Theoretical study and application of 2-phenyl-1,3,4-thiadiazole derivatives with optical and inhibitory activity against SHP1.

Src homology-2 domain-containing protein tyrosine phosphatase 1 (SHP1) is mainly restricted to hematopoietic and epithelial cells and widely accepted as a convergent node for oncogenic cell-signaling cascades. The development of efficient methods for rapidly tracing and inhibiting the SHP1 activity in complex biological systems is of considerable significance for advancing the integration of diagnosis and treatment of the related disease. With this aim, we designed and synthesized five 2-phenyl-1,3,4-thiadiazole derivatives (PT2, PT5, PT8, PT9 and PT10) here based on the reported SHP1 inhibitors (PT1, PT3, PT4, PT6 and PT7). The photophysical properties and inhibitory activities of these 2-phenyl-1,3,4-thiadiazole derivatives (PT1-PT10) against SHP1 were thoroughly studied from the theoretical simulation and experimental application aspects. The representative compound PT10 exhibited a larger quantum yield than the other molecules because of the smaller geometric relaxation and reorganization energy of the excited state, which was consistent with the results from the fluorescence experiments in organic solvents. In addition, PT10 showed a selective fluorescence response for SHP1 activity and low cytotoxicity in HeLa cells. Lastly, it indicated the potential application in two-photon cell fluorescence imaging in the future according to the calculated excellent two-photon absorption properties. In this contribution, firstly, we offered the fluorescent and activated molecule PT10 against SHP1, which achieved the integration of visualization and inhibitory activity of SHP1 preliminarily at the enzyme molecular level.

Hypoxia Impairs NK Cell Cytotoxicity through SHP-1-Mediated Attenuation of STAT3 and ERK Signaling Pathways.

Natural killer (NK) cells are innate immune effectors with potent antitumor activity. However, tumor cells can create an immunosuppressive microenvironment to escape immune surveillance. Although accumulating evidence indicates that microenvironmental hypoxia plays an important role in favoring tumor development and immune evasion, it remains unclear by what means hypoxia directly impairs NK cell antitumor activity. In this study, we confirmed that hypoxic NK cells showed significantly lower cytotoxicity against tumor cells. Consistent with this finding, we found that the reduction in NK cell cytotoxicity resulting from hypoxia correlated to the lower expression of granzyme B, IFN-γ, and degranulation marker CD107a, as well as activating receptors including NKp30, NKp46, and NKG2D expressed on the surface of NK cells. More importantly, we further demonstrated that a reduction in the phosphorylation levels of ERK and STAT3 secondary to hypoxia was strongly associated with the attenuated NK cell cytotoxicity. Focusing on the mechanism responsible for reduced phosphorylation levels of ERK and STAT3, we reveal that the activation of protein tyrosine phosphatase SHP-1 (Src homology region 2 domain-containing phosphatase-1) following hypoxia might play an essential role in this process. By knocking down SHP-1 or blocking its activity using a specific inhibitor TPI-1, we were able to partially restore NK cell cytotoxicity under hypoxia. Taken together, we demonstrate that hypoxia could impair NK cell cytotoxicity by decreasing the phosphorylation levels of ERK and STAT3 in a SHP-1-dependent manner. Therefore, targeting SHP-1 could provide an approach to enhance NK cell-based tumor immunotherapy.

SHP-2 and PD-L1 Inhibition Combined with Radiotherapy Enhances Systemic Antitumor Effects in an Anti-PD-1-Resistant Model of Non-Small Cell Lung Cancer.

Immune checkpoint inhibitors, such as anti-PD-1/PD-L1, have emerged as promising therapies for advanced non-small cell lung cancer (NSCLC). However, approximately 80% of patients do not respond to immunotherapy given alone because of intrinsic or acquired resistance. Radiotherapy (XRT) can overcome PD-1 resistance and improve treatment outcomes, but its efficacy remains suboptimal. The tyrosine phosphatase SHP-2, expressed in some cancers and in immune cells, has been shown to negatively affect antitumor immunity. Our hypothesis was that SHP-2 inhibition in combination with anti-PD-L1 would enhance immune-mediated responses to XRT and synergistically boost antitumor effects in an anti-PD-1-resistant mouse model. We treated 129Sv/Ev mice with anti-PD-1-resistant 344SQ NSCLC adenocarcinoma with oral SHP099 (a SHP-2 inhibitor) combined with XRT and intraperitoneal anti-PD-L1. Primary tumors were treated with XRT (three fractions of 12 Gy each), whereas abscopal (out-of-field) tumors were observed but not treated. XRT in combination with SHP099 and anti-PD-L1 promoted local and abscopal responses, reduced lung metastases, and improved mouse survival. XRT also increased SHP-2+ M1 tumor-associated macrophages in abscopal tumors (P = 0.019). The addition of SHP099 also associated with a higher M1/M2 ratio, greater numbers of CD8+ T cells, and fewer regulatory T cells. This triple-combination therapy had strong antitumor effects in a mouse model of anti-PD-1-resistant NSCLC and may be a novel therapeutic approach for anti-PD-1-resistant NSCLC in patients.

