-LGLL and CLPD-NK). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (Sigma Aldrich) gradient separation. LGLs were further separated from PBMCs by using immunomagnetic beads (Miltenyi Biotec). LGLs were incubated either with acetyl-11-keto-β-boswellic acid (AKBA), a pentacyclic triterpene capable of inducing SHP-1 expression, or SC-78, a regorafenib-like derivative specifically stimulating SHP-1 activity, or both at increasing concentrations and discrete time points. After such treatments, LGLs underwent annexin V-PI flow cytometry to assess the extent of apoptosis or were lysed to monitor the abundance of SHP-1 and the phosphatase activity thereof. Moreover, the phosphorylation/activation status and the protein level of factors directly involved in the constitutively activated survival pathways of LGLs were evaluated by Western blot analysis. Results: Both AKBA and SC-78 proved effective at eliciting a significant level of apoptosis in the low micromolar range, exhibiting an additive effect when used in combination. Moreover, both compounds activated SHP-1 activity, as demonstrated by in-vitro phosphatase assays. As to the phosphorylation-dependent signals affected by SHP-1 activation, we observed a marked decrease in the phosphorylation status of STAT3, resulting in the downregulation of the STAT3-induced expression of downstream target genes (Mcl-1, survivin, Cyclin D and S1P5), as well as the dephosphorylation of Akt and p65, which confirmed the role of SHP-1 as a functional antagonist of survival pathways in LGLs. Conclusion: Taken together, our results support the newly emerging evidence that the targeted activation of SHP-1, which is already recognized as a tumor suppressor in other tumor cells, may serve as a mechanism countering the aberrant pro-survival signals, thus opening new options for the treatment of LGLL. Disclosures Zambello: Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

RMC-4550, an Allosteric Inhibitor of SHP2, Displays Therapeutic Efficacy in Pre-Clinical Models of Myeloproliferative Neoplasms

Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) are JAK2-driven disorders resulting from mutations in JAK2, MPL, or CALR. Ruxolitinib, the only FDA-approved JAK2 inhibitor for MPNs, alleviates patient symptomology and improves quality of life, but has little effect on reducing mutant allele burden. This persistent survival of MPN cells in the face of ruxolitinib, as well as other JAK2 inhibitors that have been clinically tested, is a major clinical bottleneck to the development of an effective targeted therapy for MPN patients. Identifying new therapeutic targets which play critical roles in MPN cells and/or in JAK2 inhibitor persistence may lead to improved MPN therapies. SHP2 is an oncogenic tyrosine phosphatase that is an effector of growth factor and cytokine receptor signaling. SHP2 plays a critical role in the activation of the RAS-ERK pathway and regulates JAK-STAT signaling via numerous phosphatase-dependent mechanisms. Activating mutations of SHP2(PTPN11) have been identified in leukemia, including 8% of MPN patients whose disease progressed to acute myeloid leukemia (AML). In addition, SHP2 has been shown to mediate adaptive resistance to targeted therapies in several cancers. Given the role of SHP2 in cytokine and JAK-STAT signaling, we envisaged a potential role of SHP2 in MPN cell growth and/or survival and ruxolitinib persistence. Treatment of JAK2-V617F-driven MPN model cell lines (UKE1, SET2, and BaF3-JAK2-V617F) with ruxolitinib blocked constitutive tyrosine phosphorylation of SHP2, including phosphorylation of Y542, a marker for activated SHP2. This phosphorylation, however, was restored in ruxolitinib persistent cells. Combination treatment of the allosteric SHP2 inhibitor RMC-4550 (Revolution Medicines) with ruxolitinib prevented the development of ruxolitinib persistent cells and pre-established persistent cells remained sensitive to SHP2 inhibition. RMC-4550 treatment led to significantly reduced levels of pERK consistent with the role of SHP2 in RAS signaling. Interestingly, pERK levels in persistent cells were more sensitive to SHP2 inhibition compared to drug naïve cells suggesting pERK was more dependent on SHP2 in ruxolitinib persistent cells. SHP2 inhibitor treatment increased pSTAT5(Y694) in drug naïve cells but this increase was not observed in similarly treated persistent cells. Furthermore, while ruxolitinib inhibited pERK levels in UKE1 and SET2 cells, pERK levels recovered within 24 hrs of treatment. SHP2 inhibition prevented the recovery of pERK in the presence of ruxolitinib. Collectively, these data suggest that signaling pathways in MPN cells treated with ruxolitinib can become rewired, gaining greater dependence on SHP2, concomitant with sustained pERK and cell survival/growth. Interestingly, we identified a known activating SHP2 mutation (F71L) in UKE1 cells obtained from two independent sources - consistent with the presence of PTPN11 mutations in post-MPN AML. The persistent survival of UKE1 cells in ruxolitinib was antagonized by CRISPR-mediated reduction of SHP2 expression, providing further evidence that SHP2 contributes to ruxolitinib persistence. To assess the effects of a SHP2 inhibitor on MPN progression in vivo, we employed the MPLW515Lbone marrow transplant mouse model of MPN. Initial assessment of therapeutic treatment of mice with an established MPN phenotype indicated that once daily treatment of RMC-4550 (10 or 30 mg/kg) antagonized the MPN phenotype. Complete blood counts indicated a significant reduction in white blood cells, monocytes, and neutrophils compared to vehicle treated mice, while flow cytometry analysis indicated RMC-4550 diminished CD11b+ cell numbers to near that observed in mice transplanted with MPLWT-transduced bone marrow. RMC-4550 improved the overall health of diseased mice, as indicated by increased weight, and significantly reduced organomegaly of the spleen and liver compared to vehicle treated MPN mice. Finally, erythropoietin independent erythroid colony formation of JAK2V617F-positive MPN patient cells was suppressed following SHP2 inhibition, which synergized or enhanced the inhibition induced by ruxolitinib in this assay. In summary, our results suggest that SHP2 inhibition may represent a potential MPN therapy in both ruxolitinib naïve and resistant patients and is an attractive therapeutic target for future clinical investigation. Disclosures Epling-Burnette: Incyte Corporation: Research Funding; Forma Therapeutics: Research Funding; Celgene Corporation: Patents & Royalties, Research Funding. Reuther:Incyte Corporation: Research Funding.

Gain-Of-Function E76K-Mutant SHP2 Promotes Cell Proliferation, Metastasis, And Tumor Growth In Glioblastoma Through Activation Of The ERK/CREB Pathway.

PurposeThe aim of this study was to investigate the effects of gain-of-function (GOF) E76K-mutant Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) on the biological behaviors of glioblastoma (GBM) cells, and explore the molecular mechanisms of GBM progression.MethodsFirstly, a negative control vector and vectors overexpressing SHP2 and E76K-mutant SHP2 were transduced into GBM cells (U87 and A172) using a lentivirus. The effect of GOF-mutant SHP2 on proliferation was measured using the MTT assay, flow cytometry, colony formation assay, and soft agar assay. Moreover, the migration and invasion of GBM cells were determined through the transwell assay. Related proteins of the extracellular signal-regulated kinase/cAMP response element binding protein (ERK/CREB) pathway were detected by Western blotting analysis. A xenograft model was established to confirm the tumor-promoting effect of GOF-mutant SHP2 in vivo. Finally, ERK was inhibited using a mitogen-activated protein kinase/ERK kinase inhibitor (U0126) to further explore the molecular mechanism of GOF-mutant SHP2 affecting GBM cells.ResultsAfter transduction, the expression of SHP2 in the SHP2-mutant and SHP2-overexpression groups was higher than that observed in the control and normal groups. Our data indicated that GOF-mutant SHP2 enhanced the abilities of GBM cells for proliferation, migration, and invasion in vitro, and promoted tumor growth in vivo. Mechanistically, the ERK/CREB pathway was activated, and the levels of relevant proteins were increased in the SHP2-mutant group. Furthermore, following inhibition of ERK in the GOF-SHP2 mutant group, the activation of CREB was also depressed, and the malignant biological behaviors were weakened accordingly.ConclusionThe GOF-mutant SHP2 promoted GBM cell proliferation, metastasis, and tumor growth through the ERK/CREB pathway, providing a promising target for the treatment of GBM.

