SH2-containing Protein Tyrosine Phosphatase Research Articles

SHP2 Inhibition Overcomes Adaptive and Acquired Resistance to FLT3 TKI to Improve Efficacy Against FLT3/ITD AML

FMS-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase (RTK) that is frequently mutated in acute myeloid leukemia (AML), with the internal tandem mutation (FLT3/ITD) conferring a poor prognosis to AML patients. This makes FLT3/ITD AML a validated target for treatment with tyrosine kinase inhibitors (TKI), but their success has been hindered by mechanisms of resistance including rapid signal reprogramming known as adaptive resistance and acquired resistance gained through long-term treatment. Past work from our lab has shown that FLT3/ITD cells undergo adaptive resistance, through the rapid reactivation of ERK signaling within 24 hours of sustained FLT3 inhibition. To improve therapy of FLT3/ITD AML it is important to identify therapeutic targets that overcome this ERK reactivation. Since adaptive ERK reactivation is often due to the relief of negative feedback loops that result in the activation of other RTK, we performed an RTK phospho-array on the FLT3/ITD cell line MV4;11. 48-hour treatment with the FLT3 TKI sorafenib resulted in increased activation of IGF-R, HER3 and AXL. While recent work from our lab found that combining AXL and FLT3 inhibition abrogated the ERK reactivation and exerted greater antileukemic effects, the activation of multiple RTKs suggests that targeting a signaling hub that inhibits multiple RTKs at once will likely prove more efficacious in overcoming this adaptive response. Recent literature has implicated SH2-containing protein tyrosine phosphatase 2 (SHP2) as a therapeutic target in adaptive ERK reactivation in other cancers. SHP2 acts directly downstream of RTKs and is involved in the activation of the ERK pathway. Therefore, we explored whether the addition of an SHP2 inhibitorto FLT3 TKI can overcome the adaptive response and increase treatment efficacy. To explore this possibility, we first treated the FLT3/ITD cell lines Molm14 and MV4;11 as well as FLT3/ITD patient samples with FLT3 TKI alone or in combination with an SHP2 inhibitor for 24 hours. The addition of SHP099 to sorafenib decreased the expected ERK reactivation in a dose-dependent manner. Furthermore, combination treatment with synergized to decrease proliferation and increase apoptosis in the FLT3/ITD cell lines. Impressively, in a long-term assay, treatment with the combination for just 72 hours greatly reduced leukemic cell recovery. To determine whether this combination would improve efficacy in vivo, we engrafted NSG mice with Molm14 cells and treated them for 4 weeks via oral gavage. The combination of SHP099 with gilteritinib group resulted in a statistically significantly lower leukemia burden compared to either treatment alone and the vehicle control. The combination group also had prolonged survival compared to the other groups. In addition to rapid adaptive resistance, there are mechanisms of acquired resistance that render traditional FLT3 TKI treatments ineffective against FLT3/ITD AML cells. We next examined the effects of SHP2 inhibition on FLT3-TKI resistant cells models that were generated by growing and maintaining Molm14 and MV4;11 cells in the presence of FLT3 TKI. The combination of FLT3 TKI and SHP099 resensitized the cells to FLT3 TKI, resulting in decreased ERK phosphorylation, decreased proliferation and increased apoptosis of these FLT3 TKI resistant cells. We also explored the effects of treating the FLT3/ITD mutant cell lines with the combination of SHP099 and several of the cytotoxic chemotherapy agents typically used in the treatment of AML including cytarabine and daunorubicin. While daunorubicin or cytarabine alone had little effect on the level of phosphorylated ERK, the addition of SHP099 decreased ERK activation and the combination synergized to exert greater to decrease proliferation and increase apoptosis. These studies reveal that combining FLT3 TKI treatment with an allosteric SHP2 inhibitor decreases the adaptive ERK reactivation observed in FLT3/ITD cells. The finding that SHP099 synergized with both FLT3 TKI and chemotherapy agents in different FLT3-mutated AML models speaks to the versatile efficacy of SHP2 inhibition in multiple AML models. Taken together, the data suggests that SHP2 inhibition can help overcome both adaptive and acquired resistance in FLT3/ITD AML and is a candidate to try to improve patient outcomes.