Decrease in SHP-1 enhances myometrium remodeling via FAK activation leading to labor.

Preterm birth is one of the most common complications during human pregnancy and is associated with a dramatic switch within the uterus from quiescence to contractility. However, the mechanisms underlying uterine remodeling are largely unknown. Protein kinases and phosphatases play critical roles in regulating the phosphorylation of proteins involved in the smooth muscle cell functions. In the present study, we found that Src-homology phosphatase type-1 (SHP-1, PTPN6) was significantly decreased in human myometrium in labor compared with that not in labor. Timed-pregnant mice injected intraperitoneally with the specific SHP-1 inhibitor protein tyrosine phosphatase inhibitor I (PTPI-1) manifested significantly preterm labor, with enriched plasmalemmal dense plaques between myometrial cells and increased phosphorylation at Tyr397 and Tyr576/577 sites of focal adhesion kinase (FAK) in myometrial cells, which remained to the time of labor, whereas the phosphorylation levels of ERK1/2 and phosphatidylinositol 3 kinase (PI3K) showed a rapid increase upon PTPI-1 injection but fell back to normal at the time of labor. The Tyr576/577 in FAK played an important role in the interaction between FAK and SHP-1. Knockdown of SHP-1 dramatically increased the spontaneous contraction of human uterine smooth muscle cells (HUSMCs), which was reversed by coinfection of a FAK-knockdown lentivirus. PGF2α downregulated SHP-1 via PLCß-PKC-NF-κB or PI3K-NF-κB pathways, suggesting the regenerative downregulation of SHP-1 enhances the uterine remodeling and plasticity by activating FAK and subsequent focal adhesion pathway, which eventually facilitates myometrium contraction and leads to labor. The study sheds new light on understanding of mechanisms that underlie the initiation of labor, and interventions for modulation of SHP-1 may provide a potential strategy for preventing preterm birth.

Inhibition of SHP-1 Expands the Repertoire of Antitumor T Cells Available to Respond to Immune Checkpoint Blockade.

The presence and activity of CD8+ T cells within the tumor microenvironment are essential for the control of tumor growth. Utilizing B16-F10 melanoma tumors that express altered peptide ligands of chicken ovalbumin, OVA257-264, we measured high- and low-affinity OVA-specific responses following adoptive transfer of OT-I CD8+ T cell into mice subsequently challenged with tumors. T-cell receptor (TCR) affinity positively correlated with the frequency of OT-I tumor-infiltrating lymphocytes (TIL). Differences in TCR affinity inversely corresponded to in vivo tumor growth rate. Blockade of the PD-1 and CTLA-4 checkpoints preferentially increased the frequency and antitumor function of TIL responding to high-affinity antigens, while failing to enhance the antitumor activity of low-affinity T cells. To determine whether lowering the TCR activation threshold could enhance the breadth and magnitude of the antitumor T-cell response, we inhibited Src homology region 2 domain-containing phosphatase 1 (SHP-1) in OT-I T cells prior to tumor antigen exposure. SHP-1 knockdown increased the cytokine-producing potential of high- and low-affinity T cells but failed to enhance control of tumor growth. In contrast, when SHP-1 knockdown of OT-I T cells was combined with immunotherapy, we observed a significant and long-lasting suppression of tumor growth mediated by low-affinity T cells. We conclude that lowering the TCR activation threshold by targeting SHP-1 expands the repertoire of T cells available to respond to conventional checkpoint blockade, leading to enhanced control of tumor growth.

Pro-efferocytic nanoparticles are specifically taken up by lesional macrophages and prevent atherosclerosis.

