SH2 Domain-containing Phosphatase Research Articles

CEACAM1 controls the EMT switch in murine mammary carcinoma in vitro and in vivo.

We analyzed the molecular basis for carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1)-controlled inhibition of epithelial-mesenchymal transition (EMT) in a mouse model for mammary adenocarcinoma (WAP-T mice). We demonstrate that silencing of CEACAM1 in WAP-T tumor-derived G-2 cells induces epithelial-mesenchymal plasticity (EMP), as evidenced by typical changes of gene expression, morphology and increased invasion. In contrast, reintroduction of CEACAM1 into G-2 cells reversed up-regulation of genes imposing mesenchymal transition, as well as cellular invasion. We identified the Wnt-pathway as target for CEACAM1-mediated repression of EMT. Importantly, β-catenin phosphorylation status and transcriptional activity strongly depend on CEACAM1 expression: CEACAM1high G-2 cells displayed enhanced phosphorylation of β-catenin at S33/S37/T41 and decreased phosphorylation at Y86, thereby inhibiting canonical Wnt/β-catenin signaling. We identified Src-homology 2 domain-containing phosphatase 2 (SHP-2) as a critical binding partner of CEACAM1 that could modulate β-catenin Y86 phosphorylation. Hence, CEACAM1 serves as a scaffold that controls membrane proximal β-catenin signaling. In vivo, mammary tumors of WAP-T/CEACAM1null mice displayed increased nuclear translocation of β-catenin and a dramatically enhanced metastasis rate compared to WAP-T mice. Hence, CEACAM1 controls EMT in vitro and in vivo by site-specific regulation of β-catenin phosphorylation. Survival analyses of human mammary carcinoma patients corroborated these data, indicating that CEACAM1 is a prognostic marker for breast cancer survival.

SHP1 Regulates Bone Mass by Directing Mesenchymal Stem Cell Differentiation

Osteoblasts and adipocytes are derived from a common precursor, mesenchymal stem cells (MSCs). Alterations in the normal fate of differentiating MSCs are involved in the development of obesity and osteoporosis. Here, we report that viable motheaten (me(v)) mice, which are deficient in the SH2-domain-containing phosphatase-1 (SHP1), develop osteoporosis spontaneously. Consistently, MSCs from me(v)/me(v) mice exhibit significantly reduced osteogenic potential and greatly increased adipogenic potential. When MSCs were transplanted into nude mice, SHP1-deficient MSCs resulted in diminished bone formation compared with wild-type MSCs. SHP1 was found to bind to GSK3β and suppress its kinase activity by dephosphorylating pY216, thus resulting in β-catenin stabilization. Mice, in which SHP1 was deleted in MSCs using SHP1(fl/fl)Dermo1-cre, displayed significantly decreased bone mass and increased adipose tissue. Taken together, these results suggest a possible role for SHP1 in controlling tissue homeostasis through modulation of MSC differentiation via Wnt signaling regulation.

Constitutive LcK activity drives sensitivity differences between CD8+ memory T cell subsets

Abstract CD8+ T cells develop increased sensitivity following antigen experience, and differences in sensitivity exist between T cell memory subsets. How differential T cell receptor (TCR) signaling between memory subsets contributes to sensitivity differences is unclear. We show that constitutive activity of lymphocyte-specific protein tyrosine kinase (Lck) is greater in effector memory T cells (TEM) compared with central memory T cells (TCM), leading to enhanced activation signaling, including Zeta-chain-associated protein kinase 70 (Zap-70) phosphorylation and intracellular calcium influx, and resulting in increased cytotoxic effector function in TEM. We provide evidence that the differences in Lck activity between CD8+ TCM and TEM are due to differential regulation by SH2 domain-containing phosphatase-1 (Shp-1) and C-terminal Src kinase (Csk). Together, this work demonstrates a role for constitutive Lck activity in controlling antigen sensitivity, and suggests that differential activities of TCR-proximal signaling components may contribute to establishing the divergent effector properties of TCM and TEM.

Enhanced Tyrosine Phosphatase Activity Underlies Dysregulated B Cell Receptor Signaling and Promotes Survival of Human Lupus B Cells.