Phloretin attenuates STAT-3 activity and overcomes sorafenib resistance targeting SHP-1\u2013mediated inhibition of STAT3 and Akt/VEGFR2 pathway in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the most common primary liver malignancy. Phloretin (PH) possesses anticancer, antitumor, and hepatoprotective effects, however, the effects and potential mechanisms of phloretin remain elusive.MethodsFive HCC cells were tested in vitro for sensitivity to PH, Sorafenib (Sor) or both and the apoptosis, signal transduction and phosphatase activity were analyzed. To validate the role of SHP-1, we used PTP inhibitor III and SHP-1 siRNA. Further, we used purified SHP-1 proteins or HCC cells expressing deletion N-SH2 domain or D61A point mutants to study the PH efficacy on SHP-1. The `in vivo studies were conducted using HepG2 and SK-Hep1 and Sor resistant HepG2SR and Huh7SR xenografts. Molecular docking was done with Swiss dock and Auto Dock Vina.ResultsPH inhibited cell growth and induced apoptosis in all HCC cells by upregulating SHP-1 expression and downregulating STAT3 expression and further inhibited pAKT/pERK signaling. PH activated SHP-1 by disruption of autoinhibition of SHP-1, leading to reduced p-STAT3Tyr705 level. PH induced apoptosis in two Sor-resistant cell lines and overcome STAT3, AKT, MAPK and VEGFR2 dependent Sor resistance in HCCs. PH potently inhibited tumor growth in both Sor-sensitive and Sor-resistant xenografts in vivo by impairing angiogenesis, cell proliferation and inducing apoptosis via targeting the SHP-1/STAT3 signaling pathway.ConclusionOur data suggest that PH inhibits STAT3 activity in Sor-sensitive and -resistant HCCs via SHP-1–mediated inhibition of STAT3 and AKT/mTOR/JAK2/VEGFR2 pathway. Our results clearly indicate that PH may be a potent reagent for hepatocellular carcinoma and a noveltargeted therapy for further clinical investigations.Graphical abstract

Inhibitory Effect of Decitabine-inhibeting Methylation of SHP-1 Gene on Proliferation and Apoptosis of MDS Cell Line Skm-1

To investigate the phenotype and molecular mechanism of DCA on MDS cell model, and to study the response of chemotherapeutic medicines to MDS cells through multiple dimensions, such as cell proliferation, invasion, migration and apoptosis, thus revealing the molecular mechanism of DCA treatment of MDS and its relationship with SHP-1 gene methylation. MTT assay was used to determine the survival rate of MDS cells after treated by different concentrations of DCA. The effect of DCA on the invasion and migration of MDS cells was detected by Transwell assay method. Apoplexin V-FITCPI was used to detect apoptosis, the MDS treatment on the mechanism of DCA was investigated by Western blot and Real-time PCR experiment. According to the experiment, it was found that tumor proliferation could be inhibited when MDS skm-1 cells was treated by DCA, and the absorbance was lower and the inhibitory effect was more obvious in the 2.0, 5.0 μmol/L DCA group than in the 0.5 μmol/L DCA group and the negative control group. Compared with the control group, the number of MDS skm-1 cells crossing through the transwell upper chamber was significantly decreased after DCA application. After treated with 0.5, 2.0 and 5.0 μmol/L DCA, the apoptosis rate of MDS cells was 4.54%, 9.31% and 16.58% respectively, while the apoptosis rate of the control group was 3.20%, which shows the apoptosis rate increased significantly with the concentration of DCA. After treatment of MDS cell lines with different concentration of DCA, the methylation status of SHP-1 gene was decreased with the increase of drug concentration, the expression of SHP-1 was increased, the expression of STAT3 was decreased and the level of phosphorylation was decreased. By analyzing the phenotypic response of DCA treatment on MDS cells, it was found that interfere with MDS can be performed by inhibiting proliferation, metastasis, and inducing apoptosis in a dose-dependent way. It revealed that the molecular mechanism by DCA treatment can improve the methylation of SHP-1 gene and inhibit the expression of p-STAT3.

