Modulation of alpha-catenin Tyr phosphorylation by SHP2 positively effects cell transformation induced by the constitutively active FGFR3

The Src homology 2 phosphotyrosyl phosphatase (SHP2) is a nonreceptor-type phosphatase that acts as a positive transducer of receptor Tyr kinase (RTK) signaling, particularly the Ras-REK and PI3K-Akt pathways. Recently, we have demonstrated that SHP2 is required for cell transformation induced by the constitutively active fibroblast growth factor receptor 3 (K/E-FR3) (Oncogene, 22, 6909-6918). In that study, we had detected a phosphotyrosyl protein of approximately 100 KDa (p100) in cells expressing dominant-negative SHP2 (R/E-SHP2), but its identity and relevance in SHP2-meditaed transformation was not known. Here, we report the identification of p100 as alpha-catenin, a vinculin-related protein involved in adherens junction-mediated intercellular adhesion. We show that alpha-catenin becomes Tyr phosphorylated in intercellular adhesion-dependent manner and this event is counteracted by SHP2. Substrate trapping in intact cells and immunocomplex phosphatse assays confirmed that alpha-catenin is in deed an SHP2 substrate. Tyr phosphorylation of alpha-catenin enhances its translocation to the plasma membrane and its interaction with beta-catenin, leading to enhanced actin polymerization and stabilization of adherens junction-mediated intercellular adhesion, a phenomenon commensurate with loss of the transformation phenotype. Site-directed mutagenesis studies also suggested that Tyr phosphorylation of alpha-catenin enhances its inhibitory role on cell transformation. Based on our previous work and the current report, we demonstrate that mediation of cell transformation by SHP2 is a complex process that involves modulation of the Ras-ERK and PI3K-Akt signaling pathways, intercellular adhesion, focal adhesion and actin cytoskeletal reorganization. To our knowledge, this is the first report showing regulation of alpha-catenin function by Tyr phosphorylation and its inhibitory effect on cell transformation.     

FGF signaling network in the gastrointestinal tract (review)

Fibroblast growth factor (FGF) signals are transduced through FGF receptors (FGFRs) and FRS2/FRS3- SHP2 (PTPN11)-GRB2 docking protein complex to SOS-RAS-RAF-MAPKK-MAPK signaling cascade and GAB1/GAB2-PI3K-PDK-AKT/aPKC signaling cascade. The RAS approximately MAPK signaling cascade is implicated in cell growth and differentiation, the PI3K approximately AKT signaling cascade in cell survival and cell fate determination, and the PI3K approximately aPKC signaling cascade in cell polarity control. FGF18, FGF20 and SPRY4 are potent targets of the canonical WNT signaling pathway in the gastrointestinal tract. SPRY4 is the FGF signaling inhibitor functioning as negative feedback apparatus for the WNT/FGF-dependent epithelial proliferation. Recombinant FGF7 and FGF20 proteins are applicable for treatment of chemotherapy/radiation-induced mucosal injury, while recombinant FGF2 protein and FGF4 expression vector are applicable for therapeutic angiogenesis. Helicobacter pylori, a causative pathogen for peptic ulcer diseases, chronic atrophic gastritis and gastric cancer, injects bacterial proteins into gastric epithelial cells by using Type IV secretion system, which leads to FGF signaling activation through FGF2 upregulation as well as CagA-dependent SHP2 activation. FGFR2 gene is preferentially amplified and overexpressed in diffuse-type gastric cancer. PD173074 is a small-molecule inhibitor for FGFR, while RO4396686 and SU6668 are small-molecule inhibitors for FGFR and other tyrosine kinases. Cocktail therapy using multiple protein kinase inhibitors could enhance the therapeutic effects for gastrointestinal cancer through the reduction of recurrence associated with somatic mutations of drug-target genes. Single nucleotide polymorphism (SNP) and copy number polymorphism (CNP) of genes encoding FGF signaling molecules will be identified as novel risk factors of gastrointestinal cancer. Personalized prevention and personalized medicine based on the combination of genetic screening and novel therapeutic agents could dramatically improve the prognosis of cancer patients.     