Atherosclerosis is the process that underlies heart attack and stroke. A characteristic feature of the atherosclerotic plaque is the accumulation of apoptotic cells in the necrotic core. Prophagocytic antibody-based therapies are currently being explored to stimulate the phagocytic clearance of apoptotic cells; however, these therapies can cause off-target clearance of healthy tissues, which leads to toxicities such as anaemia. Here we developed a macrophage-specific nanotherapy based on single-walled carbon nanotubes loaded with a chemical inhibitor of the antiphagocytic CD47-SIRPα signalling axis. We demonstrate that these single-walled carbon nanotubes accumulate within the atherosclerotic plaque, reactivate lesional phagocytosis and reduce the plaque burden in atheroprone apolipoprotein-E-deficient mice without compromising safety, and thereby overcome a key translational barrier for this class of drugs. Single-cell RNA sequencing analysis reveals that prophagocytic single-walled carbon nanotubes decrease the expression of inflammatory genes linked to cytokine and chemokine pathways in lesional macrophages, which demonstrates the potential of 'Trojan horse' nanoparticles to prevent atherosclerotic cardiovascular disease.

TCDD-mediated suppression of naïve human B cell IgM secretion involves aryl hydrocarbon receptor-mediated reduction in STAT3 serine 727 phosphorylation and is restored by interferon-γ.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant formed as a byproduct in organic synthesis and burning of organic materials. TCDD has potent immunotoxic effects in B lymphocytes resulting in decreased cellular activation and suppressed IgM secretion following activation with CD40 ligand. Previous work from our lab demonstrated that TCDD treatment of naïve human B cells resulted in significant increases in the levels of the tyrosine phosphatase SHP-1, which corresponded with suppression of IgM secretion. STAT3 is a critical B cell transcription factor for B cell activation and secretion of immunoglobulins (Ig). STAT3 dimerizes and translocates to the nucleus following phosphorylation as a result of cytokine receptor signaling. We hypothesized that TCDD-mediated increases in SHP-1 could result in decreased STAT3 tyrosine phosphorylation. Interestingly, only modest changes in the levels of STAT3 tyrosine phosphorylation were observed. By contrast, TCDD significantly reduced levels of STAT3 serine phosphorylation as early as 12h post B cell activation. These results corresponded with decreased inhibitory phosphorylation of the serine specific phosphatase PP2a, which is regulated by SHP-1. Further, studies revealed that interferon gamma (IFNγ), which signals through the type II interferon receptor, can non-canonically induce STAT3 activation via Src kinase activity. Indeed, treatment of human B cells with IFNγ resulted in increased STAT3 serine phosphorylation and reversed TCDD-mediated suppression of the IgM response. Together, these data putatively identify a key event in the mechanism by which TCDD induces suppression of Ig secretion and demonstrate the potential of IFNγ as a means to reverse this effect in primary human B lymphocytes.

A procedure combining molecular docking and semiempirical method PM7 for identification of selective Shp2 inhibitors.

Shp2 and Shp1 make up a small family of protein tyrosine phosphatases. Finding selective inhibitors for Shp2 is useful because although its inhibition is advantageous for the treatment of some types of cancer, inhibition of Shp1 may have the opposite effect, since it acts as a suppressor of tumors. We combined molecular docking and semiempirical molecular orbital-based calculations to produce data that were effective for the identification of selective inhibitors for Shp2. After definition of the interaction modes of the inhibitors with Shp2 and Shp1 by molecular docking, the resulting interaction enthalpy values were calculated following refinement of the enzyme/inhibitor complexes' geometries with the PM7 semiempirical molecular orbital method. Despite the complexity of the thermodynamics involved in the enzyme/inhibitor interaction, the selectivity for Shp2 of a series of 76 inhibitors, divided in two groups, could be effectively correlated with the difference in their interaction enthalpy values with both enzymes. For the first group, composed by 52 Shp2-selective indoline inhibitors for which only Shp2 inhibition activity data are available, we demonstrated that the interaction enthalpy can be used as a criterion for identification of selective Shp2 inhibitors, as it is significantly more favorable for Shp2 than Shp1 at a 99% confidence level. For the second group, composed by 24 oxindole derivatives with available Shp2 and Shp1 inhibition activity data, a satisfactory correlation (R = 0.70) could be obtained between the selectivity, based on the IC50 data, and the relative percentage difference of the calculated interaction enthalpies with the two enzymes.