Shp2 expression is upregulated in cervical cancer, and Shp2 contributes to cell growth and migration and reduces sensitivity to cisplatin in cervical cancer cells

Src homology phosphotyrosine phosphatase 2 (Shp2) has been found to be overexpressed in cervical cancer tissues. However, the influence of Shp2 on the biological behavior and sensitivity to cisplatin of cervical cancer cells remains unclear. We aimed to assess Shp2 expression in cervical tissues and cell lines and to detect the influence of Shp2 knockdown and overexpression on the biological behavior and sensitivity to cisplatin in cervical cancer cells. We found that Shp2 expression was significantly upregulated in cervical cancer tissues and cell lines, and Shp2 overexpression was associated with lymph node metastasis and a high human papillomavirus (HPV) DNA load. Shp2 knockdown inhibited cell growth and migration and enhanced sensitivity to cisplatin in the HeLa and SiHa cervical cancer cell lines. In contrast, Shp2 overexpression had the opposite effects. These tumor-promoting effects of Shp2 may be partly related to Akt signaling. In conclusion, Shp2 is involved in the occurrence and development of cervical cancer and may confer cisplatin resistance in cervical cancer. Shp2 blockade may be a new strategy for cervical cancer treatment.

Upregulated SHP-2 expression in the epileptogenic zone of temporal lobe epilepsy and various effects of SHP099 treatment on a pilocarpine model.

Temporal lobe epilepsy (TLE) is defined as the sporadic occurrence of spontaneous recurrent seizures, and its pathogenesis is complex. SHP-2 (Src homology 2-containing protein tyrosine phosphatase 2) is a widely expressed cytosolic tyrosine phosphatase protein that participates in the regulation of inflammation, angiogenesis, gliosis, neurogenesis and apoptosis, suggesting a potential role of SHP-2 in TLE. Therefore, we investigated the expression patterns of SHP-2 in the epileptogenic brain tissue of intractable TLE patients and the various effects of treatment with the SHP-2-specific inhibitor SHP099 on a pilocarpine model. Western blotting and immunohistochemistry results confirmed that SHP-2 expression was upregulated in the temporal neocortex of patients with TLE. Double-labeling experiments revealed that SHP-2 was highly expressed in neurons, astrocytes, microglia and vascular endothelial cells in the epileptic foci of TLE patients. In the pilocarpine-induced C57BL/6 mouse model, SHP-2 upregulation in the hippocampus began one day after status epilepticus, reached a peak at 21days and then maintained a significantly high level until day 60. Similarly, we found a remarkable increase in SHP-2 expression at 1, 7, 21 and 60days post-SE in the temporal neocortex. In addition, we also showed that SHP099 increased reactive gliosis, the release of IL-1β, neuronal apoptosis and neuronal loss, while reduced neurogenesis and albumin leakage. Taken together, the increased expression of SHP-2 in the epileptic zone may be involved in the process of TLE.

Canine histiocytic sarcoma cell lines with SHP2 p.Glu76Gln or p.Glu76Ala mutations are sensitive to allosteric SHP2 inhibitor SHP099.

