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Purpose: This study aims to identify potential prognostic biomarkers of bladder cancer (BCa) based on large-scale multi-omics data and investigate the role of SRC in improving predictive outcomes for BCa patients and those receiving immune checkpoint therapies (ICTs)., Methods: Large-scale multi-comic data were enrolled from the Cancer Proteome Atlas, the Cancer Genome Atlas and gene expression omnibus based on machining-learning methods. Immune infiltration, survival and other statistical analyses were implemented using R software in cancers ( n  = 12,452). The predictive value of SRC was performed in 81 BCa patients receiving ICT from aa validation cohort ( n  = 81)., Results: Landscape of novel candidate prognostic protein signatures of BCa patients was identified. Differential BECLIN, EGFR, PKCALPHA, ANNEXIN1, AXL and SRC expression significantly correlated with the outcomes for BCa patients from multiply cohorts ( n  = 906). Notably, risk score of the integrated prognosis-related proteins (IPRPs) model exhibited high diagnostic accuracy and consistent predictive ability (AUC = 0.714). Besides, we tested the clinical relevance of baseline SRC protein and mRNA expression in two independent confirmatory cohorts ( n  = 566) and the prognostic value in pan-cancers. Then, we found that elevated SRC expression contributed to immunosuppressive microenvironment mediated by immune checkpoint molecules of BCa and other cancers. Next, we validated SRC expression as a potential biomarker in predicting response to ICT in 81 BCa patient from FUSCC cohort, and found that expression of SRC in the baseline tumour tissues correlated with improved survival benefits, but predicts worse ICT response., Conclusion: This study first performed the large-scale multi-omics analysis, distinguished the IPRPs ( BECLIN, EGFR, PKCALPHA, SRC, ANNEXIN1 and AXL ) and revealed novel prediction model, outperforming the currently traditional prognostic indicators for anticipating BCa progression and better clinical strategies. Additionally, this study provided insight into the importance of biomarker SRC for better prognosis, which may inversely improve predictive outcomes for patients receiving ICT and enable patient selection for future clinical treatment.

Active c-Src non-receptor tyrosine kinase localizes to the plasma membrane via N-terminal lipid modification. Membranous c-Src causes cancer initiation and progression. Even though transmembrane 4 L six family member 5 (TM4SF5), a tetraspan(in), can be involved in this mechanism, the molecular and structural influence of TM4SF5 on c-Src remains unknown. Methods: Here, we investigated molecular and structural details by which TM4SF5 regulated c-Src devoid of its N-terminus and how cell-penetrating peptides were able to interrupt c-Src activation via interference of c-Src-TM4SF5 interaction in hepatocellular carcinoma models. Results: The TM4SF5 C-terminus efficiently bound the c-Src SH1 kinase domain, efficiently to the inactively-closed form. The complex involved protein tyrosine phosphatase 1B able to dephosphorylate Tyr530. The c-Src SH1 domain alone, even in a closed form, bound TM4SF5 to cause c-Src Tyr419 and FAK Y861 phosphorylation. Homology modeling and molecular dynamics simulation studies predicted the directly interfacing residues, which were further validated by mutational studies. Cell penetration of TM4SF5 C-terminal peptides blocked the interaction of TM4SF5 with c-Src and prevented c-Src-dependent tumor initiation and progression in vivo . Conclusions: Collectively, these data demonstrate that binding of the TM4SF5 C-terminus to the kinase domain of inactive c-Src leads to its activation. Because this binding can be abolished by cell-penetrating peptides containing the TM4SF5 C-terminus, targeting this direct interaction may be an effective strategy for developing therapeutics that block the development and progression of hepatocellular carcinoma., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

To assemble the functional circuits of the nervous system, the neuronal axonal growth cones must be precisely guided to their proper targets, which can be achieved through cell-surface guidance receptor activation by ligand binding in the periphery. We investigated the function of paxillin, a focal adhesion protein, as an essential growth cone guidance intermediary in the context of spinal lateral motor column (LMC) motor axon trajectory selection in the limb mesenchyme. Using in situ mRNA detection, we first show paxillin expression in LMC neurons of chick and mouse embryos at the time of spinal motor axon extension into the limb. Paxillin loss-of-function and gain-of-function using in ovo electroporation in chick LMC neurons, of either sex, perturbed LMC axon trajectory selection, demonstrating an essential role of paxillin in motor axon guidance. In addition, a neuron-specific paxillin deletion in mice led to LMC axon trajectory selection errors. We also show that knocking down paxillin attenuates the growth preference of LMC neurites against ephrins in vitro , and erythropoietin-producing human hepatocellular (Eph)-mediated retargeting of LMC axons in vivo , suggesting paxillin involvement in Eph-mediated LMC motor axon guidance. Finally, both paxillin knockdown and ectopic expression of a nonphosphorylable paxillin mutant attenuated the retargeting of LMC axons caused by Src overexpression, implicating paxillin as a Src target in Eph signal relay in this context. In summary, our findings demonstrate that paxillin is required for motor axon guidance and suggest its essential role in the ephrin-Eph signaling pathway resulting in motor axon trajectory selection. SIGNIFICANCE STATEMENT During the development of neural circuits, precise connections need to be established among neurons or between neurons and their muscle targets. A protein family found in neurons, Eph, is essential at different stages of neural circuit formation, including nerve outgrowth and pathfinding, and is proposed to mediate the onset and progression of several neurodegenerative diseases, such as Alzheimer's disease. To investigate how Ephs relay their signals to mediate nerve growth, we investigated the function of a molecule called paxillin and found it important for the development of spinal nerve growth toward their muscle targets, suggesting its role as an effector of Eph signals. Our work could thus provide new information on how neuromuscular connectivity is properly established during embryonic development., (Copyright © 2021 the authors.)

Aside from its well-known nuclear routes of signaling, estrogen also mediates its effects through cytoplasmic signaling. Estrogen signaling involves numerous posttranslational modifications of its receptor ERα, the best known being phosphorylation. Our research group previously showed that upon estrogen stimulation, ERα is methylated on residue R260 and forms the mERα/Src/PI3K complex, central to the rapid transduction of nongenomic estrogen signals. Regulation of ERα signaling via its phosphorylation by growth factors is well recognized, and we wondered whether they could also trigger ERα methylation (mERα). Here, we found that IGF-1 treatment of MCF-7 cells induced rapid ERα methylation by the arginine methyltransferase PRMT1 and triggered the binding of mERα to IGF-1R. Mechanistically, we showed that PRMT1 bound constitutively to IGF-1R and that PRMT1 became activated upon IGF-1 stimulation. Moreover, we found that expression or pharmacological inhibition of PRMT1 impaired mERα and IGF-1 signaling. Our findings were substantiated in a cohort of breast tumors in which IGF-1R expression was positively correlated with ERα/Src and ERα/PI3K expression, hallmarks of nongenomic estrogen signaling, reinforcing the link between IGF-1R and mERα. Altogether, these results provide a new insight into ERα and IGF-1R interference, and open novel perspectives for combining endocrine therapies with PRMT1 inhibitors in ERα-positive tumors.