Predictive and Therapeutic Implications of a Novel PLCγ1/SHP2-Driven Mechanism of Cetuximab Resistance in Metastatic Colorectal Cancer.

Cetuximab is an EGFR-targeted therapy approved for the treatment of RAS wild-type (WT) metastatic colorectal cancer (mCRC). However, about 60% of these patients show innate resistance to cetuximab. To increase cetuximab efficacy, it is crucial to successfully identify responder patients, as well as to develop new therapeutic approaches to overcome cetuximab resistance. We evaluated the value of EGFR effector phospholipase C gamma 1 (PLCγ1) in predicting cetuximab responses, by analyzing progression-free survival (PFS) of a multicentric retrospective cohort of 94 treated patients with mCRC (log-rank test and Cox regression model). Furthermore, we used in vitro and zebrafish xenotransplant models to identify and target the mechanism behind PLCγ1-mediated resistance to cetuximab. In this study, levels of PLCγ1 were found increased in RAS WT tumors and were able to predict cetuximab responses in clinical samples and in vitro and in vivo models. Mechanistically, PLCγ1 expression was found to bypass cetuximab-dependent EGFR inhibition by activating ERK and AKT pathways. This novel resistance mechanism involves a noncatalytic role of PLCγ1 SH2 tandem domains in the propagation of downstream signaling via SH2-containing protein tyrosine phosphatase 2 (SHP2). Accordingly, SHP2 inhibition sensitizes PLCγ1-resistant cells to cetuximab. Our discoveries reveal the potential of PLCγ1 as a predictive biomarker for cetuximab responses and suggest an alternative therapeutic approach to circumvent PLCγ1-mediated resistance to cetuximab in patients with RAS WT mCRC. In this way, this work contributes to the development of novel strategies in the medical management and treatment of patients with mCRC.

The isoflavonoid calycosin inhibits inflammation and enhances beta cell function in gestational diabetes mellitus by suppressing RNF38 expression

Background Gestational diabetes mellitus (GDM) is a medical complication and metabolic disorder associated with pregnancy. Calycosin is a traditional Chinese herbal medicine that is used for the treatment of multiple diseases. This study focused on exploring the effects and underlying mechanisms of Calycosin on GDM. Methods The db/+ diabetic mice model of GDM was used to evaluate the effects of calycosin administration on the symptoms of GDM mice. Blood glucose, cytokine production (interleukin 6, IL-6; tumor necrosis factor-α, TNF-α), and insulin levels were measured by ELISA assay. The expression level of signal transducer and activator of transcription 3 (STAT3), ring finger protein 38 (RNF38), and SH2-containing protein tyrosine phosphatase 1 (SHP-1) were determined by Western Blot assay. Beta cell proliferation was assessed by CCK-8 assay. Results Our data indicated that administration of calycosin significantly improved the GDM symptoms in pregnant db/+ mice as demonstrated by reduced blood glucose, TNF-a, and IL-6 levels as well as increased insulin level, and body weight. Furthermore, we revealed that RNF38/SHP-1/STAT3 signaling should play a critical role in calycosin-promoted beta cell function, and forced expression of RNF38 attenuated the positive effects of calycosin on beta cells. Conclusion Our study implied that calycosin exerts favorable effects on GDM mice via rebalancing insulin sensitivity and inflammatory response.

Ligand Recognition Determines the Role of Inhibitory B Cell Co-receptors in the Regulation of B Cell Homeostasis and Autoimmunity

B cells express various inhibitory co-receptors including CD22, CD72, and Siglec-G. These receptors contain immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic region. Although many of the inhibitory co-receptors negatively regulate BCR signaling by activating SH2-containing protein tyrosine phosphatase 1 (SHP-1), different inhibitory co-receptors have distinct functional properties. CD22, Siglec-G, and CD72 preferentially regulate tonic signaling in conventional B cells, B-1 cell homeostasis, and development of lupus-like disease, respectively. CD72 recognizes RNA-related lupus self-antigen Sm/RNP as a ligand. This ligand recognition recruits CD72 to BCR in Sm/RNP-reactive B cells thereby suppressing production of anti-Sm/RNP autoantibody involved in the pathogenesis of lupus. In contrast, Siglec-G recognizes α2,3 as well as α2,6 sialic acids whereas CD22 recognizes α2,6 sialic acid alone. Because glycoproteins including BCR are dominantly glycosylated with α2,3 sialic acids in B-1 cells, Siglec-G but not CD22 recruits BCR as a ligand specifically in B-1 cells, and regulates B-1 cell homeostasis by suppressing BCR signaling in B-1 cells. Thus, recognition of distinct ligands determines functional properties of different inhibitory B cell co-receptors.