Systemic lupus erythematosus (SLE) is associated with hyperactivity of B cells and abnormalities of B cell receptor (BCR) signaling. To address the linkage between dysregulated BCR signaling and increased B cell function, we assessed immediate phosphorylation events in lupus B cells. B cells from SLE patients and healthy donors were analyzed by flow cytometry to assess phosphorylated CD22, Syk, and Akt as well as the basal expression of the BCR coreceptors CD22 and CD19. Confocal microscopy studies determined the recruitment of CD22 and the tyrosine phosphatase SH2 domain-containing phosphatase 1 to the activated BCR complex. Additionally, phosphatase activity in SLE versus healthy donor B cells was measured. B cells from SLE patients showed diminished Syk phosphorylation and reduced intracellular calcium release after BCR activation as compared to B cells from healthy donors. This was related to an enhanced activity of tyrosine, but not serine/threonine, phosphatases and was corrected by inhibition of tyrosine phosphatase activity. In contrast to reduced Syk phosphorylation after BCR activation, phosphorylation of Akt was significantly increased in SLE B cells. The disturbed balance between Syk and Akt phosphorylation was significantly correlated with B cell survival following BCR engagement. Furthermore, CD27-, but not CD27+, B cells from SLE patients displayed increased expression and phosphorylation of the inhibitory BCR coreceptor CD22. These results indicate that an imbalance between serine and tyrosine phosphatases in SLE contributes to an intrinsically disturbed balance of BCR-initiated signaling pathways, resulting in enhanced survival of lupus B cells and differentiation into plasma cells.

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.

Shp2 Associates with and Enhances Nephrin Tyrosine Phosphorylation and Is Necessary for Foot Process Spreading in Mouse Models of Podocyte Injury.

In most forms of glomerular diseases, loss of size selectivity by the kidney filtration barrier is associated with changes in the morphology of podocytes. The kidney filtration barrier is comprised of the endothelial lining, the glomerular basement membrane, and the podocyte intercellular junction, or slit diaphragm. The cell adhesion proteins nephrin and neph1 localize to the slit diaphragm and transduce signals in a Src family kinase Fyn-mediated tyrosine phosphorylation-dependent manner. Studies in cell culture suggest nephrin phosphorylation-dependent signaling events are primarily involved in regulation of actin dynamics and lamellipodium formation. Nephrin phosphorylation is a proximal event that occurs both during development and following podocyte injury. We hypothesized that abrogation of nephrin phosphorylation following injury would prevent nephrin-dependent actin remodeling and foot process morphological changes. Utilizing a biased screening approach, we found nonreceptor Src homology 2 (sh2) domain-containing phosphatase Shp2 to be associated with phosphorylated nephrin. We observed an increase in nephrin tyrosine phosphorylation in the presence of Shp2 in cell culture studies. In the human glomerulopathies minimal-change nephrosis and membranous nephropathy, there is an increase in Shp2 phosphorylation, a marker of increased Shp2 activity. Mouse podocytes lacking Shp2 do not develop foot process spreading when subjected to podocyte injury in vivo using protamine sulfate or nephrotoxic serum (NTS). In the NTS model, we observed a lack of foot process spreading in mouse podocytes with Shp2 deleted and smaller amounts of proteinuria. Taken together, these results suggest that Shp2-dependent signaling events are necessary for changes in foot process structure and function following injury.

Abstract A2-01: SHP-1 is a negative regulator of epithelial-mesenchymal transition in hepatocellular carcinoma

Abstract Epithelial-to-mesenchymal transition (EMT) is well known to involve in tumor invasion and metastasis. SH2 domain-containing phosphatase 1 (SHP-1) functions as a potent tumor suppressor and also acts as a negative regulator of p-STAT3 (Tyr705) oncogenic signaling. However, little is known about the molecular mechanism(s) through which SHP-1 regulates EMT during hepatocellular carcinoma (HCC) progression. Here, we firstly reported that endogenous SHP-1 protein levels were significantly down-regulated in cells with mesenchymal characteristics and negatively correlated with p-STAT3 (Tyr705) and vimentin but positively correlated with E-cadherin. SHP-1 overexpression abolished TGF-beta-induced p-STAT3 (Tyr705) and EMT, as well inhibited migration and invasion but further rescued by STAT3 overexpression. Constitutively overexpressed SHP-1 tyrosine phosphatase activity by D61A mutated SHP-1 markedly reduced TGF-beta-induced p-STAT3 (Tyr705) and EMT features, but not altered by C453S catalytic dead mutant SHP-1. Consequently, SHP-1 acted as a powerful suppressor in preventing EMT by exerting its activity directly targeted p-STAT3 (Tyr705). Most notably, we discovered a novel SHP-1 agonist SC-43 better than sorafenib to exert more potent anti-EMT effects in vitro as well anti-metastatic growth in vivo. In conclusion, SHP-1 is a potent suppressor of HCC EMT and metastasis, thus, highlighting that SHP-1 may serve as a potential therapeutic target for HCC EMT and metastasis. Note: This abstract was not presented at the conference. Citation Format: Li-Ching Fan, Chung-Wai Shiau, Wei-Tien Tai, Man-Hsin Hung, Pei-Yi Chu, Hang Lin, Hui-Chuan Yu, Kuen-Feng Chen. SHP-1 is a negative regulator of epithelial-mesenchymal transition in hepatocellular carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-01.