Lung cancer is the foremost cause of cancer related deaths in the U.S. It is a heterogeneous disease composed of genetically and phenotypically distinct tumor cells surrounded by heterotypic cells and extracellular matrix dynamically interacting with the tumor cells. Research in lung cancer is often restricted to patient-derived tumor specimens, in vitro cell cultures and limited animal models, which fail to capture the cellular or microenvironment heterogeneity of the tumor. Therefore, our knowledge is primarily focused on cancer-cell autonomous aberrations. For a fundamental understanding of lung cancer progression and an exploration of therapeutic options, we focused our efforts to develop an Ex Vivo Tumor platform to culture tumors in 3D matrices, which retains tumor cell heterogeneity arising due to in vivo selection pressure and environmental influences and recapitulate responses of tumor cells to external manipulations. To establish this model, implanted syngeneic murine tumors from a mutant KRAS/p53 model were harvested to yield multicellular tumor aggregates followed by culture in 3D extracellular matrices. Using this system, we identified Src signaling as an important driver of invasion and metastasis in lung cancer and demonstrate that EVTs are a robust experimental tool bridging the gap between conventional in vitro and in vivo models.

Glioblastoma (GBM) is the most aggressive type of brain tumor, with an overall survival of 17 months under the current standard of care therapy. CD99, an over-expressed transmembrane protein in several malignancies, has been considered a potential target for immunotherapy. To further understand this potentiality, we analyzed the differential expression of its two isoforms in human astrocytoma specimens, and the CD99 involved signaling pathways in glioma model U87MG cell line. CD99 was also analyzed in GBM molecular subtypes. Whole transcriptomes by RNA-Seq of CD99 -siRNA, and functional in vitro assays in CD99 -shRNA, that are found in U87MG cells, were performed. Astrocytoma of different malignant grades and U87MG cells only expressed CD99 isoform 1, which was higher in mesenchymal and classical than in proneural GBM subtypes. Genes related to actin dynamics, predominantly to focal adhesion, and lamellipodia/filopodia formation were down-regulated in the transcriptome analysis, when CD99 was silenced. A decrease in tumor cell migration/invasion, and dysfunction of focal adhesion, were observed in functional assays. In addition, a striking morphological change was detected in CD99 -silenced U87MG cells, further corroborating CD99 involvement in actin cytoskeleton rearrangement. Inhibiting the overexpressed CD99 may improve resectability and decrease the recurrence rate of GBM by decreasing tumor cells migration and invasion.

Many stress conditions including chemotherapy treatment is known to activate Src and under certain condition Src can induce the apoptotic signal via c-Jun N-terminal kinase (JNK) activation. Here we report that the newly synthesized β-phenylacrylic acid derivatives, MHY791 and MHY1036 (MHYs), bind to epidermal growth factor receptor (EGFR) tyrosine kinase domains and function as EGFR inhibitors, having anti-cancer activities selectively in wild-type KRAS colon cancer. Mechanistically, MHYs-induced Src/JNK activation which enhanced their pro-apoptotic effects and therefore inhibition of Src by the chemical inhibitor PP2 or Src siRNA abolished the response. In addition, MHYs generated reactive oxygen species and increased ER stress, and pretreatment with antioxidant-inhibited MHY-induced ER stress, Src activation, and apoptosis. Furthermore, the irreversible EGFR inhibitor PD168393 also activated Src while the reversible EGFR inhibitor gefitinib showed the opposite effect, indicating that MHYs are the irreversible EGFR inhibitor. Collectively, Src can play a key role in apoptosis induced by the novel EGFR inhibitor MHYs, suggesting that activation of Src might prove effective in treating EGFR/wild-type KRAS colon cancer.