Inhibition of STAT3 in gastric cancer: role of pantoprazole as SHP-1 inducer

BackgroundWe investigated the inhibitory effect of pantoprazole on signal transducer and activator of transcription 3 (STAT3) activity and invasiveness of gastric adenocarcinoma cells, and the role of SH2-containing protein tyrosine phosphatase 1 (SHP-1) in mediating role.MethodsWe used AGS and MKN-28 cells because of reduced SHP-1 and preserved p-STAT3 expression. Western blot, wound closure assay, Matrigel invasion assay and 3-D culture invasion assay were performed. Pharmacologic inhibitor of SHP-1 and siRNA were used for validation of the role of SHP-1.ResultsWe observed that pantoprazole at 40, 80, and 160 μg/ml upregulated SHP-1 and downregulated p-STAT3 expression in a dose-dependent manner in AGS and MKN-28 cells. Furthermore, pantoprazole significantly downregulated mesenchymal markers (Snail1 and vimentin), upregulated epithelial marker (E-cadherin), and inhibited migration and invasion of AGS and MKN-28 cells. To validate the role of SHP-1 in inhibition of STAT3 activity by pantoprazole in gastric cancer cells, we performed pharmacologic inhibition (pervanadate) or knockdown of SHP-1 before pantoprazole treatment, which significantly attenuated the suppression of p-STAT3 and anti-migration and invasion effect by pantoprazole in AGS cells. In xenograft tumor model, tumor volume was significantly reduced by intraperitoneal injection of pantoprazole, with upregulation of SHP-1 and downregulation of p-STAT3, which were attenuated by concomitant injection of pervanadate.ConclusionOur data suggest that the inhibitory effect of pantoprazole on cellular migration and invasion might be through inducing SHP-1 in gastric cancer cells.

RING finger protein 38 induces gastric cancer cell growth by decreasing the stability of the protein tyrosine phosphatase SHP-1.

The function of the E3 ligase RNF38 is still unknown in gastric cancer. Here, we found that RNF38 is upregulated in gastric cancer, and it is associated with the overall survival of gastric cancer patients. Further studies showed that RNF38 interacts with the nonreceptor tyrosine phosphatase SH2-containing protein tyrosine phosphatase 1 (SHP-1) and induces the polyubiquitination of SHP-1, which leads to destabilization of SHP-1 and promotion of STAT3 signaling in gastric cancer cells. In addition, overexpression or knockdown of RNF38 induces or suppresses gastric cancer cell growth in vitro, respectively, and silencing RNF38 delays tumor growth in vivo. These findings demonstrate that RNF38 is functional in gastric cancer and promotes STAT3 signaling by destabilizing SHP-1; thus, RNF38 could be a novel target for gastric cancer therapy.

IGF1 receptor signaling pathways.

Insulin-like growth factors (IGFs) bind specifically to the IGF1 receptor on the cell surface of targeted tissues. Ligand binding to the α subunit of the receptor leads to a conformational change in the β subunit, resulting in the activation of receptor tyrosine kinase activity. Activated receptor phosphorylates several substrates, including insulin receptor substrates (IRSs) and Src homology collagen (SHC). Phosphotyrosine residues in these substrates are recognized by certain Src homology 2 (SH2) domain-containing signaling molecules. These include, for example, an 85 kDa regulatory subunit (p85) of phosphatidylinositol 3-kinase (PI 3-kinase), growth factor receptor-bound 2 (GRB2) and SH2-containing protein tyrosine phosphatase 2 (SHP2/Syp). These bindings lead to the activation of downstream signaling pathways, PI 3-kinase pathway and Ras-mitogen-activated protein kinase (MAP kinase) pathway. Activation of these signaling pathways is known to be required for the induction of various bioactivities of IGFs, including cell proliferation, cell differentiation and cell survival. In this review, the well-established IGF1 receptor signaling pathways required for the induction of various bioactivities of IGFs are introduced. In addition, we will discuss how IGF signals are modulated by the other extracellular stimuli or by themselves based on our studies.