SHP-2 Mediates Cryptosporidium parvum Infectivity in Human Intestinal Epithelial Cells.

The parasite, Cryptosporidium parvum, induces human gastroenteritis through infection of host epithelial cells in the small intestine. During the initial stage of infection, C. parvum is reported to engage host mechanisms at the host cell-parasite interface to form a parasitophorous vacuole. We determined that upon infection, the larger molecular weight proteins in human small intestinal epithelial host cells (FHs 74 Int) appeared to globally undergo tyrosine dephosphorylation. In parallel, expression of the cytoplasmic protein tyrosine phosphatase Src homology-2 domain-containing phosphatase 2 (SHP-2) increased in a time-dependent manner. SHP-2 co-localized with the C. parvum sporozoite and this interaction increased the rate of C. parvum infectivity through SH2-mediated SHP-2 activity. Furthermore, we show that one potential target that SHP-2 acts upon is the focal adhesion protein, paxillin, which undergoes moderate dephosphorylation following infection, with inhibition of SHP-2 rescuing paxillin phosphorylation. Importantly, treatment with an inhibitor to SHP-2 and with an inhibitor to paxillin and Src family kinases, effectively decreased the multiplicity of C. parvum infection in a dose-dependent manner. Thus, our study reveals an important role for SHP-2 in the pathogenesis of C. parvum. Furthermore, while host proteins can be recruited to participate in the development of the electron dense band at the host cell-parasite interface, our study implies for the first time that SHP-2 appears to be recruited by the C. parvum sporozoite to regulate infectivity. Taken together, these findings suggest that SHP-2 and its down-stream target paxillin could serve as targets for intervention.

Molecular behavior of the aptamer HJ24 self-assembled on highly oriented pyrolytic graphite (HOPG)

Nucleic acid sensing analysis has been widely applied to the fields of food quality control, environmental monitoring, and medical diagnosis. A key step in developing effective DNA-based electrochemical biosensors is to obtain a biorecognition layer on the biosensor, which can be influenced by many factors. Hence, we constructed a series of HJ24 layers on highly oriented pyrolytic graphite to investigate the relation between the configuration of the adsorbed probe HJ24 and the redox property using atomic force microscopy and voltammetry. We used HJ24 for its diagnostic value as it specifically recognizes the SH2 domain-containing phosphatase, a critical contributor in many important signaling pathways. The results demonstrated that increasing K ions induced G-quartet oxidation peak occurrence/increase ([K+]￡400 mmol/L), and also resulted in the formation of more compact single strand sheets ([K+]￡300 mmol/L). Moreover, transitions of molecule configurations and redox currents of G-quartets were observed at low concentration [K+]=12 mmol/L, which may indicate the appearance of new configurations under this condition. Besides, by analyzing atomic force microscopy (AFM) images, it was found that the different adsorbed configurations are correlated with the HJ24 concentration, the basal configuration, and the linker group on the HJ24 sequence. This information may be useful for understanding the adsorption process of HJ24 as well as for the further development of practical applications for HJ24 films on DNA electrochemical sensors, and may ultimately help improve the diagnosis and treatment of patients with SHP2-related diseases.