Posttranslational modifications of mammalian c-Src N-terminal and C-terminal domains regulate distinct functions. Myristoylation of G 2 controls its cell membrane association and phosphorylation of Y419/Y527 controls its activation or inactivation, respectively. We provide evidence that Src-cell membrane association-dissociation and catalytic activation-inactivation are both regulated by acetylation. In EGF-treated cells, CREB binding protein (CBP) acetylates an N-terminal lysine cluster (K5, K7, and K9) of c-Src to promote dissociation from the cell membrane. CBP also acetylates the C-terminal K401, K423, and K427 of c-Src to activate intrinsic kinase activity for STAT3 recruitment and activation. N-terminal domain phosphorylation (Y14, Y45, and Y68) of STAT3 by c-Src activates transcriptionally active dimers of STAT3. Moreover, acetyl-Src translocates into nuclei, where it forms the Src-STAT3 enhanceosome for gene regulation and cancer cell proliferation. Thus, c-Src acetylation in the N-terminal and C-terminal domains play distinct roles in Src activity and regulation. Significance: CBP-mediated acetylation of lysine clusters in both the N-terminal and C-terminal regions of c-Src provides additional levels of control over STAT3 transcriptional activity. Cancer Res; 78(11); 2825-38. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Cross talk of stromal-epithelial cells plays an essential role in both normal development and tumor initiation and progression. Fibroblast growth factor (FGF)-FGF receptor (FGFR)-Src kinase axis is one of the major signal transduction pathways to mediate this cross talk. Numerous genomic studies have demonstrated that expression levels of FGFR/Src are deregulated in a variety of cancers including prostate cancer; however, the role that paracrine FGF (from stromal cells) plays in dysregulated expression of epithelial FGFRs/Src and tumor progression in vivo is not well evaluated. In this study, we demonstrate that ectopic expression of wild-type FGFR1/2 or Src kinase in epithelial cells was not sufficient to initiate prostate tumorigenesis under a normal stromal microenvironment in vivo. However, paracrine FGF10 synergized with ectopic expression of epithelial FGFR1 or FGFR2 to induce epithelial-mesenchymal transition. Additionally, paracrine FGF10 sensitized FGFR2-transformed epithelial cells to initiate prostate tumorigenesis. Next, paracrine FGF10 also synergized with overexpression of epithelial Src kinase to high-grade tumors. But loss of the myristoylation site in Src kinase inhibited paracrine FGF10-induced prostate tumorigenesis. Loss of myristoylation alters Src levels in the cell membrane and inhibited FGF-mediated signaling including inhibition of the phosphotyrosine pattern and FAK phosphorylation. Our study demonstrates the potential tumor progression by simultaneous deregulation of proteins in the FGF/FGFRs/Src signal axis and provides a therapeutic strategy of targeting myristoylation of Src kinase to interfere with the tumorigenic process., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite encouraging clinical results with sorafenib monotherapy in patients with KRAS- mutant non-small-cell lung cancer (NSCLC), the overall survival benefit of this drug is limited by the inevitable development of acquired resistance. The exact mechanism underlying acquired sorafenib resistance in KRAS -mutant NSCLC is unclear. In this study, the mechanism of acquired sorafenib resistance was explored using a biologically relevant xenograft model, which was established by using the A549 human lung adenocarcinoma cell line and an in vivo-derived, sorafenib-resistant A549 subline (A549/SRFres). Results from the initial study demonstrated that sorafenib treatment significantly decreased E-cadherin ( P < 0.05) levels but significantly increased matrix metallopeptidase 9 (MMP9) levels ( P < 0.01) in A549/SRFres tumors, whereas expression levels of phospho-protein kinase B (AKT), phospho-focal adhesion kinase (FAK), and phospho-Src were elevated in sorafenib-treated A549 and A549/SRFres tumors. We next examined whether concomitant dasatinib treatment could overcome acquired sorafenib resistance by blocking the FAK/Src escape route that mediates resistance. Despite the observed in vitro synergy between sorafenib and dasatinib, the in vivo antitumor effect of half-dose sorafenib-dasatinib combination therapy was inferior to that of the full-dose sorafenib treatment. Although the sorafenib-dasatinib combination effectively inhibited Src and AKT phosphorylation, it did not block the Y576/577-FAK phosphorylation, nor did it decrease vimentin protein expression; unexpectedly, it increased Y397-FAK phosphorylation and MMP9 protein expression in tumors. These results suggest that acquired sorafenib resistance in KRAS -mutant A549 xenografts involves the compensatory activation of FAK and Src, and Src inhibition alone is insufficient to diminish sorafenib-promoted epithelial-mesenchymal transition process and invasive potentials in tumors., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Purpose: The FAK/Src/Paxillin (PXN) axis has been implicated in malignant transformation, tumor growth, progression and metastasis. The present study aimed to assess FAK/Src/PXN protein expression in both primary and liver metastatic sites of colorectal adenocarcinoma (CRC)., Methods: FAK, Src and p-PXN expression was assessed immunohistochemically on 32 primary CRCs and their corresponding liver metastases, being also analyzed in relation with clinicopathological characteristics and patient survival., Results: FAK, Src and p-PXN expression was significantly decreased in liver metastasis compared to matched paired primary CRCs (p<0.01). Increased FAK expression in primary CRCs was significantly associated with poor histological grade and advanced disease stage (p=0.0330 and p=0.0204, respectively). Increased Src expression in primary colorectal tumors was significantly associated with the presence of lymph node metastasis (p=0.0325), while elevated p-PXN expression with poor histological grade (p=0.0284)., Conclusions: FAK, Src and p-PXN appear to play a role in the pathophysiological aspects of CRC. The lower expression of these proteins in liver metastasis compared to the primary CRC could significantly impact the choice of a novel therapeutic agent according to the disease stage.

Transgenic mice overexpressing growth hormone (GH) show increased hepatic protein content of the epidermal growth factor receptor (EGFR), which is broadly associated with cell proliferation and oncogenesis. However, chronically elevated levels of GH result in desensitization of STAT-mediated EGF signal and similar response of ERK1/2 and AKT signaling to EGF compared to normal mice. To ascertain the mechanisms involved in GH attenuation of EGF signaling and the consequences on cell cycle promotion, phosphorylation of signaling mediators was studied at different time points after EGF stimulation, and induction of proteins involved in cell cycle progression was assessed in normal and GH-overexpressing transgenic mice. Results from kinetic studies confirmed the absence of STAT3 and 5 activation and comparable levels of ERK1/2 phosphorylation upon EGF stimulation, which was associated with diminished or similar induction of c-MYC, c-FOS, c-JUN, CYCLIN D1 and CYCLIN E in transgenic compared to normal mice. Accordingly, kinetics of EGF-induced c-SRC and EGFR phosphorylation at activating residues demonstrated that activation of these proteins was lower in the transgenic mice with respect to normal animals. In turn, EGFR phosphorylation at serine 1046/1047, which is implicated in the negative regulation of the receptor, was increased in the liver of GH-overexpressing transgenic mice both in basal conditions and upon EGF stimulus. Increased basal phosphorylation and activation of the p38-mitogen-activated protein kinase might account for increased Ser 1046/1047 EGFR. Hyperphosphorylation of EGFR at serine residues would represent a compensatory mechanism triggered by chronically elevated levels of GH to mitigate the proliferative response induced by EGF., (© 2017 Society for Endocrinology.)

Epithelial-mesenchymal transition (EMT) is a biological process that allows epithelial cells to assume a mesenchymal cell phenotype. EMT is considered as a therapeutic target for several persistent inflammatory airway diseases related to tissue remodeling. Herein, we investigated the role of endoplasmic reticulum (ER) stress and c-Src in TGF- β 1-induced EMT. A549 cells, primary nasal epithelial cells (PNECs), and inferior nasal turbinate organ cultures were exposed to 4-phenylbutylic acid (4PBA) or PP2 and then stimulated with TGF- β 1. We found that E-cadherin, vimentin, fibronectin, and α -SMA expression was increased in nasal polyps compared to inferior turbinates. TGF- β 1 increased the expression of EMT markers such as E-cadherin, fibronectin, vimentin, and α -SMA and ER stress markers (XBP-1s and GRP78), an effect that was blocked by PBA or PP2 treatment. 4-PBA and PP2 also blocked the effect of TGF- β 1 on migration of A549 cells and suppressed TGF- β 1-induced expression of EMT markers in PNECs and organ cultures of inferior turbinate. In conclusion, we demonstrated that 4PBA inhibits TGF- β 1-induced EMT via the c-Src pathway in A549 cells, PNECs, and inferior turbinate organ cultures. These results suggest an important role for ER stress and a diverse role for TGF- β 1 in upper airway chronic inflammatory disease such as CRS.