Negative regulation of B cell responses and self-tolerance to RNA-related lupus self-antigen.

The antibody response to RNA-related antigens such as Sm/RNP requires the endosomal RNA sensor TLR7, and this process is crucial in the development of systemic lupus erythematosus at least in animal models. The inhibitory B cell receptor CD72 is unique because it recognizes Sm/RNP and specifically inhibits the activation of Sm/RNP-reactive B cells by activating SH2-containing protein tyrosine phosphatase 1 (SHP-1). In the normal immune system, Sm/RNP-reactive B cells are tolerized by a unique mechanism that probably involves SHP-1. These self-reactive B cells appear in the peripheral lymphoid organs, differentiate into marginal zone B cells, and then undergo apoptosis without further maturation into plasma cells. Thus, CD72 is involved in the suppression of TLR7-mediated response to RNA in complexes with nuclear proteins that are resistant to nucleases, whereas free RNAs are degraded by nucleases before they encounter the endosomal RNA sensor.

Effect of arsenic trioxide on epithelial-mesenchymal transition via induction of SH2?containing protein tyrosine phosphatase 1.

76 Background: Arsenic trioxide (ATO) is known to inhibit epithelial-mesenchymal transition (EMT) in hepatolocellular carcinoma and breast cancer cells, however, little has been reported in gastric cancer cells. In this study, we aimed to investigate the mechanism of ATO to inhibit signal transducer and activator of transcription 3 (STAT3) activity and EMT in gastric cancer cells. Methods: We performed wound closure assay and Matrigel invasion assay for functional studies of EMT, and western blot for measurement of protein markers using AGS gastric cancer cells. Results: Compared with control, 5 and 10 μM of ATO significantly inhibited cellular migration and inhibition in a dose-dependent manner. Furthermore, ATO significantly downregulated snail expression, a mesenchymal marker, and upregulated E-cadherin expression, an epithelial marker. We could observe that ATO induced SH2-containing protein tyrosine phosphatase 1 (SHP1), a non-receptor type protein tyrosine phosphatase, and subsequently downregulated phospho-STAT3 in a dose-dependent manner. To validate the molecular link between ATO and SHP1 to inhibit EMT in gastric cancer cell, we pre-treated 50 μM of pervanadate, a phosphatase inhibitor, before treatment of 10 μM ATO, and this significantly abolished anti-invasive effect by ATO in AGS cells. In xenograft tumor model, intraperitoneal injection of ATO significantly reduced the tumor volume and upregulated SHP-1 expression by immunohistochemistry stain compared with vehicle, which were reversed by ATO with pervanadate injection. Conclusions: Our findings suggest that ATO may show anti-EMT effects via induction of SHP1 and inhibition of STAT3 activity in gastric cancer cells.

A missense methionine mutation augments catalytic activity but reduces thermal stability in two protein tyrosine phosphatases

Recent data sets that catalog the missense mutations in thousands of human genomes have revealed a vast and largely unexplored world of non-canonical protein sequences that are expressed in humans. The functional consequences of most human missense mutations, however, are unknown, and the accuracy with which their effects can be predicted by computational algorithms remains unclear. Among humans of European descent, the most common missense mutation in the catalytic domain of SH2-containing protein tyrosine phosphatase 1 (SHP-1) converts the enzyme's canonical valine 451 to methionine (V451M). The V451M mutation lies in a conserved motif adjacent to the protein tyrosine phosphatase (PTP) consensus sequence and is predicted to compromise catalytic function. In this study it is shown that, counter to prediction, V451M SHP-1 possesses increased catalytic activity as compared to the wild-type enzyme. Additionally, a PTP-wide search of missense-mutation data revealed a variant of one other PTP, Fas-associated PTP (FAP-1), that contains a methionine mutation at the position corresponding to 451 of SHP-1 (T2406M FAP-1). It is shown here that the T2406M mutation increases FAP-1's PTP activity, to a degree that is comparable to the activation deriving from the V451M mutation in SHP-1. Although the two non-canonical methionine residues confer increased activity at moderate temperatures, both V451M SHP-1 and T2406M FAP-1 are less thermally stable than their canonical counterparts, as demonstrated by the mutants' strongly reduced activities at high temperatures. These results highlight the challenges in predicting the functional consequences of missense mutations, which can differ under varying conditions, and suggest that, with regard to position 451/2406, canonical PTP domains have “chosen” stability over optimized activity during the course of evolution.