Concomitant activation of the PI3K-Akt and the Ras-ERK signaling pathways is essential for transformation by the V-SEA tyrosine kinase oncogene

V-SEA is the transforming component of S13 Avian Erythroblastosis Retrovirus that causes erythroblastosis and anemia in chicken. Like all members in the family (MET, RON, SEA), its cytosolic domain possesses two tyrosine autophosphorylation sites in the tandemly arranged bidentate motif that serve as docking sites for SH2 domain-containing proteins. Here, we investigated phosphotyrosine-dependent activation of signaling pathways and their significance in V-SEA-induced transformation and/or proliferation. We demonstrated that V-SEA activates the PI3K-Akt signaling pathway primarily in Y557- and secondarily in Y564-dependent manner. V-SEA was also shown to induce the tyrosine phosphorylation of the Gab2 protein, leading to PI3K association and thus providing an alternative route for PI3K activation. On the other hand, activation of the Ras-ERK pathway is primarily via Y564 and secondarily via Y557. A dominant-negative form of Ras inhibited V-SEA-induced ERK phosphorylation in concentration dependent manner suggesting the importance of the Grb2-Ras signaling axis in V-SEA-induced ERK activation. The biological significance of activation of the PI3K-Akt and the Ras-ERK pathways in V-SEA-induced transformation was analysed in the V-SEA-RAT1 and V-SEA-3T3 cell lines by employing specific inhibitors, LY294002 and PD98059 compounds. Both the PD and LY compounds inhibited cell growth, but only the PD compound caused reversion of the transformed phenotype. In addition, both compounds inhibited focal colony formation by the transformants in soft agar. Thus, transformation by the V-SEA oncogene is a function of the concomitant activation of, at least, the PI3K-Akt and Ras-ERK signaling pathways that regulate cell growth and morphology.     

Src activation plays an important key role in lymphomagenesis induced by FGFR1 fusion kinases

Chromosomal translocations and activation of the fibroblast growth factor (FGF) receptor 1 (FGFR1) are a feature of stem cell leukemia-lymphoma syndrome (SCLL), an aggressive malignancy characterized by rapid transformation to acute myeloid leukemia and lymphoblastic lymphoma. It has been suggested that FGFR1 proteins lose their ability to recruit Src kinase, an important mediator of FGFR1 signaling, as a result of the translocations that delete the extended FGFR substrate-2 (FRS2) interacting domain that Src binds. In this study, we report evidence that refutes this hypothesis and reinforces the notion that Src is a critical mediator of signaling from the FGFR1 chimeric fusion genes generated by translocation in SCLL. Src was constitutively active in BaF3 cells expressing exogenous FGFR1 chimeric kinases cultured in vitro as well as in T-cell or B-cell lymphomas they induced in vivo. Residual components of the FRS2-binding site retained in chimeric kinases that were generated by translocation were sufficient to interact with FRS2 and activate Src. The Src kinase inhibitor dasatinib killed transformed BaF3 cells and other established murine leukemia cell lines expressing chimeric FGFR1 kinases, significantly extending the survival of mice with SCLL syndrome. Our results indicated that Src kinase is pathogenically activated in lymphomagenesis induced by FGFR1 fusion genes, implying that Src kinase inhibitors may offer a useful option to treatment of FGFR1-associated myeloproliferative/lymphoma disorders.     

Regulation of growth factor signaling by FRS2 family docking/scaffold adaptor proteins