Some canine cases of histiocytic sarcoma (HS) carry an activating mutation in the src homology two domain-containing phosphatase 2 (SHP2) encoded by PTPN11. SHP099 is an allosteric inhibitor of SHP2 that stabilizes SHP2 in a folded, auto-inhibited conformation. Here, we examined the expression and mutation status of SHP2 in five canine HS cell lines and evaluated the growth inhibitory properties of SHP099 against these cell lines. All five of the canine HS cell lines expressed SHP2, with three of the lines each harbouring a distinct mutation in PTPN11/SHP2 (p.Glu76Gln, p.Glu76Ala and p.Gly503Val). In silico analysis suggested that p.Glu76Gln and p.Glu76Ala, but not p.Gly503Val, promote shifting of the SHP2 conformation from folded to open-active state. SHP099 potently suppressed the growth of two of the mutant cell lines (harbouring SHP2 p.Glu76Gln or p.Glu76Ala) but not that of the other three cell lines. In addition, SHP099 suppressed ERK activation in the cell line harbouring the SHP2 p.Glu76Ala mutation. The SHP2 p.Glu76Gln and p.Glu76Ala mutations are considered to be activating mutations, and the signal from SHP2 p.Glu76Ala is inferred to be transduced primarily via the ERK pathway. Moreover, SHP099-sensitive HS cells, including those with SHP2 p.Glu76Gln or p.Glu76Ala mutations, may depend on these mutations for growth. Therefore, targeting cells harbouring SHP2 p.Glu76Gln and p.Glu76Ala with SHP099 may be an approach for the treatment of canine HS.

Structure based design of selective SHP2 inhibitors by De novo design, synthesis and biological evaluation.

SHP2 phosphatase, encoded by the PTPN11 gene, is a non-receptor PTP, which plays an important role in growth factor, cytokine, integrin, hormone signaling pathways, and regulates cellular responses, such as proliferation, differentiation, adhesion migration and apoptosis. Many studies have reported that upregulation of SHP2 expression is closely related to human cancer, such as breast cancer, liver cancer and gastric cancer. Hence, SHP2 has become a promising target for cancer immunotherapy. In this paper, we reported the identification of compound 1 as SHP2 inhibitor. Fragment-based ligand design, De novo design, ADMET and Molecular docking were performed to explore potential selective SHP2 allosteric inhibitors based on SHP836. The results of docking studies indicated that the selected compounds had higher selective SHP2 inhibition than existing inhibitors. Compound 1 was found to have a novel selectivity against SHP2 with an in vitro enzyme activity IC50 value of 9.97μM. Fluorescence titration experiment confirmed that compound 1 directly bound to SHP2. Furthermore, the results of binding free energies demonstrated that electrostatic energy was the primary factor in elucidating the mechanism of SHP2 inhibition. Dynamic cross correlation studies also supported the results of docking and molecular dynamics simulation. This series of analyses provided important structural features for designing new selective SHP2 inhibitors as potential drugs and promising candidates for pre-clinical pharmacological investigations.

The prognostic significance of SHP2 and its binding protein Hook1 in non-small cell lung cancer.

BackgroundWe previously reported that Hook1 inhibits the phosphatase activity of SHP2 in the regulation of the epithelial-mesenchymal transition (EMT) in lung cancer. In this study, we performed a comprehensive analysis of SHP2 and Hook1 expression and relationships with the prognosis of patients with non-small cell lung cancer (NSCLC).Materials and methodsA total of 121 patients with NSCLC were included in this study. Expression of SHP2 and Hook1 was assessed by immunohistochemistry and Western blot analysis. The overall survival rate of NSCLC patients was analysed using Cox’s ratio hazard multivariate analysis and the log-rank test.ResultsIn tumour tissue specimens, positive expression rates of SHP2 proteins were 58.4% by immunohistochemical analysis. A significant correlation between expression of SHP2 and that of Hook1 was observed. Based on Western blot analysis, we found that Hook1 was downregulated and that SHP2 has a tendency to overexpress without statistical significance in NSCLC tissues compared with their levels in normal lung tissues. The median overall survival (OS) of NSCLC patients who presented low levels of SHP2 expression were better (40 vs 24 months, p=0.004) than those of patients who exhibited high levels of SHP2 expression. The results of multivariate analysis showed that the level of SHP2 expression was an independent prognostic factor for OS.ConclusionSHP2 might play an important role in NSCLC and has the potential to serve as a clinical biomarker or NSCLC.

