Mapping of protein signaling networks within tumors can identify new targets for therapy and provide a means to stratify patients for individualized therapy. Despite advances in combination chemotherapy, the overall survival for childhood rhabdomyosarcoma remains f60%. A critical goal is to identify functionally important protein signaling defects associated with treatment failure for the 40% nonresponder cohort. Here, we show, by phosphoproteomic network analysis of microdissected tumor cells, that interlinked components of the Akt/mammalian target of rapamycin (mTOR) pathway exhibited increased levels of phosphorylation for tumors of patients with short-term survival. Specimens (n = 59) were obtained from the Children's Oncology Group Intergroup Rhabdomyosarcoma Study (IRS) IV, D9502 and D9803, with 12-year follow-up. High phosphorylation levels were associated with poor overall and poor disease-free survival: Akt Ser 473 (overall survival P < 0.001, recurrence-free survival P < 0.0009), 4EBP1 Thr 37/46 (overall survival P < 0.0110, recurrence-free survival P < 0.0106), eIF4G Ser 1108 (overall survival P < 0.0017, recurrence-free survival P < 0.0072), and p70S6 Thr 389 (overall survival P < 0.0085, recurrence-free survival P < 0.0296). Moreover, the findings support an altered interrelationship between the insulin receptor substrate (IRS-1) and Akt/mTOR pathway proteins (P < 0.0027) for tumors from patients with poor survival. The functional significance of this pathway was tested using CCI-779 in a mouse xenograft model. CCI-779 suppressed phosphorylation of mTOR downstream proteins and greatly reduced the growth of two different rhabdomyosarcoma (RD embryonal P = 0.00008; Rh30 alveolar P = 0.0002) cell lines compared with controls. These results suggest that phosphoprotein mapping of the Akt/mTOR pathway should be studied further as a means to select patients to receive mTOR/IRS pathway inhibitors before administration of chemotherapy. [Cancer Res 2007;67(7):3431-40]

Cranioplasty appears to affect postural blood flow regulation, CVR capacity, and cerebral glucose metabolism markedly. Thus, early cranioplasty is warranted to facilitate rehabilitation in patients after decompressive craniectomy.

Interferon‐alpha (IFN alpha) induces rapid tyrosine phosphorylation of its receptors, two JAK kinases and three STAT transcription factors. One kinase, p135tyk2, is complexed with the IFNaR1 receptor, and may catalyze some of these phosphorylation events. We demonstrate that, in vitro, p135tyk2 phosphorylates two tyrosines on IFNaR1. A phosphopeptide corresponding to the major phosphorylation site (Tyr466) binds STAT2, but not STAT1, in an SH‐2‐dependent manner. Furthermore, only latent, non‐phosphorylated STAT2 interacts with this phosphopeptide. When this phosphopeptide is introduced into permeabilized cells, the IFN alpha‐dependent tyrosine phosphorylation of both STATs is blocked. Finally, mutant versions of IFNaR1, in which Tyr466 is changed to phenylalanine, can act in a dominant negative manner to inhibit phosphorylation of STAT2. These observations are consistent with a model in which IFNaR1 mediates the interaction between JAK kinases and the STAT transcription factors.

Binding of type I interferons (IFNs) to their receptors induces rapid tyrosine phosphorylation of multiple proteins, including the a and jB subunits of the receptor, the polypeptides that form the transcriptional activator ISGF3a (Statll3, Stat84, and Stat9l), and the pl35'yk2 and Jak-1 tyrosine kinases. In this report, we demonstrate that the a subunit of the type I IFN receptor (IFN-R) corresponds to the product of a previously cloned receptor subunit cDNA and, further, that the pl35cyk2 tyrosine kinase directly binds and tyrosine phosphorylates this receptor subunit. Glutathione S-transferase (GST) fusion proteins encoding the different regions of the cytoplasmic domain of the a subunit can bind the pl35'yk2 contained in human cell lysates. The association between the a subunit and Tyk2 was demonstrated by immunoblotting with anti-Tyk2 and antiphosphotyrosine antibodies and by using an in vitro kinase assay. Analogous experiments were then performed with recombinant baculoviruses encoding constitutively active Jak family tyrosine kinases. In this case, pl35tk2, but not Jak-1 or Jak-2 protein, binds to the GST-IFN-R proteins, suggesting that the interaction between these two proteins is both direct and specific. We also demonstrate that Tyk2, from extracts of either IFNa-treated human cells or insect cells infected with the recombinant baculoviruses, can catalyze in vitro phosphorylation of GST-IFN-R protein in a specific manner. Deletion mutants of the GST-IFN-R protein were used to localize both the binding and tyrosine phosphorylation site(s) to a 46-amino-acid juxtamembrane region of the a subunit, which shows sequence homology to functionally similar regions of other cytokine receptor proteins. These data support the hypothesis that the Tyk2 protein functions as part of a receptor complex to initiate intracellular signaling in response to type I IFNs.