The interactions of Src family kinases (SFKs) with the plasma membrane are crucial for their activity. They depend on their fatty-acylated N-termini, containing N-myristate and either a polybasic cluster (in Src) or palmitoylation sites (e.g., Fyn). To investigate the roles of these moieties in SFK membrane association, we used fluorescence recovery after photobleaching beam-size analysis to study the membrane interactions of c-Src-GFP (green fluorescent protein) or Fyn-GFP fatty-acylation mutants. Our studies showed for the first time that the membrane association of Fyn is more stable than that of Src, an effect lost in a Fyn mutant lacking the palmitoylation sites. Unexpectedly, Src-S3C/S6C (containing cysteines at positions 3/6, which are palmitoylated in Fyn) exhibited fast cytoplasmic diffusion insensitive to palmitoylation inhibitors, suggesting defective fatty acylation. Further replacement of the charged Lys-5 by neutral Gln to resemble Fyn (Src-S3C/S6C/K5Q) restored Fyn-like membrane interactions, indicating that Lys-5 in the context of Src-S3C/S6C interferes with its myristoylation/palmitoylation. This was validated by direct myristoylation and palmitoylation studies, which indicated that the residue at position 5 regulates the membrane interactions of Src versus Fyn. Moreover, the palmitoylation levels correlated with targeting to detergent-resistant membranes (rafts) and to caveolin-1. Palmitoylation-dependent preferential containment of Fyn in rafts may contribute to its lower transformation potential., (© 2016 Gottlieb-Abraham et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Background: The elusiveness of pain mechanisms is a major impediment in developing effective clinical treatments. We examined whether the signal regulatory protein α1 (SIRPα1)-activated spinal Src homology-2 domain-containing protein tyrosine phosphatase 2 (SHP2)/Src cascade and the downstream GluN2B phosphorylation play a role in inflammatory pain., Methods: At hour 3 and days 1, 3, 5, and 10 after the intraplantar injection of complete Freund adjuvant (CFA), we assessed paw withdrawal latency using the Hargreaves test and analyzed dorsal horn samples (L4-L5) by Western blotting and immunoprecipitation., Results: Intraplantar CFA injection provoked the behavioral hyperalgesia in the ipsilateral hind-paw along with SIRPα1, phosphorylated SHP2 (pSHP2), phosphorylated Src (pSrc), and phosphorylated GluN2B expressions and total SHP2 (tSHP2)-SIRPα1/pSHP2/pSrc and total Src (tSrc)-SIRPα1/pSHP2/pSrc coprecipitation in the ipsilateral dorsal horn. Although both of them failed to show an effect on CFA-enhanced SIRPα1 expression, spinal administration with SIRPα1-neutralizing antibody (10, 50, and 100 μg, 10 μL) and 8-Hydroxy-7-[(6-sulfo-2-naphthyl)azo]-5-quinolinesulfonic acid (NSC 8787; an SHP2 antagonist, 1, 10, and 100 μM, 10 μL) dose-dependently attenuated the behavioral hyperalgesia, SHP2 and Src phosphorylation, and tSHP2-SIRPα1/pSHP2/pSrc coprecipitation at day 1 after CFA injection. Intrathecal application of 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2; a Src-family kinase inhibitor, 10, 30, and 50 nM, 10 μL) exhibited a similar effect as these agents, except that it failed to ameliorate CFA-enhanced SHP2 phosphorylation and tSHP2-SIRPα1/pSHP2 coprecipitation., Conclusions: CFA-induced spinal SIRPα1 expression, which triggers SHP2, and Src phosphorylation, which subsequently induced pSrc-GluN2B interaction to mediate the GluN2B activation, contribute to spinal plasticity underlying the maintenance of inflammatory pain. These findings provide a possible strategy for pain relief by targeting to spinal SIRPα1-SHP2 coupling.

To understand the cytogenetic characteristics of acute fibrosarcoma in chickens infected with the subgroup J avian leukosis virus associated with the v-src oncogene, we performed a karyotype analysis of fibrosarcoma cell cultures. Twenty-nine of 50 qualified cell culture spreads demonstrated polyploidy of some macrochromosomes, 21 of which were trisomic for chromosome 7, and others were trisomic for chromosomes 3, 4, 5 (sex chromosome w), and 10. In addition, one of them was trisomic for both chromosome 7 and the sex chromosome 5 (w). In contrast, no aneuploidy was found for 10 macrochromosomes of 12 spreads of normal chicken embryo fibroblast cells, although aneuploidy for some microchromosomes was demonstrated in five of the 12 spreads. The cytogenetic mosaicism or polymorphism of the aneuploidy in the acute fibrosarcoma described in this study suggests that the analysed cells are polyclonal.

Background: Bone metastases (BMs) are associated with poor outcome in metastatic clear-cell renal carcinoma (m-ccRCC) treated with anti-vascular endothelial growth factor tyrosine kinase inhibitors (anti-VEGFR-TKIs). We aimed to investigate whether expression in the primary tumour of genes involved in the development of BM is associated with outcome in m-ccRCC patients treated with anti-VEGFR-TKIs., Methods: Metastatic clear-cell renal cell carcinoma patients with available fresh-frozen tumour and treated with anti-VEGFR-TKIs. Quantitative real-time PCR (qRT-PCR) for receptor activator of NF-kB (RANK), RANK-ligand (RANKL), osteoprotegerin (OPG), the proto-oncogene SRC and DKK1 (Dickkopf WNT signalling pathway inhibitor-1). Time-to-event analysis by Kaplan-Meier estimates and Cox regression., Results: We included 129 m-ccRCC patients treated between 2005 and 2013. An elevated RANK/OPG ratio was associated with shorter median time to metastasis (HR 0.50 (95% CI 0.29-0.87); P=0.014), shorter time to BM (HR 0.54 (95% CI 0.31-0.97); P=0.037), shorter median overall survival (mOS) since initial diagnosis (HR 2.27 (95% CI 1.44-3.60); P=0.0001), shorter median progression-free survival (HR 0.44 (95% CI 0.28-0.71); P=0.001) and mOS (HR 0.31 (95% CI 0.19-0.52); P<0.0001) on first-line anti-VEGFR-TKIs in the metastatic setting. Higher RANK expression was associated with shorter mOS on first-line anti-VEGFR-TKIs (HR 0.46 (95% CI 0.29-0.73); P=0.001)., Conclusions: RANK/OPG ratio of expression in primary ccRCC is associated with BM and prognosis in patients treated with anti-VEGFR-TKIs. Prospective validation is warranted.