SH2 domain-containing phosphatase 1 regulates pyruvate kinase M2 in hepatocellular carcinoma

Pyruvate kinase M2 (PKM2) is known to promote tumourigenesis through dimer formation of p-PKM2Y105. Here, we investigated whether SH2-containing protein tyrosine phosphatase 1 (SHP-1) decreases p-PKM2Y105 expression and, thus, determines the sensitivity of sorafenib through inhibiting the nuclear-related function of PKM2. Immunoprecipitation and immunoblot confirmed the effect of SHP-1 on PKM2Y105 dephosphorylation. Lactate production was assayed in cells and tumor samples to determine whether sorafenib reversed the Warburg effect. Clinical hepatocellular carcinoma (HCC) tumor samples were assessed for PKM2 expression. SHP-1 directly dephosphorylated PKM2 at Y105 and further decreased the proliferative activity of PKM2; similar effects were found in sorafenib-treated HCC cells. PKM2 was also found to determine the sensitivity of targeted drugs, such as sorafenib, brivanib, and sunitinib, by SHP-1 activation. Significant sphere-forming activity was found in HCC cells stably expressing PKM2. Clinical findings suggest that PKM2 acts as a predicting factor of early recurrence in patients with HCC, particularly those without known risk factors (63.6%). SHP-1 dephosphorylates PKM2 at Y105 to inhibit nuclear function of PKM2 and determines the efficacy of targeted drugs. Targeting PKM2 by SHP-1 might provide new therapeutic insights for patients with HCC.

Recent updates of precision therapy for gastric cancer: Towards optimal tailored management.

Signaling pathways of gastric carcinogenesis and gastric cancer progression are being avidly studied to seek optimal treatment of gastric cancer. Among them, hepatocyte growth factor (HGF)/c-MET, phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathways have been widely investigated. Their aberrant expression or mutation has been significantly associated with advanced stage or poor prognosis of gastric cancer. Recently, aberrations of immune checkpoints including programmed cell death-1/programmed cell death ligand-1 (PD-1/PD-L1) have been suggested as an important step in the formation of a microenvironment favorable for gastric cancer. Accomplishments in basic research have led to the development of novel agents targeting these signaling pathways. However, phase III studies of selective anti-HGF/c-MET antibodies and mTOR inhibitor failed to show significant benefits in terms of overall survival and progression-free survival. Few agents directly targeting STAT3 have been developed. However, this target is still critical issue in terms of chemoresistance, and SH2-containing protein tyrosine phosphatase 1 might be a significant link to effectively inhibit STAT3 activity. Inhibition of PD-1/PD-L1 showed durable efficacy in phase I studies, and phase III evaluation is warranted. Therapeutic strategy to concurrently inhibit multiple tyrosine kinases is a reasonable option, however, lapatinib needs to be further evaluated to identify good responders. Regorafenib has shown promising effectiveness in prolonging progression-free survival in a phase II study. In this topic highlight, we review the biologic roles and outcomes of clinical studies targeting these signaling pathways.

New and Unexpected Biological Functions for the Src-Homology 2 Domain-Containing Phosphatase SHP-2 in the Gastrointestinal Tract.