Regulation of Microtubule Nucleation in Mouse Bone Marrow-Derived Mast Cells by Protein Tyrosine Phosphatase SHP-1.

The antigen-mediated activation of mast cells initiates signaling events leading to their degranulation, to the release of inflammatory mediators, and to the synthesis of cytokines and chemokines. Although rapid and transient microtubule reorganization during activation has been described, the molecular mechanisms that control their rearrangement are largely unknown. Microtubule nucleation is mediated by γ-tubulin complexes. In this study, we report on the regulation of microtubule nucleation in bone marrow-derived mast cells (BMMCs) by Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 1 (SHP-1; Ptpn6). Reciprocal immunoprecipitation experiments and pull-down assays revealed that SHP-1 is present in complexes containing γ-tubulin complex proteins and protein tyrosine kinase Syk. Microtubule regrowth experiments in cells with deleted SHP-1 showed a stimulation of microtubule nucleation, and phenotypic rescue experiments confirmed that SHP-1 represents a negative regulator of microtubule nucleation in BMMCs. Moreover, the inhibition of the SHP-1 activity by inhibitors TPI-1 and NSC87877 also augmented microtubule nucleation. The regulation was due to changes in γ-tubulin accumulation. Further experiments with antigen-activated cells showed that the deletion of SHP-1 stimulated the generation of microtubule protrusions, the activity of Syk kinase, and degranulation. Our data suggest a novel mechanism for the suppression of microtubule formation in the later stages of mast cell activation.

KU70 Inhibition Impairs Both Non-Homologous End Joining and Homologous Recombination DNA Damage Repair Through SHP-1 Induced Dephosphorylation of SIRT1 in T-Cell Acute Lymphoblastic Leukemia (T-ALL) [corrected].

BACKGROUND/AIMS: T-Cell Acute Lymphoblastic Leukemia (T-ALL) [corrected] is an aggressive disease which is highly resistant to chemotherapy. Studies show that enhanced ability of DNA damage repair (DDR) in cancer cells plays a key role in chemotherapy resistance. Here, we suggest that defect in DDR related genes might be a promising target to destroy the genome stability of tumor cells. METHODS: Since KU70 is highly expressed in Jurkat cells, one of the most representative cell lines of ATL, we knocked down KU70 by shRNA and analyzed the impact of KU70 deficiency in Jurkat cells as well as in NOD-SCID animal models by western blot, immunofluorescence, flow cytometry and measuring DNA repair efficiency. RESULTS: It is observed that silencing of KU70 resulted in accumulated DNA damage and impaired DDR in Jurkat cells, resulting in more apoptosis, decreased cell proliferation and cell cycle arrest. DNA damage leads to DNA double-strand breaks (DSBs), which are processed by either non-homologous end joining(NHEJ) or homologous recombination(HR). In our study, both NHEJ and HR are impaired because of KU70 defect, accompanied with increased protein level of SHP-1, a dephosphorylation enzyme. In turn, SHP-1 led to dephosphorylation of SIRT1, which further impaired HR repair efficiency. Moreover, KU70 deficiency prolonged survival of Jurkat-xenografted mice. CONCLUSION: These findings suggest that targeting KU70 is a promising target for ATL and might overcome the existing difficulties in chemotherapy.

Targeting SHP-1, 2 and SHIP Pathways: A Novel Strategy for Cancer Treatment?

Well-balanced levels of tyrosine phosphorylation, maintained by the reversible and coordinated actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), are critical for a wide range of cellular processes including growth, differentiation, metabolism, migration, and survival. Aberrant tyrosine phosphorylation, as a result of a perturbed balance between the activities of PTKs and PTPs, is linked to the pathogenesis of numerous human diseases, including cancer, suggesting that PTPs may be innovative molecular targets for cancer treatment. Two PTPs that have an important inhibitory role in haematopoietic cells are SHP-1 and SHP-2. SHP-1, 2 promote cell growth and act by both upregulating positive signaling pathways and by downregulating negative signaling pathways. SHIP is another inhibitory phosphatase that is specific for the inositol phospholipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). SHIP acts as a negative regulator of immune response by hydrolysing PIP3, and SHIP deficiency results in myeloproliferation and B-cell lymphoma in mice. The validation of SHP-1, 2 and SHIP as oncology targets has generated interest in the development of inhibitors as potential therapeutic agents for cancers; however, SHP-1, 2 and SHIP have proven to be an extremely difficult target for drug discovery, primarily due to the highly conserved and positively charged nature of their PTP active site, and many PTP inhibitors lack either appro-priate selectivity or membrane permeability. To overcome these caveats, novel techniques have been employed to synthesise new inhibitors that specifically attenuate the PTP-dependent signaling inside the cell and amongst them; some are already in clinical development which are discussed in this review.