Human monopolar spindle-one-binder 2 (hMOB2) is a member of the hMOB family of proteins, and it has been reported to regulate the nuclear-Dbf2-related kinase (NDR) activation. However, the function of hMOB2 expression in tumor cell adhesion and motility has not been addressed. Herein, the lentiviral-mediated overexpression and the knockdown of hMOB2 in HepG2 and SMMC-7721 cells was established. It was demonstrated that overexpression of hMOB2 significantly reduced the cell motility and enhanced the cell-matrix adhesion, while the hMOB2 knockdown decreased not only the cell motility, but also the cell-matrix adhesion. Immunofluorescence results showed that both hMOB2 overexpression and knockdown altered assembly of the focal adhesions and the actin cytoskeleton rearrangement. Furthermore, the focal adhesion kinase (FAK)-Src-paxillin signal pathway activated by hMOB2 was confirmed to be involved in controlling the cell motility and the cell-matrix adhesion. These results demonstrated that the altered cell-matrix adhesion and cell motility induced by hMOB2 expression was caused by the assembly of focal adhesions as well as the actin cytoskeleton rearrangement through the activation of the FAK-Src-paxillin signal pathway, unveiling a novel mechanism of cell motility and cell-matrix adhesion regulation induced by hMOB2 expression.

To characterize the molecular mechanism and map the response element used by progesterone (P) to upregulate tissue factor (TF) in breast cancer cells. TF expression and mRNA levels were analyzed in breast cancer ZR-75 and T47D cells, using Western blot and real-time PCR, respectively. Mapping of the TF promoter was performed using luciferase vectors. Progesterone receptor (PR) and specificity protein 1 (Sp1) binding to the TF promoter were analyzed by chromatin immuno precipitation assay. Specific or selective inhibitors were used for the MEK1/2 and the c-Src pathways (UO126 and PP2, respectively). TF mRNA increase peaks at 18 h following P treatment in ZR-75 and T47D cells. P upregulation occurs via a transcriptional mechanism that depends on PR and MEK1/2 activation, PR and Sp1 transcription factors bind to a region in the TF promoter that contains three Sp1 sites. TF mRNA upregulation requires an intact PR proline-rich site (mPRO), but it is independent from c-Src. TF upregulation by P is mediated by Sp1 sites in the TF promoter region. Transcriptional upregulation in breast cancer cells occurs via a new mechanism that requires MEK1/2 activation and the mPRO site but independent of c-Src activity. PR Phosphorylation at serine 294 and 345 is not essential.

Obesity and type 2 diabetes (T2D) are associated with increased breast cancer incidence and mortality, whereas carbohydrate-restricted ketogenic diets ameliorate T2D and suppress breast cancer. These observations suggest an inherent efficacy of nonesterified long-chain fatty acids (LCFA) in suppressing T2D and breast tumorigenesis. In this study, we investigated novel antidiabetic MEDICA analogues consisting of methyl-substituted LCFA that are neither β-oxidized nor esterified to generate lipids, prompting interest in their potential efficacy as antitumor agents in the context of breast cancer. In the MMTV-PyMT oncomouse model of breast cancer, in which we confirmed that tumor growth could be suppressed by a carbohydrate-restricted ketogenic diet, MEDICA treatment suppressed tumor growth, and lung metastasis, promoting a differentiated phenotype while suppressing mesenchymal markers. In human breast cancer cells, MEDICA treatment attenuated signaling through the STAT3 and c-Src transduction pathways. Mechanistic investigations suggested that MEDICA suppressed c-Src-transforming activity by elevating reactive oxygen species production, resulting in c-Src oxidation and oligomerization. Our findings suggest that MEDICA analogues may offer therapeutic potential in breast cancer and overcome the poor compliance of patients to dietary carbohydrate restriction., (©2014 American Association for Cancer Research.)

The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in the mammalian body. However, it undergoes cyclic restructuring during the epithelial cycle of spermatogenesis in which the "old" BTB located above the preleptotene spermatocytes being transported across the immunological barrier is "disassembled," whereas the "new" BTB found behind these germ cells is rapidly "reassembled," i.e., mediated by endocytic vesicle-mediated protein trafficking events. Thus, the immunological barrier is maintained when preleptotene spermatocytes connected in clones via intercellular bridges are transported across the BTB. Yet the underlying mechanism(s) in particular the involving regulatory molecules that coordinate these events remains unknown. We hypothesized that c-Src and c-Yes might work in contrasting roles in endocytic vesicle-mediated trafficking, serving as molecular switches, to effectively disassemble and reassemble the old and the new BTB, respectively, to facilitate preleptotene spermatocyte transport across the BTB. Following siRNA-mediated specific knockdown of c-Src or c-Yes in Sertoli cells, we utilized biochemical assays to assess the changes in protein endocytosis, recycling, degradation and phagocytosis. c-Yes was found to promote endocytosed integral membrane BTB proteins to the pathway of transcytosis and recycling so that internalized proteins could be effectively used to assemble new BTB from the disassembling old BTB, whereas c-Src promotes endocytosed Sertoli cell BTB proteins to endosome-mediated protein degradation for the degeneration of the old BTB. By using fluorescence beads mimicking apoptotic germ cells, Sertoli cells were found to engulf beads via c-Src-mediated phagocytosis. A hypothetical model that serves as the framework for future investigation is thus proposed., (Copyright © 2014 the American Physiological Society.)

Resistance to receptor tyrosine kinase (RTK) blockade in breast cancer is often mediated by activation of bypass pathways that sustain growth. Src and mammalian target of rapamycin (mTOR) are two intrinsic targets that are downstream of most RTKs. To date, limited clinical efficacy has been observed with either Src or mTOR inhibitors when used as single agents. Resistance to mTOR inhibitors is associated with loss of negative feedback regulation, resulting in phosphorylation and activation of AKT. Herein, we describe a novel role for Src in contributing to rapalog-induced AKT activation. We found that dual activation of Src and the mTOR pathway occurs in nearly half of all breast cancers, suggesting potential cross-talk. As expected, rapamycin inhibition of mTOR results in feedback activation of AKT in breast cancer cell lines. Addition of the Src/c-Abl inhibitor, dasatinib, completely blocks this feedback activation, confirming convergence between Src and the mTOR pathway. Analysis in vivo revealed that dual Src and mTOR inhibition is highly effective in two mouse models of breast cancer. In a luminal disease model, combined dasatinib and rapamycin is more effective at inducing regression than either single agent. Furthermore, the combination of dasatinib and rapamycin delays tumor recurrence following the cessation of treatment. In a model of human EGFR-2-positive (HER2(+)) disease, dasatinib alone is ineffective, but potentiates the efficacy of rapamycin. These data suggest that combining mTOR and Src inhibitors may provide a new approach for treating multiple breast cancer subtypes that may circumvent resistance to targeted RTK therapies., (©2014 American Association for Cancer Research.)