The FRS2 family of adaptor/scaffold proteins has two members, FRS2α and FRS2β. Both proteins contain N-terminal myristylation sites for localization on the plasma membrane and a PTB domain for binding to limited species of receptor tyrosine kinases (RTKs), including the FGF receptor, the neurotophin receptor, RET, and ALK. Activation of these RTKs allows FRS2 proteins to become phosphorylated of tyrosine residues and then bind to Grb2 and Shp2, a SH2 domain-containing adaptor and a tyrosine phosphatase, respectively. Subsequently, Shp2 activates a Ras/ERK pathway and Grb2 activates a Ras/ERK, phosphatidyl inositol (PI)-3 kinase and ubiquitination/degradation pathways by binding to SOS, Gab1, and Cbl via the SH3 domains of Grb2. FRS2α acts as 'a conning center' in FGF signaling mainly because it induces sustained levels of activation of ERK via Shp2-binding sites and Grb2-binding sites, though the contribution of the former is greater. Indeed, FRS2α knockout mice and mice with mutated Shp2-binding sites exhibit a variety of phenotypes due to defects in FGF signaling in vivo . Although FRS2β binds to the EGF receptor, it does not induce tyrosine phosphorylation on the receptor. Instead, it inhibits EGF signaling, resulting in inhibition of EGF-induced cell proliferation and cell transformation. Based on these findings, the involvement of FRS2 proteins in tumorigenesis should be studied extensively to be validated as candidate biomarkers for the effectiveness of treatments targeting RTKs such as the FGF receptor and EGF receptor. ( Cancer Sci 2008; 99: 1319–1325)     

SHP2作为抗肿瘤治疗靶点的价值及其在肿瘤免疫治疗中的应用

包含Src同源2结构域的蛋白酪氨酸磷酸酶2(Src homology region 2-containing protein tyrosine phosphatase 2,SHP2)是目前唯一被证实具有促癌作用的细胞质蛋白酪氨酸磷酸酶,在多种恶性肿瘤中高表达。SHP2可以通过介导受体酪氨酸激酶(Receptor tyrosine kinase,RTK)下游的RAS/ERK、PI3K/Akt和JAK/STAT等信号通路促进肿瘤发生发展,影响肿瘤预后。同时,SHP2参与调控PD-1/PD-L1、CTLA-4、BLTA和TIGIT等免疫检查点信号通路,对于肿瘤微环境中各种免疫细胞都有重要的调节作用。靶向SHP2不仅可以通过抑制RTK下游信号通路,还可以通过改善免疫微环境治疗恶性肿瘤。因此,SHP2具有免疫与靶向双重治疗肿瘤的功能,作为抗肿瘤治疗的靶点展现出极高的潜能与价值。     

HPV16 E5 and KGFR/FGFR2b interplay in differentiating epithelial cells

The E5 oncogenic protein of the human papillomavirus type 16 (HPV16 E5) cooperates in epithelial transformation perturbing the behaviour of differentiating suprabasal cells. Among the receptor tyrosine kinases deregulated by 16E5 expression, the key paracrine mediator of epithelial homeostasis keratinocyte growth factor receptor (KGFR/FGFR2b) is altered in its signaling and endocytic traffic in undifferentiated keratinocytes expressing 16E5 and it would represent a major target of the viral protein in differentiated cells. With the aim to specifically address the possible interplay of 16E5 with KGFR/FGFR2b in cells already committed to differentiation, we took advantage of an in vitro model for forced overexpression or depletion of KGFR in E5 expressing human keratinocytes under synchronous waves of differentiation. Quantitative RT-PCR, biochemical and immunofluorescence analysis showed that KGFR down-modulation is responsible for a E5-mediated decrease of the early differentiation marker K1 and that the receptor re-expression as well as triggering of its kinase activity and signaling are able to efficiently counteract the impairment of differentiation, providing a further demonstration of the tumor-suppressive role of KGFR in the new unexplored context of HPV16 E5-mediated carcinogenesis. In addition, KGFR induced a ligand-dependent decrease of p63 through a miR-203 independent mechanism and this effect was blocked by inhibition of the PI3K/Akt signaling, which is the main pathway involved in KGFR-dependent keratinocyte differentiation, suggesting that alterations of the KGFR/p63 crosstalk are responsible for the impairment of keratinocyte differentiation induced by 16E5 and that the opposite tumor-suppressive action of KGFR and oncogenic role of E5 might both involve p63.     