Allicin Inhibits Proliferation and Invasion in Vitro and in Vivo via SHP-1-Mediated STAT3 Signaling in Cholangiocarcinoma.

BACKGROUND/AIMS: Cholangiocarcinoma (CCA) is a malignant tumor that is resistant to chemotherapy, so new therapeutic agents are needed. Allicin which is rapidly converted from allin by allinase, is one of the most biologically active compounds in freshly crushed garlic and has been shown to have strong anti-tumor effects. Our aim was to explore the molecular mechanism by which allicin affects the cell proliferation and invasion of CCA. METHODS: Cell viability and apoptosis were measured using the CCK-8 assay, colony formation assay, and flow cytometry. Cell migration and invasion were evaluated by wound healing and Transwell assays, respectively. The expression of several proteins involved in cell apoptosis and invasion were assessed by Western blot. The activation of STAT3 signaling was detected by Western blot and immunofluorescence staining. The involvement of SHP-1 was determined using small interfering RNA (siRNA). Moreover, a nude mouse model of human CCA was established to assess the anti-tumor effects of allicin in vivo. RESULTS: Allicin significantly suppressed CCA cell proliferation by activating the caspase cascade, inducing apoptosis, and reducing the expression of proteins downstream of STAT3, such as B-cell lymphoma 2 (Bcl-2), while upregulating Bcl-2-associated X (Bax) protein. In addition, allicin inhibited the migration, invasion, and epithelial-mesenchymal transition (EMT) of CCA cells. Moreover, the protein expression of MMP-2 and MMP-9 was significantly downregulated in CCA cells treated with allicin compared with CCA cells treated with control. Mechanistic investigations indicated that allicin upregulated SHP-1 expression in CCA, and pervanadate treatment reversed the allicin-induced downregulation of STAT3. Moreover, suppression of SHP-1 by siRNA overturned the effect of allicin on the induction of SHP-1 and inhibition of STAT3 activation. Additionally, treatment with allicin attenuated tumor growth in the nude mouse model of CCA. CONCLUSIONS: Our findings suggest that allicin suppresses cell proliferation and invasion via STAT3 signaling and may be a potential therapeutic agent for CCA.

Alteration of SHP-1/p-STAT3 Signaling: A Potential Target for Anticancer Therapy.

The Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 1 (SHP-1), a non-receptor protein tyrosine phosphatase, has been reported as a negative regulator of phosphorylated signal transducer and activator of transcription 3 (STAT3) and linked to tumor development. In this present review, we will discuss the importance and function of SHP-1/p-STAT3 signaling in nonmalignant conditions as well as malignancies, its cross-talk with other pathways, the current clinical development and the potential role of inhibitors of this pathway in anticancer therapy and clinical relevance of SHP-1/p-STAT3 in cancers. Lastly, we will summarize and highlight work involving novel drugs/compounds targeting SHP-1/p-STAT3 signaling and combined strategies that were/are discovered in our and our colleagues' laboratories.

Inhibition of Src homology 2 domain containing protein tyrosine phosphatase as the possible mechanism of metformin-assisted amelioration of obesity induced insulin resistance in high fat diet fed C57BL/6J mice.

SHP-1 (Src homology 2 domain containing protein tyrosine phosphatase) is a known negative regulator of insulin signaling and inflammation. To date, the molecular mechanism of metformin in modulating SHP-1 expression has remained elusive. In the present study, we have investigated the role of SHP-1 in relation to anti-hyperglycemic and anti-inflammatory actions of metformin in an obese phenotype mouse model. We observed that metformin treatment significantly reduced SHP-1 activity in obese mice, leading to improved insulin sensitivity. Additionally, metformin down regulated inflammatory markers like TLR2, TLR4, CD80, CD86, NF-κB, STAT1 and suppressed adipose tissue inflammation by efficiently polarizing adipose tissue macrophages toward anti-inflammatory state by way of indirect inhibition of SHP-1 mRNA and protein expressions. Our study suggests that metformin exerts its insulin sensitizing effects via inhibition of SHP-1 activity and expression.