SHP-2 is a tyrosine phosphatase expressed in most embryonic and adult tissues. SHP-2 regulates many cellular functions including growth, differentiation, migration, and survival. Genetic and biochemical evidence show that SHP-2 is required for rat sarcoma viral oncogene/extracellular signal-regulated kinases mitogen-activated protein kinase pathway activation by most tyrosine kinase receptors, as well as by G-protein–coupled and cytokine receptors. In addition, SHP-2 can regulate the Janus kinase/signal transducers and activators of transcription, nuclear factor-κB, phosphatidyl-inositol 3-kinase/Akt, RhoA, Hippo, and Wnt/β-catenin signaling pathways. Emerging evidence has shown that SHP-2 dysfunction represents a key factor in the pathogenesis of gastrointestinal diseases, in particular in chronic inflammation and cancer. Variations within the gene locus encoding SHP-2 have been associated with increased susceptibility to develop ulcerative colitis and gastric atrophy. Furthermore, mice with conditional deletion of SHP-2 in intestinal epithelial cells rapidly develop severe colitis. Similarly, hepatocyte-specific deletion of SHP-2 induces hepatic inflammation, resulting in regenerative hyperplasia and development of tumors in aged mice. However, the SHP-2 gene initially was suggested to be a proto-oncogene because activating mutations of this gene were found in pediatric leukemias and certain forms of liver and colon cancers. Moreover, SHP-2 expression is up-regulated in gastric and hepatocellular cancers. Notably, SHP-2 functions downstream of cytotoxin-associated antigen A (CagA), the major virulence factor of Helicobacter pylori, and is associated with increased risks of gastric cancer. Further compounding this complexity, most recent findings suggest that SHP-2 also coordinates carbohydrate, lipid, and bile acid synthesis in the liver and pancreas. This review aims to summarize current knowledge and recent data regarding the biological functions of SHP-2 in the gastrointestinal tract.

Epigenetic regulation and anti-tumorigenic effects of SH2-containing protein tyrosine phosphatase 1 (SHP1) in human gastric cancer cells.

SH2-containing protein tyrosine phosphatase 1 (SHP1) is an important negative regulator in cytokine-mediated signal transduction and cell cycling. Recent studies have demonstrated that SHP1 promoter methylation is frequently observed in gastric adenocarcinoma tissues. In this in vitro study, we attempted to reveal promoter hypermethylation and to investigate effects of SHP1 in gastric carcinoma cell lines. We observed that both gene and protein expression of SHP1 were negative in 8 of 10 gastric cancer cell lines (SNU-1, SNU-5, SNU-16, SNU-638, SNU-719, MKN-28, MKN-45, AGS). Methylation-specific PCR (MSP) showed a methylation-specific band only in the 10 gastric cancer lines. Bisulfite pyrosequencing in AGS, MKN-28, and SNU-719 cells indicated that methylation frequency was as high as 94.4, 92.6, and 94.5%, respectively, in the three cell lines. Treatment of SNU-719, MKN-28, and AGS cells with 5-Aza-2'-deoxycytidine (5-Aza-dc) led to re-expression of SHP1 in these cells. Introduction of exogenous SHP1 in SNU-719 and MKN-28 cells by transient transfection substantially downregulated protein expression of constitutive phosphor-Janus kinase 2 (JAK2) (tyrosine 1007/1008) and phosphor-signal transducers and activators of transcription 3 (STAT3) (tyrosine 705), which in turn decreased expression of STAT3 target genes including those encoding cyclin D1, MMP-9, VEGF-1, and survivin. Induction of SHP1 significantly inhibited cell proliferation, migration and invasion in SNU-719 and MKN-28 cells. Taken together, epigenetic silencing of SHP1 is frequently caused by promoter hypermethylation in gastric carcinoma cells. Overexpression of SHP1 downregulates the JAK2/STAT3 pathway to modulate various target genes and inhibit cell proliferation, migration, and invasion in gastric cancer cells.

Antitumorigenic effect of plumbagin by induction of SH2-containing protein tyrosine phosphatase 1 in human gastric cancer cells.