Protein kinases have long been reported to regulate connexins; however, little is known about the involvement of phosphatases in the modulation of intercellular communication through gap junctions and the subsequent downstream effects on cellular processes. Here, we identify an interaction between the T-cell protein tyrosine phosphatase (TC-PTP, officially known as PTPN2) and the carboxyl terminus of connexin43 (Cx43, officially known as GJA1). Two cell lines, normal rat kidney (NRK) cells endogenously expressing Cx43 and an NRK-derived cell line expressing v-Src with temperature-sensitive activity, were used to demonstrate that EGF and v-Src stimulation, respectively, induced TC-PTP to colocalize with Cx43 at the plasma membrane. Cell biology experiments using phospho-specific antibodies and biophysical assays demonstrated that the interaction is direct and that TC-PTP dephosphorylates Cx43 residues Y247 and Y265, but does not affect v-Src. Transfection of TC-PTP also indirectly led to the dephosphorylation of Cx43 S368, by inactivating PKCα and PKCδ, with no effect on the phosphorylation of S279 and S282 (MAPK-dependent phosphorylation sites). Dephosphorylation maintained Cx43 gap junctions at the plaque and partially reversed the channel closure caused by v-Src-mediated phosphorylation of Cx43. Understanding dephosphorylation, along with the well-documented roles of Cx43 phosphorylation, might eventually lead to methods to modulate the regulation of gap junction channels, with potential benefits for human health., (© 2014. Published by The Company of Biologists Ltd.)

The heterotrimeric G protein Gα13 transduces signals from G protein-coupled receptors (GPCRs) to induce cell spreading, differentiation, migration, and cell polarity. Here, we describe a novel GPCR-independent function of Gα13 in regulating the stability of endothelial cell adherens junctions (AJs). We observed that the oxidant H2O2, which is released in response to multiple proinflammatory mediators, induced the interaction of Gα13 with VE-cadherin. Gα13 binding to VE-cadherin in turn induced Src activation and VE-cadherin phosphorylation at Tyr 658, the p120-catenin binding site thought to be responsible for VE-cadherin internalization. Inhibition of Gα13-VE-cadherin interaction using an interfering peptide derived from the Gα13 binding motif on VE-cadherin abrogated the disruption of AJs in response to inflammatory mediators. These studies identify a unique role of Gα13 binding to VE-cadherin in mediating VE-cadherin internalization and endothelial barrier disruption and inflammation.

T-lymphocyte adhesion plays a critical role in both inflammatory and autoimmune responses. The small GTPase Rap1 is the key coordinator mediating T-cell adhesion to endothelial cells, antigen-presenting cells, and virus-infected cells. We describe a signaling pathway, downstream of the cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor, leading to Rap1-mediated adhesion. We identified a role for the Rap1 guanine nucleotide exchange factor C3G in the regulation of T-cell adhesion and showed that this factor is required for both T-cell receptor (TCR)-mediated and CTLA-4-mediated T-cell adhesion. Our data indicated that C3G translocates to the plasma membrane downstream of TCR signaling, where it regulates activation of Rap1. We also showed that CTLA-4 receptor signaling mediates tyrosine phosphorylation in the C3G protein, and that this is required for augmented activation of Rap1 and increased adhesion mediated by leukocyte function-associated antigen type 1 (LFA-1). Zap70 is required for C3G translocation to the plasma membrane, whereas the Src family member Hck facilitates C3G phosphorylation. These findings point to C3G and Hck as promising potential therapeutic targets for the treatment of T-cell-dependent autoimmune disorders.

Background/aims: Functional membrane androgen receptors (mARs) have recently been described in colon tumor cells and tissues. Their activation by specific testosterone albumin conjugates (TAC) down-regulates the PI-3K/Akt pro-survival signaling and triggers potent pro-apoptotic responses both, in vitro and in vivo. The present study explored the mAR-induced regulation of gene products implicated in the tumorigenic activity of Caco2 colon cancer cells., Methods: In Caco2 human colon cancer cells transcript levels were determined by RT-PCR, protein abundance and phosphorylation by Western blotting and confocal microscopy, as well as cytoskeletal architecture by confocal microscopy., Results: We report time dependent significant decrease in Tyr-416 phosphorylation of c-Src upon mAR activation. In line with the reported late down-regulation of the PI-3K/Akt pathway in testosterone-treated colon tumors, GSK-3beta was phosphorylated at Tyr-216 after long term stimulation of the cells with TAC, a finding supporting the role of this kinase to promote apoptosis. PCR analysis revealed significant decrease of beta-catenin and cyclin D1 transcript levels following TAC treatment. Moreover, confocal laser scanning microscopic analysis disclosed co-localization of beta-catenin with actin cytoskeleton. It is thus conceivable that beta-catenin may participate in the reported modulation of cytoskeletal dynamics in mAR stimulated Caco2 cells., Conclusions: Our results provide strong evidence that mAR activation regulates late expression and/or activity of the tumorigenic gene products c-Src, GSK-3beta, and beta-catenin thus facilitating the pro-apoptotic response in colon tumor cells., (© 2014 S. Karger AG, Basel.)