Pharmacological Targeting SHP-1-STAT3 Signaling Is a Promising Therapeutic Approach for the Treatment of Colorectal Cancer

STAT3 activation is associated with poor prognosis in human colorectal cancer (CRC). Our previous data demonstrated that regorafenib (Stivarga) is a pharmacological agonist of SH2 domain-containing phosphatase 1 (SHP-1) that enhances SHP-1 activity and induces apoptosis by targeting STAT3 signals in CRC. This study aimed to find a therapeutic drug that is more effective than regorafenib for CRC treatment. Here, we showed that SC-43 was more effective than regorafenib at inducing apoptosis in vitro and suppressing tumorigenesis in vivo. SC-43 significantly increased SHP-1 activity, downregulated p-STAT3Tyr705 level, and induced apoptosis in CRC cells. An SHP-1 inhibitor or knockdown of SHP-1 by siRNA both significantly rescued the SC-43–induced apoptosis and decreased p-STAT3Tyr705 level. Conversely, SHP-1 overexpression increased the effects of SC-43 on apoptosis and p-STAT3Tyr705 level. These data suggest that SC-43–induced apoptosis mediated through the loss of p-STAT3Tyr705 was dependent on SHP-1 function. Importantly, SC-43–enhanced SHP-1 activity was because of the docking potential of SC-43, which relieved the autoinhibited N-SH2 domain of SHP-1 and inhibited p-STAT3Tyr705 signals. Importantly, we observed that a significant negative correlation existed between SHP-1 and p-STAT3Tyr705expression in CRC patients (P = .038). Patients with strong SHP-1 and weak p-STAT3Tyr705 expression had significantly higher overall survival compared with patients with weak SHP-1 and strong p-STAT3Tyr705 expression (P = .029). In conclusion, SHP-1 is suitable to be a useful prognostic marker and a pharmacological target for CRC treatment. Targeting SHP-1-STAT3 signaling by SC-43 may serve as a promising pharmacotherapy for CRC.

An HNF1α-regulated feedback circuit modulates hepatic fibrogenesis via the crosstalk between hepatocytes and hepatic stellate cells.

Hepatocytes are critical for the maintenance of liver homeostasis, but its involvement in hepatic fibrogenesis remains elusive. Hepatocyte nuclear factor 1α (HNF1α) is a liver-enriched transcription factor that plays a key role in hepatocyte function. Our previous study revealed a significant inhibitory effect of HNF1α on hepatocellular carcinoma. In this study, we report that the expression of HNF1α is significantly repressed in both human and rat fibrotic liver. Knockdown of HNF1α in the liver significantly aggravates hepatic fibrogenesis in either dimethylnitrosamine (DMN) or bile duct ligation (BDL) model in rats. In contrast, forced expression of HNF1α markedly alleviates hepatic fibrosis. HNF1α regulates the transcriptional expression of SH2 domain-containing phosphatase-1 (SHP-1) via directly binding to SHP-1 promoter in hepatocytes. Inhibition of SHP-1 expression abrogates the anti-fibrotic effect of HNF1α in DMN-treated rats. Moreover, HNF1α repression in primary hepatocytes leads to the activation of NF-κB and JAK/STAT pathways and initiates an inflammatory feedback circuit consisting of HNF1α, SHP-1, STAT3, p65, miR-21 and miR-146a, which sustains the deregulation of HNF1α in hepatocytes. More interestingly, a coordinated crosstalk between hepatocytes and hepatic stellate cells (HSCs) participates in this positive feedback circuit and facilitates the progression of hepatocellular damage. Our findings demonstrate that impaired hepatocytes play an active role in hepatic fibrogenesis. Early intervention of HNF1α-regulated inflammatory feedback loop in hepatocytes may have beneficial effects in the treatment of chronic liver diseases.

Reversal of Arthritis by Human Monomeric IgA Through the Receptor-Mediated SH2 Domain-Containing Phosphatase 1 Inhibitory Pathway.