THEMIS enhances TCR signaling and enables positive selection by selective inhibition of the phosphatase SHP-1.

THEMIS, a T cell-specific protein with high expression in CD4+CD8+ thymocytes, has a crucial role in positive selection and T cell development. THEMIS lacks defined catalytic domains but contains two tandem repeats of a distinctive module of unknown function (CABIT). Here we found that THEMIS directly regulated the catalytic activity of the tyrosine phosphatase SHP-1. This action was mediated by the CABIT modules, which bound to the phosphatase domain of SHP-1 and promoted or stabilized oxidation of SHP-1's catalytic cysteine residue, which inhibited the tyrosine-phosphatase activity of SHP-1. Deletion of SHP-1 alleviated the developmental block in Themis-/- thymocytes. Thus, THEMIS facilitates thymocyte positive selection by enhancing the T cell antigen receptor signaling response to low-affinity ligands.

The NF-kB regulates the SHP-1 expression in monocytes in congestive heart failure.

It has been shown that functional recovery of patients with acute congestive heart failure (ACHF) after treatment with conventional drugs (CD) is mediated by suppression of inflammation in peripheral blood mononuclear cells. Here, we analyzed gene expression profiles of monocytes from symptomatic ACHF patients (NYHA Class III-IV) before and after pharmacological treatment with CD. The treatment was associated with selective down-regulation of "TNFR signaling" and pro-inflammatory mediators CCL5, MIP-1α receptor, CD14, ITGAM, and significant up-regulation of "TNFR signaling" as evidenced by increase in anti-inflammatory factors including NF-kBIA, TNFAIP3 and SHP-1. In monocyte TNF-alpha-stimulated there is a down-regulation of the phosphatase SHP-1 which induces a significant activation of TAK-1/IKK/NF-kB signaling. These findings suggest that the therapeutic impact of CD treatment in symptomatic ACHF includes negative regulation of the NF-kB signaling in monocytes and the improvement of the SHP-1 activity.

Host SHP1 phosphatase antagonizes Helicobacter pylori CagA and can be downregulated by Epstein-Barr virus.

Most if not all gastric cancers are associated with chronic infection of the stomach mucosa with Helicobacter pylori cagA-positive strains(1-4). Approximately 10% of gastric cancers also harbour Epstein-Barr virus (EBV) in the cancer cells(5,6). Following delivery into gastric epithelial cells via type IV secretion(7,8), the cagA-encoded CagA protein undergoes tyrosine phosphorylation on the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs initially by Src family kinases (SFKs) and then by c-Abl(9,10). Tyrosine-phosphorylated CagA binds to the pro-oncogenic protein tyrosine phosphatase SHP2 and thereby deregulates the phosphatase activity(11,12), which has been considered to play an important role in gastric carcinogenesis(13). Here we show that the SHP2 homologue SHP1 interacts with CagA independently of the EPIYA motif. The interaction potentiates the phosphatase activity of SHP1 that dampens the oncogenic action of CagA by dephosphorylating the CagA EPIYA motifs. In vitro infection of gastric epithelial cells with EBV induces SHP1 promoter hypermethylation, which strengthens phosphorylation-dependent CagA action via epigenetic downregulation of SHP1 expression. Clinical specimens of EBV-positive gastric cancers also exhibit SHP1 hypermethylation with reduced SHP1 expression. The results reveal that SHP1 is the long-sought phosphatase that can antagonize CagA. Augmented H. pylori CagA activity, via SHP1 inhibition, might also contribute to the development of EBV-positive gastric cancer.

Dovitinib Acts As a Novel Radiosensitizer in Hepatocellular Carcinoma by Targeting SHP-1/STAT3 Signaling.