Liraglutide Attenuates Nonalcoholic Fatty Liver Disease through Adjusting Lipid Metabolism via SHP1/AMPK Signaling Pathway.

A glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide (LR) had been experimentally and clinically shown to ameliorate nonalcoholic fatty liver disease (NAFLD). This study aimed to investigate the beneficial effect of LR on NAFLD in vivo and in vitro and its underlying molecular mechanism. The effects of LR were examined on the high-fat diet-induced in vivo model in mice and in vitro model of NAFLD in human HepG2 cells. Liver tissues and HepG2 cells were procured for measuring lipid metabolism, histological examination, and western blot analysis. LR administration significantly lowered the serum lipid profile and lipid disposition in vitro and in vivo because of the altered expression of enzymes on hepatic gluconeogenesis and lipid metabolism. Moreover, LR significantly decreased Src homology region 2 domain-containing phosphatase-1 (SHP1) and then increased the expression of phosphorylated-AMP-activated protein kinase (p-AMPK). However, the overexpression of SHP1 mediated by lentivirus vector reversed LR-induced improvement in lipid deposition. Moreover, SHP1 silencing could further increase the expression of p-AMPK to ameliorate lipid metabolism and relative lipogenic gene induced by LR. In addition, abrogation of AMPK by Compound C eliminated the protective effects of LR on lipid metabolism without changing the expression of SHP1. LR markedly prevented NAFLD through adjusting lipid metabolism via SHP1/AMPK signaling pathway.

Distinct heat shock protein-mediated immune responses are induced by macrophages and dendritic cells

Abstract Heat shock proteins (HSPs) induce potent immune responses against their chaperoned antigens and thus have potential as cancer immunotherapy. APCs, T cells, and NK cells are all required in HSP-mediated immune responses. NK cells are activated indirectly via APCs, but the mechanism of this activation is poorly understood. Interestingly, MΦ are capable of inducing IFNγ production by NK cells, however, DC are not. This is observed in response to multiple HSPs including gp96, calreticulin and HSP90. Thus, APC production of potential mediators of NK cell activation were investigated. Gp96 induces CXCL10 production in MΦ but not DC and this chemokine is necessary for NK cell activation. Since CXCL10 is STAT1 induced, STAT1 phosphorylation (STAT1p) was examined. A unique pattern of STAT1p is observed in MΦ where S727 is phosphorylated early, followed by delayed phosphorylation of Y701. In contrast, DC exhibit delayed phosphorylation of S727, and phosphorylation of Y701 is not observed. Upstream, p38 MAPK is phosphorylated to comparable levels in both APCs. However, basal expression of the phosphatase SHP2 is higher in DC. Blocking SHP2 in DC leads to earlier phosphorylation of S727 but no recovery of Y701 phosphorylation or subsequent induction of IFNγ production by NK cells. Thus, additional phosphatases and inhibitors are likely contributing to these signaling events. Collectively, these data demonstrate that targeting both MΦ and DC in vivo is critical for optimal HSP-mediated immune responses.

TRIM14 Promotes Breast Cancer Cell Proliferation by Inhibiting Apoptosis.