A recent study reported that plumbagin downregulated the activity of Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway to show various antitumor effects in multiple myeloma cells. We aimed in this in vitro study to demonstrate the inhibition of JAK2/STAT3 pathway by plumbagin through inducing SH2-containing protein tyrosine phosphatase 1 (SHP1) expression in the MKN-28 gastric cancer cell line. We performed western blot analysis to measure SHP1, phosphor-JAK2/STAT3 level, and observed that plumbagin induced SHP1 expression and simultaneously downregulated phosphor-JAK2/STAT3 in MKN-28 cells, with negative SHP1 expression. This effect was consistent when JAK2/STAT3 signaling was activated by interleukin-6 (IL-6), and ameliorated when cells were treated with prevanadate, a protein tyrosin phosphatase inhibitor. Furthermore, plumbagin significantly reduced gene expression of cyclin D1, vascular endothelial growth factor (VEGF)-1, Bcl-xL, survivin and matrix metalloproteinase-9 (MMP-9), known target products of STAT3 activation in gastric carcinogenesis by reverse transcription-polymerase chain reaction (RT-PCR). Several functional studies such as water soluble tetrazolium salt-1 (WST-1) assay, wound closure assay, Matrigel invasion assay and Annexin V assay were also performed, and we validated the functional effect of plumbagin for inhibition of cell proliferation, migration and invasion, and induction of apoptosis. Collectively, our findings suggest that plumbagin is a potential regulator of cellular growth, migration, invasion and apoptosis by inhibiting both constitutive and inducible STAT3 activity through induction of SHP1 in gastric cancer cells.

Epigenetic regulation and functional roles of SHP1 in gastric carcinoma cell lines.

61 Background: The SH2-containing protein tyrosine phosphatase 1 (SHP1) is an important negative regulator in cytokine-mediated signal transduction and cell cycling. Recent studies demonstrated that promoter methylation of SHP1 is frequently observed in gastric adenocarcinoma tissues. We tried in this in vitrostudy to reveal promoter hypermethylation and to investigate the effects of SHP1 in gastric carcinoma cell lines. Methods: We performed RT-PCR, Western blot, methylation specific PCR (MSP) and bisulfite pyrosequencing to demonstrate promoter hypermethylation of SHP1. To evaluate functional roles of SHP1, we transfected SHP1 plasmid and analyzed WST-1 assay, wound closure assay and Matrigel invasion assay. Results: We observed that both gene and protein expression of SHP1 were negative in 8 of 10 gastric cancer cell lines (SNU-1, SNU-5, SNU-16, SNU-638, SNU-719, MKN-28, MKN-45, AGS). MSP showed methylation-specific band only in all 10 gastric cancer lines. Bisulfite pyrosequencing revealed 96.5% and 97.3% of methylation frequency in AGS and SNU-719 cells. When treating SNU-719, MKN-28 and AGS cells with 5-Aza-2’-deoxycytidine (5-Aza-dc), SHP1 was re-expressed in all three cells. SHP1 expression is known to be correlated with Janus kinase (JAK) and signal transducers and activators of transcription (STAT) signaling pathway in epithelial cells. When introducing exogenous SHP1 in SNU-719 and MKN-28 cells by transient transfection, protein expression of constitutive phospho-JAK2 (Tyr 1007/1008) and phospho-STAT3 (Tyr 705) were substantially down-regulated both, which in turn decreased target gene expression of STAT3, including cyclin D1, MMP-9, VEGF and survivin. Induction of SHP1 significantly inhibited cell proliferation, migration and invasion in SNU-719 and MKN-28 cells. Conclusions: Epigenetic silencing of SHP1 is frequently caused by promoter hypermethylation in gastric carcinoma cell lines. Overexpression of SHP1 down-regulates JAK2/STAT3 pathway to modulate various target genes and inhibit cell proliferation, migration and invasion in gastric cancer cells.

Antitumorigenic effect of plumbagin by induction of SHP1 in human gastric carcinoma cell lines.

74 Background: Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is a plant-drived natural agent extracted from the root of Plumbago zeylanic. A recent study reported that plumbagin down-regulated the activity of Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway to show various anti-tumor effects. We aimed in this in vitrostudy to demonstrate the inhibition of JAK2-STAT3 pathway by plumbagin through inducing SH2-containing protein tyrosine phosphatase 1 (SHP1) expression in gastric cancer cell line. Methods: We performed Wetern blot to measure SHP1, phospho-JAK2/STAT3 level, and reverse transcriptase-polymerase chain reaction (RT-PCR) to evaluate target gene expression of STAT3. Several functional studies such as water soluble tetrazolium-1 (WST-1) assay, wound closure assay and matrigel invasion assay were also performed. Results: Plumbagin induced SHP1 expression and simultaneously down-regulated phospho-JAK2/STAT3 level via dose-and time-dependant manner in MKN28 cell, a gastric carcinoma cell line which has negative SHP1 expression. This effect was consistent when JAK2-STAT3 signaling was activated by interleukin-6, and ameliorated when cells were treated with prevanadate, a protein tyrosin phosphatase inhibitor. Furthermore, plumbagin significantly reduced gene expression of cyclin D1, VEGF1, survivin, MMP9, known target products of STAT3 activation in gastric cancinogenesis. The functional effect of plumbagin could be validated as inhibition of cell proliferation, migration and invasion, which are the results of activation of JAK2-STAT3 pathway in gastric cancer cells. Conclusions: Plumbagin is a potential negative regulator of cellular growth, migration and invasion by inhibiting both constitutive and inducible STAT3 activity through induction of SHP1 in gastric cancer cells.