PURPOSE: Hepatocellular carcinoma (HCC) is among the most lethal human malignancies, and curative therapy is not an option for most patients. There is growing interest in the potential benefit of combining targeted therapies with radiation therapy (RT). This study aimed to characterize the efficacy and mechanism of an investigational drug, dovitinib, used in combination with RT. METHODS AND MATERIALS: HCC cell lines (PLC5, Hep3B, SK-Hep1, HA59T, and Huh-7) were treated with dovitinib, RT, or both, and apoptosis and signal transduction were analyzed. RESULTS: Dovitinib treatment resulted in Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1)-mediated downregulation of p-STAT3 and promoted potent apoptosis of HCC cells. Ectopic expression of STAT3, or inhibition of SHP-1, diminished the effects of dovitinib on HCC cells. By ectopic expression and purified recombinant proteins of various mutant forms of SHP-1, the N-SH2 domain of SHP-1 was found to be required for dovitinib treatment. Overexpression of STAT3 or catalytic-dead mutant SHP-1 restored RT-induced reduction of HCC cell survival. Conversely, ectopic expression of SHP-1 or activation of SHP-1 by dovitinib enhanced the effects of RT against HCC in vitro and in vivo. CONCLUSIONS: SHP-1/STAT3 signaling is critically associated with the radiosensitivity of HCC cells. Combination therapy with RT and the SHP-1 agonist, such as dovitinib, resulted in enhanced in vitro and in vivo anti-HCC effects.

Honokiol Inhibits Constitutive and Inducible STAT3 Signaling via PU.1-Induced SHP1 Expression in Acute Myeloid Leukemia Cells.

Constitutive and inducible activation of signal transducer and activator of transcription 3 (STAT3) signaling facilitates the carcinogenesis in most human cancers including acute myeloid leukemia (AML). Negative regulators, such as protein tyrosine phosphatases SHP1, inhibit the activated STAT3 signaling. In this study, we investigated the effect of honokiol (HNK), a constituent of Magnolia officinalis, on the STAT3 signaling. STAT3 signaling and SHP1 expression were measured by quantitative real-time PCR and western blotting in leukemic cell lines and primary AML blasts treated with HNK. HNK decreased the phosphorylated STAT3 but not the total STAT3 through increasing the expression of SHP1. In addition, HNK inhibited transcription activity of STAT3, reduced nuclear translocation of STAT3, and decreased the expression of STAT3 target genes. Knockdown of SHP1 by small hairpin RNA (shRNA) or treatment with vanadate, a protein tyrosine phosphatases inhibitor, abolished HNK-induced STAT3 inhibition, suggesting that SHP1 plays an important role in the inhibition of STAT3 signaling by HNK. Further, HNK increased the expression of transcript factor PU.1, which had been reported to activate the expression of SHP1 via binding SHP1 promoter region. Knockdown of PU.1 reversed HNK-induced upregulation of SHP1 and inactivation of STAT3 signaling. Finally, HNK increased the expression of PU.1 and SHP1 in hematopoietic progenitors isolated from patients with AML. In conclusion, our data have shown a regulatory mechanism underlying the inhibition of STAT3 signaling by HNK. Therefore, as a relative non-toxic compound, HNK may offer a therapeutic advantage in the clinical treatment for AML.

9-cis-retinoic acid improves sensitivity to platelet-derived growth factor-BB via RXRα and SHP-1 in diabetic retinopathy.

Diabetic retinopathy (DR) is one of the most common complications of diabetes mellitus. But few efficient therapeutic methods have been reported. This study discussed the functions of 9-cis-retinoic acid (9-cis-RA) in sensitizing retinal pericytes to platelet-derived growth factor (PDGF)-BB. Using streptozotocin (STZ)-induced diabetic mice and high glucose-treated bovine retinal pericytes (BRPC), we analyzed the impacts of 9-cis-RA by detecting cell apoptosis via DNA fragmentation assay and detecting related factors through adenovirus or lentivirus infection and western blot. Results showed that in retinas of STZ-induced diabetic mice, 9-cis-RA significantly inhibited expression of SHP-1 (P < 0.01), thus promoting p-AKT and p-ERK1/2, which reflected the improved sensitivity to PDGF-BB. In BRPC, 9-cis-RA also improved sensitivity to PDGF-BB and suppressed cell apoptosis (P < 0.01) via down-regulating SHP-1. Further mechanism analyses showed that the efficient functioning of 9-cis-RA relied on the existence of its receptor, retinoic X receptor α (RXRα), independent of the previous reported protein kinase C delta (PKCδ)/SHP-1 axis. Because 9-cis-RA could not inhibit SHP-1 or improve sensitivity to PDGF-BB when RXRα was knocked down, while it still suppressed SHP-1 after overexpression of PKCδ. Taken together, these results indicated the vital roles of 9-cis-RA in improving sensitivity to PDGF-BB of retinal pericytes in DR, and provided basic evidences of new therapeutic targets like RXRα for further DR treatment.