Identification of SNT/FRS2 docking site on RET receptor tyrosine kinase and its role for signal transduction

SNT/FRS2 is a lipid anchored docking protein that contains an amino-terminal myristylation signal, followed by a phosphotyrosine-binding (PTB) domain and a carboxy-terminal region with multiple tyrosine residues. Here we show that the SNT/FRS2 PTB domain binds to RET receptor tyrosine kinase activated by glial cell line-derived neurotrophic factor (GDNF) or multiple endocrine neoplasia (MEN) 2 mutations. Analyses by site directed-mutagenesis revealed that it binds to tyrosine 1062 in RET that is also known to be a binding site for the SHC adaptor protein. Whereas SHC bound to RET was associated with GRB2 and GAB1 proteins, SNT/FRS2 was associated with GRB2 only, suggesting that SNT/FRS2 is involved mainly in the activation of the RAS/mitogen activated protein kinase (MAPK) pathway but not the phosphatidylinositol 3-kinase (PI3-K)/AKT pathway. In addition, phosphorylated SNT/FRS2 appeared to directly complex with SHP-2 tyrosine phosphatase. These results suggest that tyrosine 1062 in RET provides a site for the interaction of multiple signaling molecules and that the balance of SHC and SNT/FRS2 binding may affect the nature of the intracellular signaling for cell proliferation, differentiation and survival induced by activated RET.     

Blocking of FGFR signaling inhibits breast cancer cell proliferation through downregulation of D-type cyclins

Overexpression of fibroblast growth factor receptor (FGFR) tyrosine kinases has been found in many human breast cancers and has been associated with poor patient prognosis. In order to understand the mechanism by which FGFR mediates breast cancer cell proliferation, we used a low molecular weight compound, PD173074, that selectively inhibits FGFR tyrosine kinase activity and autophosphorylation. This potential anticancer agent caused a G1 growth arrest of MDA-MB-415, MDA-MB-453 and SUM 52 breast cancer cells. Our analyses revealed that FGFR signaling links to the cell cycle machinery via D-type cyclins. PD173074-mediated inhibition of FGFR activity caused downregulation of cyclin D1 and cyclin D2 expression, inhibition of cyclin D/cdk4 activity and, as a consequence, reduction of pRB phosphorylation. Retroviral-mediated ectopic expression of cyclin D1 prevented pRB hypophosphorylation and the cell cycle G1 block in PD173074-treated cells, suggesting a central role for D cyclins in proliferation of FGFR-driven breast cancer cells. The repression of FGFR activity caused downregulation of MAPK in MDA-MB-415 and MDA-MB-453 cells. In SUM 52 cells, both MAPK and PI3K signaling pathways were suppressed. In conclusion, results shown here describe a mechanism by which FGFR promotes proliferation of breast cancer cells.     

Scaffolding protein Gab2 mediates fibroblast transformation by the SEA tyrosine kinase

Transformation of fibroblasts by V-SEA involves activation of the ERK and phosphatidylinositol 3-kinase (PI3K) pathways. Effector proteins that are key mediators of the ERK and PI3K pathways, namely Grb2, the tyrosine phosphatase, SHP2 and PI3K, interact with the two phosphotyrosines found in the bidentate motif in the carboxy-terminal region of V-SEA. Genetic analysis demonstrated that while Y557 was a primary binding site and thus activator of the PI3K-Akt pathway, Y564 also contributed to the activation of this pathway. Y564 was located within a Grb2-binding motif, this raised the possibility that a protein that associated with Grb2 might be important for this PI3K activation. The scaffolding proteins Gab1 and/or Gab2 were candidates for this role. In this report, we demonstrate that V-SEA preferentially interacts with Gab2. Furthermore by using Gab2 null fibroblasts, we demonstrate that Gab2 is essential for fibroblast transformation by V-SEA. Using mutant forms of Gab2, we show that activation of the PI3K-Akt pathway via Gab2 is required for V-SEA-induced transformation. However, efficient fibroblast transformation also requires the SHP2 interaction site on Gab2.     