Elevated expression of p130Cas (Crk-associated substrate)/BCAR1 (breast cancer antiestrogen resistance 1) in human breast tumors is a marker of poor prognosis and poor overall survival. p130Cas is a downstream target of the tyrosine kinase c-Src. Signaling mediated by p130Cas through its phosphorylated substrate domain (SD) and interaction with effector molecules directly promotes tumor progression. We previously developed a constitutively phosphorylated p130Cas SD molecule, Src*/SD (formerly referred to as Src*/CasSD), which acts as decoy molecule and attenuates the transformed phenotype in v-crk-transformed murine fibroblasts and human breast cancer cells. To test the function of this molecule in vivo, we established mouse mammary tumor virus (MMTV)-long terminal repeat-Src*/SD transgenic mice in which mammary gland development and tumor formation were analyzed. Transgenic expression of the Src*/SD molecule under the MMTV-long terminal repeat promoter did not interfere with normal mammary gland development or induce tumors in mice observed for up to 11 months. To evaluate the effects of the Src*/SD molecule on tumor development in vivo, we utilized the MMTV-polyoma middle T-antigen (PyMT) murine breast cancer model that depends on c-Src. PyMT mice crossed with Src*/SD mice displayed accelerated tumor formation. The earlier onset of tumors can be explained by the interaction of the Src* domain with PyMT and targeting the fused phosphorylated SD to the membrane. At membrane compartments, it might integrate membrane-associated active signaling complexes leading to increased proliferation measured by phospho-Histone H3 staining. Although these results were unexpected, they emphasize the importance of preventing the membrane association of Src*/SD when employed as decoy molecule.

A fundamental property of tumor cells is to defy anoikis, cell death caused by a lack of cell-matrix interaction, and grow in an anchorage-independent manner. How tumor cells organize signaling molecules at the plasma membrane to sustain oncogenic signals in the absence of cell-matrix interactions remains poorly understood. Here, we describe a role for phosphatidylinositol 4-phosphate 5-kinase (PIPK) Iγi2 in controlling anchorage-independent growth of tumor cells in coordination with the proto-oncogene Src. PIPKIγi2 regulated Src activation downstream of growth factor receptors and integrins. PIPKIγi2 directly interacted with the C-terminal tail of Src and regulated its subcellular localization in concert with talin, a cytoskeletal protein targeted to focal adhesions. Co-expression of PIPKIγi2 and Src synergistically induced the anchorage-independent growth of nonmalignant cells. This study uncovers a novel mechanism where a phosphoinositide-synthesizing enzyme, PIPKIγi2, functions with the proto-oncogene Src, to regulate oncogenic signaling.

The use of a caged G-quadruplex ligand allows for transcriptional control of quadruplex-containing genes using UV light as an external trigger. An important oncogene, SRC, involved in the initiation and proliferation of epithelial tumours is shown to be significantly downregulated in cells treated by the caged ligand in synergy with UV light treatment.

Previously, our in vivo studies demonstrated that folic acid (FA) could inhibit angiogenesis and in vitro studies showed that FA reduced vascular endothelial cell proliferation through activating the cSrc/ERK-2/NFκB/p53 pathway mediated by FA receptor (FR). Here, we further examined the effect of FA on endothelial cell migration. Our results showed that FA (10 μM) inhibited the formation of lamellipodia, migration and capillary-like tube formation of human umbilical venous endothelial cells (HUVEC). These inhibition effects induced by FA treatment were not due to reduction of cell survival and cell adhesion on the collagen-coated plate. Treatment of HUVEC with FA (10 μM) increased the activity of cSrc and p190RhoGAP and decreased the activity of RhoA. Over-expression of the constitutively active RhoA construct (RhoA V14) prevented the FA-induced inhibition of migration and capillary-like tube formation in HUVEC. However, these preventive effects were abolished by pretreatment of HUVEC with a ROCK inhibitor, Y27632. Pretreatment with a cSrc inhibitor, PP2, prevented the FA-induced activation of p190GAP, reduction of the RhoA activity and migration inhibition in HUVEC. Moreover, pre-transfection with p190RhoGAP siRNA abolished the FA-induced reduction in the RhoA activity and migration inhibition in HUVEC. Taken together, our results suggest that FA might inhibit endothelial cell migration through inhibiting the RhoA activity mediated by activating the FR/cSrc/p190RhoGAP-signaling pathway. These findings further support the anti-angiogenic activity of FA., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Phosphatase of Regenerating Liver (PRL) family members have emerged as molecular markers that significantly correlate to the ability of many cancers to metastasize. However, contradictory cellular responses to PRL expression have been reported, including the inhibition of cell cycle progression. An obvious culprit for the discrepancy is the use of dozens of different cell lines, including many isolated from tumors or cultured cells selected for immortalization which may have missing or mutated modulators of PRL function. We created transgenic Drosophila to study the effects of PRL overexpression in a genetically controlled, organismal model. Our data support the paradigm that the normal cellular response to high levels of PRL is growth suppression and furthermore, that PRL can counter oncogenic activity of Src. The ability of PRL to inhibit growth under normal conditions is dependent on a CAAX motif that is required to localize PRL to the apical edge of the lateral membrane. However, PRL lacking the CAAX motif can still associate indiscriminately with the plasma membrane and retains its ability to inhibit Src function. We propose that PRL binds to other membrane-localized proteins that are effectors of Src or to Src itself. This first examination of PRL in a model organism demonstrates that PRL performs as a tumor suppressor and underscores the necessity of identifying the conditions that enable it to transform into an oncogene in cancer.

The miRNAs have great potential as biomarkers and therapeutic agents owing to their ability to control multiple genes and potential to influence cellular behavior. Here, we identified that miR-23b is a methylation-silenced tumor suppressor in prostate cancer. We showed that miR-23b expression is controlled by promoter methylation and has great promise as a diagnostic and prognostic biomarker in prostate cancer. High levels of miR-23b expression are positively correlated with higher overall and recurrence-free survival in patients with prostate cancer. Furthermore, we elucidated the tumor suppressor role of miR-23b using in vitro and in vivo models. We showed that proto-oncogene Src kinase and Akt are direct targets of miR-23b. Increased expression of miR-23b inhibited proliferation, colony formation, migration/invasion, and triggered G(0)-G(1) cell-cycle arrest and apoptosis in prostate cancer. Overexpression of miR-23b inhibited epithelial-to-mesenchymal transition (EMT) causing a decline in mesenchymal markers Vimentin and Snail and increasing the epithelial marker, E-cadherin. Depletion of Src by RNA interference conferred similar functional effects as that of miR-23b reconstitution. miR-23b expression caused a dramatic decrease in tumor growth in nude mice and attenuated Src expression in excised tumors compared with a control miR. These findings suggest that miR-23b is a methylation-silenced tumor suppressor that may be a useful biomarker in prostate cancer. Loss of miR-23b may confer proliferative advantage and promote prostate cancer migration and invasion, and reexpression of miR-23b may contribute to the epigenetic therapy for prostate cancer.