Leptin signaling.

Leptin is secreted by adipose tissue and regulates energy homeostasis, glucose and lipid metabolism, immune function, and other systems. The binding of leptin to its specific receptor activates various intracellular signaling pathways, including Janus kinase 2 (JAK2)/ signal transducer and activator of transcription 3 (STAT3), insulin receptor substrate (IRS)/phosphatidylinositol 3 kinase (PI3K), SH2-containing protein tyrosine phosphatase 2 (SHP2)/mitogen-activated protein kinase (MAPK), and 5' adenosine monophosphate-activated protein kinase (AMPK)/ acetyl-CoA carboxylase (ACC), in the central nervous system and peripheral tissues. Understanding of leptin signaling provides insights into its roles in health and disease.

Mechanical activation of mammalian target of rapamycin pathway is required for cartilage development.

Mechanical stress regulates development by modulating cell signaling and gene expression. However, the cytoplasmic components mediating mechanotransduction remain unclear. In this study, elimination of muscle contraction during chicken embryonic development resulted in a reduction in the activity of mammalian target of rapamycin (mTOR) in the cartilaginous growth plate. Inhibition of mTOR activity led to significant inhibition of chondrocyte proliferation, cartilage tissue growth, and expression of chondrogenic genes, including Indian hedgehog (Ihh), a critical mediator of mechanotransduction. Conversely, cyclic loading (1 Hz, 5% matrix deformation) of embryonic chicken growth plate chondrocytes in 3-dimensional (3D) collagen scaffolding induced sustained activation of mTOR. Mechanical activation of mTOR occurred in serum-free medium, indicating that it is independent of growth factor or nutrients. Treatment of chondrocytes with Rapa abolished mechanical activation of cell proliferation and Ihh gene expression. Cyclic loading of chondroprogenitor cells deficient in SH2-containing protein tyrosine phosphatase 2 (Shp2) further enhanced mechanical activation of mTOR, cell proliferation, and chondrogenic gene expression. This result suggests that Shp2 is an antagonist of mechanotransduction through inhibition of mTOR activity. Our data demonstrate that mechanical activation of mTOR is necessary for cell proliferation, chondrogenesis, and cartilage growth during bone development, and that mTOR is an essential mechanotransduction component modulated by Shp2 in the cytoplasm.

Gab Docking Proteins in Cardiovascular Disease, Cancer, and Inflammation

The docking proteins of the Grb2-associated binder (Gab) family have emerged as crucial signaling compartments in metazoans. In mammals, the Gab proteins, consisting of Gab1, Gab2, and Gab3, are involved in the amplification and integration of signal transduction evoked by a variety of extracellular stimuli, including growth factors, cytokines, antigens, and other molecules. Gab proteins lack the enzymatic activity themselves; however, when phosphorylated on tyrosine residues, they provide binding sites for multiple Src homology-2 (SH2) domain-containing proteins, such as SH2-containing protein tyrosine phosphatase 2 (SHP2), phosphatidylinositol 3-kinase regulatory subunit p85, phospholipase Cγ, Crk, and GC-GAP. Through these interactions, the Gab proteins transduce signals from activated receptors into pathways with distinct biological functions, thereby contributing to signal diversification. They are known to play crucial roles in numerous physiological processes through their associations with SHP2 and p85. In addition, abnormal Gab protein signaling has been linked to human diseases including cancer, cardiovascular disease, and inflammatory disorders. In this paper, we provide an overview of the structure, effector functions, and regulation of the Gab docking proteins, with a special focus on their associations with cardiovascular disease, cancer, and inflammation.