支架蛋白Gab2与肿瘤关系的研究进展

Gab2是Gabs家族蛋白中一种至关重要的信号中介分子,该家族蛋白通过接受多种生长因子、细胞因子及抗原等胞外刺激,参与信号转导的放大及整合。Gab2蛋白被蛋白酪氨酸激酶磷酸化激活后,招募富含SH2结构域的信号效应蛋白如SHP2、 PI3K的p85亚基、Crk和GC-GAP,主要介导SHP2/RAS/ERK和PI3K/AKT两条经典的信号通路,在多种生理学及病理学过程中发挥重要作用。近年研究发现,异常的Gab2及其信号与乳腺癌、白血病及黑色素瘤等其他人类肿瘤密切相关。本文就Gab2蛋白的结构、信号调控及在肿瘤中的研究进展进行综述。     

Functional characterization of a novel FGFR1OP‐RET rearrangement in hematopoietic malignancies

The RET (REarranged during Transfection) receptor tyrosine kinase is targeted by oncogenic rearrangements in thyroid and lung adenocarcinoma. Recently, a RET (exon 12) rearrangement with FGFR1OP [fibroblast growth factor receptor 1 (FGFR1) oncogene partner] (exon 12) was identified in one chronic myelomonocytic leukemia (CMML) patient. We report the molecular cloning and functional characterization of a novel FGFR1OP (exon 11)‐RET (exon 11) gene fusion event (named FGFR1OP‐RET), mediated by a reciprocal translocation t(6; 10)(q27; q11), in a patient affected by primary myelofibrosis (PMF) with secondary acute myeloid leukemia (AML). The FGFR1OP‐RET fusion protein displayed constitutive tyrosine kinase and transforming activity in NIH3T3 fibroblasts, and induced IL3‐independent growth and activation of PI3K/STAT signaling in hematopoietic Ba/F3 cells. FGFR1OP‐RET supported cytokine‐independent growth, protection from stress and enhanced self‐renewal of primary murine hematopoietic progenitor and stem cells in vitro. In vivo, FGFR1OP‐RET caused a spectrum of disease phenotypes, with >50% of mice showing a fatal myeloproliferative disorder (MPD). Other phenotypes were leukemia transplantable in secondary recipients, dramatic expansion of the mast cell lineage, and reduction of repopulating activity upon lethal irradiation. In conclusion, FGFR1OP‐RET chimeric oncogenes are endowed with leukemogenic potential and associated to myeloid neoplasms (CMML and PMF/AML).     

The tyrosine phosphatase SHP2 is required for cell transformation by the receptor tyrosine kinase mutants FIP1L1-PDGFRα and PDGFRα D842V

Activated forms of the platelet derived growth factor receptor alpha (PDGFRα) have been described in various tumors, including FIP1L1-PDGFRα in patients with myeloproliferative diseases associated with hypereosinophilia and the PDGFRα^D842V mutant in gastrointestinal stromal tumors and inflammatory fibroid polyps.To gain a better insight into the signal transduction mechanisms of PDGFRα oncogenes, we mutated twelve potentially phosphorylated tyrosine residues of FIP1L1-PDGFRα and identified three mutations that affected cell proliferation. In particular, mutation of tyrosine 720 in FIP1L1-PDGFRα or PDGFRα^D842V inhibited cell growth and blocked ERK signaling in Ba/F3 cells. This mutation also decreased myeloproliferation in transplanted mice and the proliferation of human CD34⁺ hematopoietic progenitors transduced with FIP1L1-PDGFRα. We showed that the non-receptor protein tyrosine phosphatase SHP2 bound directly to tyrosine 720 of FIP1L1-PDGFRα. SHP2 knock-down decreased proliferation of Ba/F3 cells transformed with FIP1L1-PDGFRα and PDGFRα^D842V and affected ERK signaling, but not STAT5 phosphorylation. Remarkably, SHP2 was not essential for cell proliferation and ERK phosphorylation induced by the wild-type PDGF receptor in response to ligand stimulation, suggesting a shift in the function of SHP2 downstream of oncogenic receptors.In conclusion, our results indicate that SHP2 is required for cell transformation and ERK activation by mutant PDGF receptors.     