The value of rapid prototype models of the skull in our craniofacial and neurosurgical practice was analyzed. Individual skull models of 52 patients were produced by means of rapid prototyping techniques and used in various procedures. Patients were divided into three groups as follows: group I (26 patients) requiring corrective cranioplasty 1) after resection of osseous tumors (15 patients) and 2) with congenital and posttraumatic craniofacial deformities (11 patients), group II (10 patients) requiring reconstructive cranioplasty, and group III (16 patients) requiring planning of difficult skull base approaches. The utility of the stereolithographic models was assessed using the Gillespie scoring system. The esthetic and clinical outcomes were assessed by means of the esthetic outcome score and the Glasgow Outcome Score, respectively. Simulation of osteotomies for advancement plasty and craniofacial reassembly in the model before surgery in group I reduced operating time and intraoperative errors. In group II, the usefulness of the models depended directly on the size and configuration of the cranial defect. The planning of approaches to uncommon and complex skull base tumors (group III) was significantly influenced by the stereolithographic models. The esthetic outcome was pleasing. The indications for the manufacture of individual three-dimensional models could be cases of craniofacial dysmorphism that require meticulous preoperative planning and skull base surgery with difficult anatomical and reconstructive problems. The stereolithographic models provide 1) better understanding of the anatomy, 2) presurgical simulation, 3) intraoperative accuracy in localization of lesions, 4) accurate fabrication of implants, and 5) improved education of trainees.

Recent reports on the role of the membrane-cytoskeleton linker protein ezrin in sarcomas showed an effect on the formation of metastases, dependent on the level of ezrin expression. In this study, we explore the role of ezrin in Ewing's sarcoma, a frequently fatal mesenchymal neoplasm of children and young adults. Through both immunohistochemistry and Western immunoblot studies we find ubiquitous, high-level expression of ezrin in Ewing's sarcoma. In contrast to the observations in osteosarcoma and rhabdomyosarcoma, we demonstrate that inhibition of ezrin-mediated signal transduction, through the expression of a non-phosphorylatable T567A mutant, slows primary growth of Ewing's sarcoma cells in vitro. This reduction in growth is a result of increased apoptosis in the mutant expressing cells. We further show that expression of this mutant reduces the ability of Ewing's sarcoma cells to form experimental metastases in vivo. Molecular examination reveals that the action of ezrin in Ewing's sarcoma is dependent on the AKT/mTOR signal transduction cascade, but not MAP Kinase. These results, therefore, demonstrate that, in Ewing's sarcoma, the biology of ezrin is distinct from that described in other sarcomas. This study further validates ezrin as a potential therapeutic target.

Binding of alpha interferon (IFN␣) to its receptors induces rapid tyrosine phosphorylation of the receptor subunits IFNaR1 and IFNaR2, the TYK2 and JAK1 tyrosine kinases, and the Stat1 and Stat2 transcription factors. Previous studies have demonstrated that TYK2 directly and specifically binds to and tyrosine phosphorylates IFNaR1 in vitro. We now report a detailed analysis of the TYK2 binding domain on the IFNaR1 subunit. First, we used an in vitro binding assay to identify the TYK2 binding motif in IFNaR1 as well as the critical residues within this region. The most striking feature is the importance of a number of hydrophobic and acidic residues. A minor role is also ascribed to a region resembling the proline-rich ''box 1'' sequence. In addition, mutations which disrupt in vitro binding also disrupt the coimmunoprecipitation of the receptor and TYK2. We also provide direct evidence that the binding region is both necessary and sufficient to activate TYK2 in vivo. Specifically, mutations in the binding domain act in a dominant-negative fashion to inhibit the IFN␣-induced tyrosine phosphorylation of TYK2 and Stat2. Further, introduction of dimerized glutathione S-transferase-IFNaR1 fusion proteins into permeabilized cells is sufficient to induce phosphorylation of TYK2 and the receptor, confirming the role of the binding domain in IFN␣ signal transduction. These studies provide clues to the sequences determining the specificity of the association between JAK family tyrosine kinases and cytokine receptors as well as the functional role of these kinases in cytokine signal transduction.A number of polypeptide hormones initiate signal transduction by binding to their cognate receptors and stimulating intracellular protein tyrosine kinase activity (43). Although cytokine receptors do not possess intrinsic tyrosine kinase activity, rapid tyrosine phosphorylation occurs in response to cytokine treatment of cells (reviewed in references 10, 22, 40, and 46). In a number of cases, JAK family tyrosine kinases are found associated with cytokine receptors and become activated following ligand binding (3,8,54). The JAKs are a structurally distinct subfamily of nonreceptor tyrosine kinases composed of TYK2 (14), JAK1 (53), JAK2 (19), JAK3 (6,23,39,45,55), and the gene product of the Drosophila hopscotch locus (5). These kinases are proteins of 115 to 135 kDa that contain a bona fide carboxy-terminal kinase domain along with an adjacent kinase-like domain and lack both SH2 and SH3 domains. The kinase-like domain contains a number of the sequence motifs characteristic of protein kinases but lacks some of the conserved amino acids thought to be essential for protein kinase activity (18). The amino-terminal half of the JAK kinases contains five additional regions of homology and a relatively unique region present at the extreme amino terminus of the protein (19,55). Some of these amino-terminal domains are apparently involved in the association of the JAKs with members of the cytokine receptor superfamily (47, 58). However, ther...

In Abelson murine leukemia virus (A-MuLV