Rheumatoid arthritis (RA), one of the most frequent chronic inflammatory rheumatic disorders, is characterized by the presence of autoantibodies and joint infiltration by activated immune cells, leading to cartilage and bone destruction. IgA occurs predominantly as monomers (mIgA) in plasma and regulates many cell responses through interaction with the Fcα receptor type I (FcαRI). FcαRI targeting by anti-FcαRI Fab inhibits activating receptors by inducing an inhibitory immunoreceptor tyrosine-based activation motif (ITAMi) configuration through SH2 domain-containing phosphatase 1 (SHP-1) recruitment. The aim of this study was to investigate the potential utility of mIgA for the treatment of arthritis by acting as an inducer of ITAMi signaling. The effect of plasma-derived human mIgA on inhibition of multiple heterologous receptors was evaluated on FcαRI+ cell transfectants, blood phagocytes from healthy individuals, and synovial cells from RA patients. FcαRI-transgenic mice and wild-type mice treated with mIgA were studied in models of collagen antibody-induced arthritis (CAIA) and collagen-induced arthritis (CIA). The mice were assessed for development of arthritis using an arthritis score, and joint tissue samples were evaluated for the extent of leukocyte infiltration and expression of phosphatase. Treatment with mIgA impaired cell activation in an FcαRI-FcRγ-dependent manner, involving ITAMi signaling. Human mIgA or anti-FcαRI Fab were strongly effective in either preventing or attenuating CAIA or CIA in FcαRI-transgenic mice. Administration of mIgA markedly inhibited the recruitment of leukocytes to the inflamed joints of mice, which was associated with induction of SHP-1 phosphorylation in joint tissue cells. Moreover, mIgA reversed the state of inflammation in the synovial fluid of RA patients by inducing an ITAMi configuration. These results demonstrate a therapeutic potential of human mIgA in experimental arthritis. The findings support future clinical exploration of mIgA for the treatment of RA.

BIM and SHP2 expression levels to predict clinical outcome to EGFR tyrosine kinase inhibitors (TKI) in EGFR-mutant non-small-cell lung cancer (NSCLC) patients (p).

e19078 Background: EGFR mutations are among the most common driver mutations, responding well, albeit transiently, to EGFR TKIs. We were able to identify high levels of BIM mRNA expression as a predictive marker of response, progression free survival (PFS) and overall survival (OS) in erlotinib-treated NSCLC p. The Src-homology 2 domain-containing phosphatase 2 (SHP2), encoded by PTPN11, is downstream of both EGFR and several other receptor tyrosine kinases (RTK). SHP2 is required for sustained activation of extracellular signal-regulated kinase (ERK) and BIM downregulation. Methods: We assessed the static levels of BIM and SHP2 mRNA expression, by quantitative real time polymerase chain reaction, and correlated our findings with PFS and OS in 30 EGFR-mutant NSCLC p treated with EGFR TKIs like erlotinib, gefitinib or afatinib. Results: Median age 68; 73.3% female; 73.3% never-smokers; 83.3% adenocarcinoma; 80% erlotinib treated; 66.7% response rate to EGFR TKIs. PFS was 22.3 months (m) for p with intermed...

EGF augments TGFβ-induced epithelial-mesenchymal transition by promoting SHP2 binding to GAB1.

In many epithelial cells, epidermal growth factor (EGF) augments the epithelial-mesenchymal transition (EMT) that occurs when cells are treated with transforming growth factor β (TGFβ). We demonstrate that this augmentation requires activation of SH2 domain-containing phosphatase-2 (SHP2; also known as PTPN11), a proto-oncogene. In lung and pancreatic cancer cell lines, reductions in E-cadherin expression, increases in vimentin expression and increases in cell scatter rates were larger when cells were treated with TGFβ and EGF versus TGFβ or EGF alone. SHP2 knockdown promoted epithelial characteristics basally and antagonized EMT in response to TGFβ alone or in combination with EGF. Whereas EGF promoted SHP2 binding to tyrosine phosphorylated GAB1, which promotes SHP2 activity, TGFβ did not induce SHP2 association with phosphotyrosine-containing proteins. Knockdown of endogenous SHP2 and reconstitution with an SHP2 mutant with impaired phosphotyrosine binding ability eliminated the EGF-mediated EMT augmentation that was otherwise restored with wild-type SHP2 reconstitution. These results demonstrate roles for basal and ligand-induced SHP2 activity in EMT and further motivate efforts to identify specific ways to inhibit SHP2, given the role of EMT in tumor dissemination and chemoresistance.

Leukemogenic Ptpn11 allele causes defective erythropoiesis in mice.

Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by PTPN11, regulates signaling networks and cell fate in many tissues. Expression of oncogenic PTPN11 in the hematopoietic compartment causes myeloproliferative neoplasm (MPN) in humans and mice. However, the stage-specific effect(s) of mutant Ptpn11 on erythroid development have remained unknown. We found that expression of an activated, leukemogenic Ptpn11 allele, Ptpn11D61Y, specifically in the erythroid lineage causes dyserythropoiesis in mice. Ptpn11D61Y progenitors produce excess cKIT+CD71+Ter119− cells and aberrant numbers of cKITl°CD71+ erythroblasts. Mutant erythroblasts show elevated activation of ERK, AKT and STAT3 in response to EPO stimulation, and MEK inhibitor treatment blocks Ptpn11D61Y-evoked erythroid hyperproliferation in vitro. Thus, the expression of oncogenic Ptpn11 causes dyserythropoiesis in a cell-autonomous manner in vivo.

LEOPARD syndrome-associated SHP2 mutation confers leanness and protection from diet-induced obesity.

LEOPARD syndrome (multiple Lentigines, Electrocardiographic conduction abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormal genitalia, Retardation of growth, sensorineural Deafness; LS), also called Noonan syndrome with multiple lentigines (NSML), is a rare autosomal dominant disorder associating various developmental defects, notably cardiopathies, dysmorphism, and short stature. It is mainly caused by mutations of the PTPN11 gene that catalytically inactivate the tyrosine phosphatase SHP2 (Src-homology 2 domain-containing phosphatase 2). Besides its pleiotropic roles during development, SHP2 plays key functions in energetic metabolism regulation. However, the metabolic outcomes of LS mutations have never been examined. Therefore, we performed an extensive metabolic exploration of an original LS mouse model, expressing the T468M mutation of SHP2, frequently borne by LS patients. Our results reveal that, besides expected symptoms, LS animals display a strong reduction of adiposity and resistance to diet-induced obesity, associated with overall better metabolic profile. We provide evidence that LS mutant expression impairs adipogenesis, triggers energy expenditure, and enhances insulin signaling, three features that can contribute to the lean phenotype of LS mice. Interestingly, chronic treatment of LS mice with low doses of MEK inhibitor, but not rapamycin, resulted in weight and adiposity gains. Importantly, preliminary data in a French cohort of LS patients suggests that most of them have lower-than-average body mass index, associated, for tested patients, with reduced adiposity. Altogether, these findings unravel previously unidentified characteristics for LS, which could represent a metabolic benefit for patients, but may also participate to the development or worsening of some traits of the disease. Beyond LS, they also highlight a protective role of SHP2 global LS-mimicking modulation toward the development of obesity and associated disorders.

Leptin induces upregulation of sphingosine kinase 1 in oestrogen receptor-negative breast cancer via Src family kinase-mediated, janus kinase 2-independent pathway.

IntroductionObesity is a known risk factor for breast cancer. Sphingosine kinase 1 (SK1) is an oncogenic lipid kinase that is overexpressed in breast tumours and linked with poor prognosis, however, its role in obesity-driven breast cancer was never elucidated.MethodsHuman primary and secondary breast cancer tissues were analysed for SK1 and leptin receptor expression using quantitative real-time polymerase chain reaction (qRT-PCR) assay. Leptin-induced signalling was analysed in human oestrogen receptor (ER)-positive and negative breast cancer cells using Western blotting, qRT-PCR and radiolabelling assays.ResultsOur findings show for the first time that human primary breast tumours and associated lymph node metastases exhibit a strong correlation between SK1 and leptin receptor expression (Pearson R = 0.78 and R = 0.77, respectively, P <0.001). Both these genes are elevated in metastases of ER-negative patients and show a significant increase in patients with higher body mass index (BMI). Leptin induces SK1 expression and activation in ER-negative breast cancer cell lines MDAMB-231 and BT-549, but not in ER-positive cell lines. Pharmacological inhibition and gene knockdown showed that leptin-induced SK1 activity and expression are mediated by activation of extracellular signal-regulated kinases 1/2 (ERK1/2) and Src family kinase (SFK) pathways, but not by the major pathways downstream of leptin receptor (LEPR) - janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). Src-homology 2 domain-containing phosphatase 2 (SHP2) appeared to be key to SK1 activation, and may function as an adaptor protein between SFKs and LEPR. Importantly, leptin-induced breast cancer cell proliferation was abrogated by SK1-specific small interfering RNA (siRNA).ConclusionsOverall, our findings demonstrate a novel SFK/ERK1/2-mediated pathway that links leptin signalling and expression of oncogenic enzyme SK1 in breast tumours and suggest the potential significance of this pathway in ER-negative breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0426-6) contains supplementary material, which is available to authorized users.