Regulation of CXCR4-mediated chemotaxis and chemoinvasion of breast cancer cells

The chemokine-CXCL12 and its receptor, CXCR4, have recently been shown to play an important role in regulating the directional migration of breast cancer cells to sites of metastasis. In the present study, we showed that CXCL12 enhanced the chemotaxis, chemoinvasion and adhesive properties of breast cancer cells; parameters that are critical for development of metastasis. We have also evaluated the signaling mechanisms that regulate CXCL12-induced and CXCR4-mediated breast cancer cell motility and invasion. These studies revealed that CXCL12 induces the tyrosine phosphorylation of focal adhesion kinase (FAK) at residues 397 and 577, and of RAFTK/Pyk2 at residues 402 and 579/580. The cytoskeletal proteins paxillin and Crk, as well as tyrosine phosphatase SHP2 and adaptor protein Cbl, were also phosphorylated. CXCL12 induced the activation of PI 3-kinase, and increased its association with Cbl and SHP2. PI 3-kinase, RAFTK/Pyk2 and tyrosine phosphatase inhibitors significantly blocked CXCL12-induced chemotaxis and chemoinvasion. The role of SHP2 and Cbl in CXCL12-induced chemotaxis and chemoinvasion in breast cancer cells was further defined by transiently overexpressing wild-type SHP2, wild-type Cbl, dominant-negative SHP2, Cbl mutants 70Z/3 and G306E or double transfectants of the Cbl and SHP2 constructs. We found a novel role of Cbl in CXCL12-induced chemotaxis, which may be mediated through the activation and formation of a multimeric complex comprised of Cbl, SHP2 and PI 3-kinase. We also observed the activation of matrix metalloproteinases 2 and 9 upon CXCL12 stimulation. These studies provide new information regarding signaling pathways that may regulate CXCL12-induced metastasis in breast cancer cells.     

Inhibition of EGFR-mediated phosphoinositide-3-OH kinase (PI3-K) signaling and glioblastoma phenotype by signal-regulatory proteins (SIRPs)

Several growth factors and cytokines, including EGF, are known to induce tyrosine phosphorylation of Signal Regulatory Proteins (SIRPs). Consistent with the idea that increased phosphorylation activates SIRP function, we overexpressed human SIRPalpha1 in U87MG glioblastoma cells in order to examine how SIRPalpha1 modulates EGFR signaling pathways. Endogenous EGFR proteins are overexpressed in U87MG cells and these cells exhibit survival and motility phenotypes that are influenced by EGFR kinase activity. Overexpression of the SIRPalpha1 cDNA diminished EGF-induced phosphoinositide-3-OH kinase (PI3-K) activation in U87MG cells. Reduced EGF-stimulated activation of PI3-K was mediated by interactions between carboxyl terminus of SIRPalpha1 and the Src homology-2 (SH2)-containing phosphotyrosine phosphatase, SHP2. SIRPalpha1 overexpression also reduced the EGF-induced association between SHP2 and the p85 regulatory subunit of PI3-K. Inhibition of transformation and enhanced apoptosis following gamma-irradiation were observed in SIRPalpha1-overexpressing U87MG cells, and enhanced apoptosis was associated with reduced levels of bcl-xL protein. Furthermore, SIRPalpha1-overexpressing U87MG cells displayed reduced cell migration and cell spreading that was mediated by association between SIRPalpha1 and SHP2. However, SIRPalpha1-overexpressing U87MG clonal derivatives exhibited no differences in cell growth or levels of mitogen-activated protein kinase (MAPK) activation. These data reveal a pathway that negatively regulates EGFR-induced PI3-K activation in glioblastoma cells and involves interactions between SHP2 and tyrosine phosphorylated SIRPalpha1. These results also suggest that negative regulation of PI3-K pathway activation by the SIRP family of transmembrane receptors may diminish EGFR-mediated motility and survival phenotypes that contribute to transformation of glioblastoma cells. Oncogene (2000) 19, 3999 - 4010.     

靶向FGFR4的抗肿瘤药物研究进展

 成纤维细胞生长因子受体4（FGFR4）通过启动STAT3、MAPK和PI3K/AKT等信号级联,调节细胞的增殖、分化和转移。其基因突变、过表达或其配体分子过表达等异常信号转导在某些肿瘤的发生发展中发挥着重要的作用,表明FGFR4是此类肿瘤潜在的治疗靶标。靶向FGFR4的抗肿瘤药物或方法主要包括小分子抑制剂、单克隆抗体、配体捕获蛋白、短链RNA寡核苷酸适配体（shRNA）。本文回顾FGFR4异常的肿瘤及靶向FGFR4抗肿瘤药物的研究进展。