Tripartite motif-containing 14 (TRIM14) is abnormally expressed in several human cancers. However, the function and expression of TRIM14 in human breast cancer are still largely unknown. To understand the biological function of TRIM14 in breast cancer, we measured the expression level of TRIM14. Cell proliferation and cell apoptosis were measured after TRIM14 overexpression or knockdown. Upregulation of TRIM14 was found in human breast cancer specimens and cell lines. Reduction of TRIM14 inhibited cell proliferation but increased cell apoptosis in the BT474 and MDA-MB-231 cell lines. Further study showed that knockdown of TRIM14 upregulated the expression of BAX while downregulating the expression of BCL2. In addition, the expression of SHP-1 was increased, and the phosphorylation of STAT3 (p-STAT3) was inhibited. Conversely, overexpression of TRIM14 had the opposite effects. Additionally, cryptotanshinone, a STAT3 inhibitor, inhibited cell proliferation but increased cell apoptosis in the BT474 and MDA-MB-231 cell lines. In conclusion, TRIM14 may act as an oncogene in human breast cancer and may be a novel strategy for human breast cancer.

TRIM52 promotes colorectal cancer cell proliferation through the STAT3 signaling

BackgroundThe tripartite motif (TRIM) family proteins are implicated in the pathogenesis of various human malignancies. The up-regulation and oncogenic roles of TRIM52 have been reported in hepatocellular carcinoma. In the current study, we aimed to examine its expression and possible function in colorectal cancer (CRC).MethodImmunohistochemical staining or immunoblotting analysis was carried out to detect protein expression. Cell proliferation and apoptosis was evaluated by Cell Counting Kit-8 (CCK-8) and flow cytometry assay, respectively.ResultsTRIM52 expression was increased in 67.5% of CRC tissues (54/80) compared to matched normal colonic mucosa. TRIM52 expression was closely related with tumor size (p = 0.0376), tumor stage (p = 0.0227) and overall survival (p = 0.0177). Short hairpin RNAs (shRNAs) targeting TRIM52 had the potential anti-proliferative effects on CRC cell lines, SW480 and LoVo, by inducing cell apoptosis. In addition, an in vivo xenograft experiment confirmed the in vitro results. In addition, TRIM52 shRNAs decreased the phosphorylation of STAT3, but increased the protein expression of SHP2, a negative regulator of STAT3 phosphorylation. TRIM52 formed a complex with SHP2 and promoted the ubiquitination of SHP2. Furthermore, inhibition of the STAT3 signaling by AG490 in RKO cells significantly abolished the effects of TRIM52 overexpression on cell proliferation, apoptosis and STAT3 activation.ConclusionsTRIM52 might exert oncogenic role in CRC via regulating the STAT3 signaling pathway.

Association of nuclear localization of SHP2 and YAP1 with unfavorable prognosis in non-small cell lung cancer

Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2) is ubiquitously expressed in cytoplasmic localization, which in turn confers tumor malignancy and poor prognosis in various human cancers. YAP1 interacts with SHP2 to promote translocation of SHP2 to nucleus, which consequently promotes Wnt target activation. However, the oncogenic role of the nuclear localization of SHP2 in human cancers remains unclear. We hypothesized that nuclear SHP2 localization, in combination with nuclear YAP1 expression, could be associated with poor overall survival (OS) and relapse free survival (RFS) due to an increase in cyclin D1 and c-Myc mRNA expression following activation of Wnt/ß-catenin signaling. Immunohistochemical analysis of SHP2 and YAP1 protein expression in 102 tumors resected from patients with NSCLC revealed that nuclear SHP2 expression was well correlated with nuclear YAP1 expression (P < 0.001). Evaluation of cyclin D1 and c-Myc mRNA levels by the real-time reverse-phase polymerase chain reaction (RT-PCR) revealed that patients with high cyclin D1 and high c-Myc mRNA expressing tumors more commonly showed high nuclear YAP1 and high nuclear SHP2 (high/high) rather than the high/low, low/high, or low/low combinations (P < 0.001 for cyclin D1 and c-Myc). Kaplan-Meier and Cox-regression models showed OS and RFS to be poorer in patients in the high/high subgroup than in the low/low subgroup (OS: HR = 2.85, 95% CI, 1.52–5.35, P = 0.001; RFS: HR = 2.55, 95% CI, 1.37–4.72, P = 0.003). No prognostic significance was observed for the other two subgroups (low/high and high/low) when compared to the low/low subgroup in this study population. Therefore, we suggest that the prognostic value of SHP2 could reflect the nuclear localization of SHP2 and its interaction with nuclear YAP1, which led to subsequent upregulation of cyclin D1 and c-Myc mRNA expression via activation of the Wnt/ß-catenin signaling pathway.