SHP-1 is a target of regorafenib in colorectal cancer.

Regorafenib is an inhibitor of multiple protein kinases which exerts antitumor and antimetastatic activities in metastatic colorectal cancer (CRC). SH2 domain-containing phosphatase 1 (SHP-1) is reported to have tumor suppressive potential because it acts as a negative regulator of p-STAT3(Tyr705) signaling. However, little is known about the mechanism regarding regorafenib affects SHP-1 tyrosine phosphatase activity and leads to apoptosis and tumor suppression in CRC. Here, we found that regorafenib triggered apoptotic cell death and significantly enhanced SHP-1 activity, which dramatically decreased the phosphorylated form of STAT3 at Tyr705 (p-STAT3(Tyr705)). Importantly, regorafenib augmented SHP-1 activity by direct disruption of the association between N-SH2 and catalytic PTP domain of SHP-1. Deletion of the N-SH2 domain (dN1) or point mutation (D61A) of SHP-1 blocked the effect of regorafenib-induced SHP-1 activity, growth inhibition and a decrease of p-STAT3(Tyr705) expression, suggesting that regorafenib triggers a conformational change in SHP-1 by relieving its autoinhibition. In vivo assay showed that regorafenib significantly inhibited xenograft growth and decreased p-STAT3(Tyr705) expression but induced higher SHP-1 activity. Collectively, regorafenib is a novel SHP-1 agonist exerts superior anti-tumor effects by enhancing SHP-1 activity that directly targets p-STAT3(Tyr705). Small molecule-enhancement of SHP-1 activity may be a promising therapeutic approach for CRC treatment.

Tyrosine phosphatase SHP2 increases cell motility in triple-negative breast cancer through the activation of SRC-family kinases.

Tumor cell migration has a fundamental role in early steps of metastasis, the fatal hallmark of cancer. In the present study, we investigated the effects of the tyrosine phosphatase, SRC-homology 2 domain-containing phosphatase 2 (SHP2), on cell migration in metastatic triple-negative breast cancer (TNBC), an aggressive disease associated with a poor prognosis for which a targeted therapy is not yet available. Using mouse models and multiphoton intravital imaging, we have identified a crucial effect of SHP2 on TNBC cell motility in vivo. Further, analysis of TNBC cells revealed that SHP2 also influences cell migration, chemotaxis and invasion in vitro. Unbiased phosphoproteomics and biochemical analysis showed that SHP2 activates several SRC-family kinases and downstream targets, most of which are inducers of migration and invasion. In particular, direct interaction between SHP2 and c-SRC was revealed by a fluorescence resonance energy transfer assay. These results suggest that SHP2 is a crucial factor during early steps of TNBC migration to distant organs.

SC-60, a Dimer-Based Sorafenib Derivative, Shows a Better Anti–Hepatocellular Carcinoma Effect than Sorafenib in a Preclinical Hepatocellular Carcinoma Model

Sorafenib is the first approved targeted therapeutic reagent for hepatocellular carcinoma. Here, we report that SC-60, a dimer-based sorafenib derivative, overcomes the resistance of sorafenib and shows a better anti-hepatocellular carcinoma effect in vitro and in vivo. SC-60 substantially increased SH2 domain-containing phosphatase 1 (SHP-1) phosphatase activity in hepatocellular carcinoma cells and purified SHP-1 proteins, suggesting that SC-60 affects SHP-1 directly. Molecular docking and truncated mutants of SHP-1 further confirmed that SC-60 interferes with the inhibitory N-SH2 domain to relieve the closed catalytic protein tyrosine phosphatase domain of SHP-1. Deletion of N-SH2 domain (dN1) or point mutation (D61A) of SHP-1 abolished the effect of SC-60 on SHP-1, p-STAT3, and apoptosis. Importantly, SC-60 exhibited significant survival benefits compared with sorafenib in a hepatocellular carcinoma orthotopic model via targeting the SHP-1/STAT3-related signaling pathway. In summary, dimer derivative of sorafenib, SC-60, is a SHP-1 agonist and may be a potent reagent for hepatocellular carcinoma-targeted therapy.