Interleukin-6 (IL-6) expression is strongly correlated with the degree of human glioma malignancy and necessary for tumor formation in a mouse model of spontaneous astrocytomas. Yet, exactly how IL-6 contributes to malignant progression of these brain tumors is still unclear. We have scrutinized the mechanism of transcriptional activation of vascular endothelial growth factor (VEGF) expression by IL-6 in the mouse brain and in glioblastoma cells. We demonstrate here that IL-6 drives transcriptional upregulation of VEGF in astrocytes in vivo using glial fibrillary acidic protein (GFAP)-IL-6/VEGF-green fluorescent protein (GFP) double transgenic mice. We further show that IL-6-induced VEGF transcription and VEGF secretion by human glioblastoma cells is dependent on signal transducer and activator of transcription 3 (STAT3). By progressive 5 0 -deletion analysis we defined the minimal VEGF promoter region for IL-6-responsiveness to nucleotides 288/250. Surprisingly, this promoter region is rich in GC-boxes and does not contain STAT3 binding elements. Electrophoretic mobility shift and supershift assays revealed binding of Sp1 and Sp3 to the 288/250 element upon IL-6 stimulation. Interestingly, preincubation with STAT3 antibody prevented the binding of Sp1 and Sp3 to the 288/250 element, indicating that STAT3 is involved in IL-6-driven Sp1/Sp3 protein-DNA complex formation. Physical interaction of STAT3 and Sp1 was demonstrated by coimmunoprecipitation. The functional relevance of the STAT3/ Sp1 association was corroborated by transient transfection experiments, which showed that overexpression of constitutively active STAT3 increased the minimal VEGF promoter activity. Taken together, our study suggests that IL-6 promotes tumor angiogenesis in gliomas and describes a novel transcriptional activation mechanism for STAT3 in the context of a STAT3 binding element (SBE)-free promoter. ' 2005 Wiley-Liss, Inc.

The pleiotropic cytokine interleukin-6 (IL-6) contributes to malignant progression and apoptosis resistance of various cancer types. Although IL-6 is elevated in malignant gliomas, and glioma cells respond to IL-6, its functional role in gliomagenesis is unclear. We have investigated this role of IL-6 in a mouse model of spontaneous astrocytoma by crossbreeding glial fibrillary acidic protein (GFAP)-viral src oncogene transgenic mice with IL-6-deficient mice. We show here that ablation of IL-6 prevents tumour formation in these predisposed animals, but did not affect preneoplastic astrogliosis. Moreover, we demonstrate phosphorylation and nuclear translocation of the transcription factor signal transducer and activator of transcription (STAT)3, a prerequisite for IL-6 signalling, in 51 human gliomas WHO grade II-IV and all experimental mouse tumours investigated. Together with the observation that STAT3 activation increases with malignancy, these findings indicate an important role for IL-6 in the development and malignant progression of astrocytomas.

Neural cell adhesion molecule (NCAM) mediates cell-cell adhesion and signaling in the nervous system, yet NCAM is also expressed in non-neural tissues, in which its function has in most parts remained elusive. We have previously reported that NCAM stimulates cell-matrix adhesion and neurite outgrowth by activating fibroblast growth factor receptor (FGFR) signaling. Here, we investigated whether the interplay between NCAM and FGFR has any impact on the response of FGFR to its classical ligands, FGFs. To this end, we employed two fibroblast cell lines, NCAM-negative L cells and NCAM-positive NIH-3T3 cells, in which the expression of NCAM was manipulated by means of transfection or RNAi technologies, respectively. The results demonstrate that NCAM expression reduces FGF-stimulated ERK1/2 activation, cell proliferation and cell-matrix adhesion, in both L and NIH-3T3 cells. Furthermore, our data show that NCAM inhibits the binding of FGF to its high-affinity receptor in a competitive manner, providing the mechanisms for the NCAM-mediated suppression of FGF function. In this context, a small peptide that mimics the binding of NCAM to FGFR was sufficient to block FGF-dependent cell proliferation. These findings point to NCAM as being a major regulator of FGF-FGFR interaction, thus introducing a novel type of control mechanism for FGFR activity and opening new therapeutic perspectives for those diseases characterized by aberrant FGFR function.

The neurotrophin brain-derived neurotrophic factor (BDNF) binds to two cell surface receptors: TrkB receptors that promote neuronal survival and differentiation and p75NTR that induces apoptosis or survival. BDNF, as well as the other members of the neurotrophin family, is synthesized as a larger precursor, pro-BDNF, which undergoes posttranslational modifications and proteolytic processing by furin or related proteases. Both mature neurotrophins and uncleaved proneurotrophins are secreted from cells. The bioactivities of proneurotrophins could differ from those of mature, cleaved neurotrophins; therefore, we wanted to test whether pro-BDNF would differ from mature BDNF in its neurotrophin receptor binding and activation. A furin-resistant pro-BDNF, secreted from COS-7 cells, bound to TrkB-Fc and p75NTR-Fc, but not to TrkA-Fc or TrkC-Fc. Likewise, pro-BDNF elicited prototypical TrkB responses in biological assays, such as TrkB tyrosine phosphorylation, activation of ERK1/2, and neurite outgrowth. Moreover, mutation of the R103 residue of pro-BDNF abrogated its binding to TrkB-Fc but not to p75NTR-Fc. Taken together, these data indicate that pro-BDNF binds to and activates TrkB and could be involved in TrkB-mediated neurotrophic activity in vivo.

Objective: We sought to define in colorectal cancer (CRC) the function of Shoca-2 (a.k.a. SH2D4A), an until now uncharacterized SH2 domain-containing protein. Methods: Tandem affinity purification, immunoprecipitation, site-specific mutagenesis, shRNA expression targeting and gene expression analysis were used to characterize binding partners of human Shoca-2 and identify the signaling pathway(s) where this adapter protein is involved. To assess Shoca-2 function on cell growth, colony formation assays and cell cycle analyses were carried out. CRC biopsies were analyzed by immunohistochemistry and molecular genetic methods for the presence of Shoca-2. Results: Here we demonstrate that Shoca-2 forms a complex with EGFR and STAT3. In response to EGF signaling, a Shoca-2/STAT3 dimer dissociates from EGFR and recruits the serine/threonine phosphatase PP1beta to the complex. Consequently, STAT3 activity is repressed and the EGF signaling pathway ceases to be activated. A lack of Shoca-2 expression secondary to a genomic loss of the locus is frequently observed in metastatic colorectal cancer and correlates with an increase in phosphorylated STAT3 in these cells. Conversely, the ectopic expression of wild type Shoca-2 but not of a mutant form defective in its ability to bind to PP1beta suppresses STAT3-dependent tumor cell growth. Conclusion: Our results identify Shoca-2 as a tumor suppressor that acts upon EGF stimulation as an adaptor protein to target STAT3 for dephosphorylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3081.