Phosphatase Shp2 exacerbates intestinal inflammation by disrupting macrophage responsiveness to interleukin-10.

Inflammatory cytokines produced by activated macrophages largely contribute to the pathological signs of inflammatory bowel disease (IBD). Interleukin-10 (IL-10) is the predominant anti-inflammatory cytokine in the intestine, and its therapeutic efficacy for IBD has been clinically tested. Nevertheless, how the function of IL-10 is regulated in the intestinal microenvironment remains unknown, which largely hinders the further development of IL-10-based therapeutic strategies. Here, we found that the expression of phosphatase Shp2 was increased in colonic macrophages and blood monocytes from IBD patients compared with those from healthy controls. Shp2 deficiency in macrophages protects mice from colitis and colitis-driven colon cancer. Mechanistically, Shp2 disrupts IL-10-STAT3 signaling and its dependent anti-inflammatory response in human and mouse macrophages. Furthermore, a Shp2-inducing role of TNF-α is unveiled in our study. Collectively, our work identifies Shp2 as a detrimental factor for intestinal immune homeostasis and hopefully will be helpful in the future exploitation of IL-10 immunotherapy for IBD.

A Ce(iii) complex potently inhibits the activity and expression of tyrosine phosphatase SHP-2.

Four new Ce(iii) complexes 1-4 with tridentate NNO-donor Schiff base ligands have been designed and successfully synthesized. These complexes were characterized by elemental analysis, IR, and ESI-MS, with formulas of [Ce(HL1)2(NO3)3]·2CH3OH (1), [Ce(L2)2(NO3)]·3H2O (2), [Ce(HL3)(L3)(NO3)Br]·H2O (3) and [Ce(L4)2(NO3)]·3H2O (4), in which ligands HL1-HL4 are respectively N'-[(1E)-pyridin-2-ylmethylidene]pyrazine-2-carbohydrazide (HL1), 2-(1-(salicyloylhydrazono)ethyl)pyrazine (HL2), N'-[(1E)-pyridin-2-ylmethylidene]pyridine-2-carbohydrazide (HL3) and 2-(1-(salicyloylhydrazono)ethyl) pyridine (HL4). X-ray single crystal diffraction analysis indicates that complex 1 crystallizes in the monoclinic system with the space group C2/c and the structure of complex 1 consists of a monomeric Ce(iii) species with a Ce(iii) moiety bonded to two tridentate Schiff base ligands, three nitrates and solvents. These complexes effectively inhibit the enzyme activities of PTPs (SHP-1, SHP-2, TCPTP and PTP1B), among which complex 3 shows the most potent inhibition of SHP-2 with the lowest IC50 value of 0.61 μM and displays obvious selectivity towards SHP-2. Its inhibition potency against SHP-2 was approximately 17, 4, and 5 fold higher than that against SHP-1, TCPTP and PTP1B, respectively. Further study discloses that complex 3 inhibits SHP-2 in a competitive manner. Fluorescence measurements indicate that complex 3 tightly binds to SHP-2 with a molar ratio of 1 : 1 and a binding constant of 5.45 × 105 M-1. Western blot experiments show that complex 3 promotes the phosphorylation of the SHP-2 substrate by the combination of the inhibition of the activity and expression of SHP-2. Moreover, complex 3 decreases the survival rate of A549 cells to 35.12% at 100 μM and induces apoptosis with an apoptosis rate of 12.06% at 50 μM. All these results suggest that complex 3 is a potential bi-functional inhibitor of the activity and expression of tyrosine phosphatase SHP-2.