Aim: The aim of this work was to determine the pathways implicated in the mechanosensing of chondrocytes., Methods: Rat chondrocytes were cultured in collagen hydrogels of different stiffness (2-20 Pa) in normoxia and hypoxia, in monolayer and embedded inside hydrogels. First, chondrocyte were cultured on hydrogels in the presence of antibodies to block integrins. Second, custom RT-PCR array plates and western blot were used to detect changes in expression of genes implicated in downstream signalling pathways., Results: The results allowed us to demonstrate the mechanosensing of chondrocytes for changes in stiffness in the range of Pascals. We also identified Non-Muscle Myosin II (NMMII) and integrins α1, β1 and β3 as participants in the mechanosensing, since their blockade inhibits the sensing of the stiffness, and they are up-regulated in the process. RT-PCR arrays and western blot detected up-regulation of Paxillin, RhoA, Fos, Jun and Sox9. We detected no expression of Src in the monolayer cultures, but we found a role for this protein in 3D. The expression of HIF-1α was not modified under normoxia but was found to participate under hypoxia. Focal Adhesion Kinase (FAK), showed a direct relationship with the expression of Aggrecan in hypoxia and an inverse one in normoxia. Finally, immunofluorescence analysis located the expression of factors AP-1, Sox-9 and HIF-1α inside the cell nuclei and RhoA, Src, Paxillin and FAK close to the cytoplasmic membrane., Conclusions: We determined here some of the genes that are up-regulated during the process of chondrocyte mechanosensing., (Copyright © 2012 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

The present study was to determine the effect of c-SRC on the viability of human cervical cancer HeLa cells and the expression of phosphorylated signal transducer and activator of transcription-3 (p-STAT3) of the cell. Post-transfection of c-SRC RNA interference vector, RT-PCR and Western blot were utilized to observe the contents of c-SRC mRNA and protein, respectively, in HeLa cells. The MTT was used to observe the viability of the cells. Cell cycle was observed by flow cytometry. The content of p-STAT3 in the cells was also investigated after knockdown of c-SRC. Knockdown of c-SRC significantly decreased the contents of c-SRC mRNA and protein in the cells. The viability of the cells decreased by 23.1%, 29.3%, 38.6% and 45.0% (all P < 0.05), respectively, after the cells were transfected with c-SRC RNA interference vector for 24, 48, 72, and 96 h. The number of S-phase cells decreased by 5.6%, 10.0%, 15.2% and 19.9% (all P < 0.05), respectively, after transfection of c-SRC RNA interference vector for 24, 48, 72, and 96 h. The content of p-STAT3 also decreased when c-SRC was knockdowned. Compared with the control group, after treatment of HeLa cells with STAT3 inhibitor Piceatannol for 24, 48, 72, and 96 h, the cell viability decreased by 23.8%, 29.7%, 37.3% and 45.4% (all P < 0.05), respectively, while increase of c-SRC content could not reverse the inhibitory effect. These results suggest that the inhibited viability of HeLa cells caused by knockdown of c-SRC is associated with the decreased content of p-STAT3 protein.

Enterovirus 71 (EV71) has been shown to induce cyclooxygenase-2 (COX-2) expression in human neuroblastoma SK-N-SH cells through the action of MAPKs, NF-κB, and AP-1. On the other hand, the transcription factor CREB has also been implicated in the expression of COX-2 in other cell lines. Here, we report that EV71-induced COX-2 expression and PGE(2) production were both inhibited by pretreatment with the PKA inhibitor H89 or by transfection with CREB siRNA. In addition, EV71-induced COX-2 expression and c-Src/EGFR phosphorylation were both attenuated by transfection with c-Src siRNA or pretreatment with the inhibitors of c-Src (PP1) or EGF receptor (EGFR) (AG1478 and EGFR-neutralizing antibody). We also observed that EV71-induced p42/p44 MAPK phosphorylation was decreased following pretreatment with AG1478. Moreover, EV71-induced COX-2 expression was blocked by pretreatment with the p300 inhibitor GR343 or by transfection with p300 siRNA. Using immunoprecipitation and chromatin immunoprecipitation assays, we observed that EV71 stimulated the association of CREB and p300 with the COX-2 promoter region. Notably, we also demonstrated that EV71-induced COX-2 expression and PGE(2) production promoted viral replication via cAMP signaling. In summary, this study demonstrates that EV71 activates the c-Src/EGFR/p42/p44 MAPK pathway in human SK-N-SH cell, which leads to the activation of CREB/p300, and stimulates COX-2 expression and PGE(2) release., (Copyright © 2010 Wiley-Liss, Inc.)

Increasing evidence has pointed to activated type I interferon signaling in tumors. However, the molecular basis for such activation and its role in tumorigenesis remain unclear. In the current studies, we report that activation of type I interferon (IFN) signaling in tumor cells is primarily due to elevated secretion of the type I interferon, IFN-β. Studies in oncogene-transformed cells suggest that oncogenes such as Ras and Src can activate IFN-β signaling. Significantly, elevated IFN-β signaling in Ras-transformed mammary epithelial MCF-10A cells was shown to contribute to Ras transformation as evidenced by morphological changes, anchorage-independent growth, and migratory properties. Our results demonstrate for the first time that the type I IFN, IFN-β, contributes to Ras transformation and support the notion that oncogene-induced cytokines play important roles in oncogene transformation.

One of the characteristics of gliomas is a decrease in the expression of connexin43, a protein that forms gap junctions. Restoring connexin43 expression in glioma cells reduces their exacerbated rate of cell growth, although it is not yet known how connexin43 modifies the expression of genes involved in cell proliferation. Here, we show that restoring connexin43 to C6 glioma cells impedes their progression from G0/G1 to the S phase of the cell cycle by reducing retinoblastoma phosphorylation and cyclin E expression through the upregulation of p21 and p27. Interestingly, connexin43 diminishes the oncogenic activity of c-Src exhibited by glioma cells. By studying a Tyr247 and Tyr265 mutant connexin43, we show that these residues are required for connexin43 to inhibit c-Src activity and cell proliferation. In conclusion, by acting as a substrate of c-Src, connexin43 reduces its oncogenic activity and decreases the rate of glioma cell proliferation, potentially an early step in the antiproliferative effects of connexin43. Although c-Src is known to phosphorylate connexin43, this study provides the first evidence that connexin43 can also inhibit c-Src activity.

The scaffolding postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domain-containing protein melanoma differentiation associated gene-9 (MDA-9)/syntenin is a tandem PDZ protein overexpressed in human melanoma, and breast and gastric cancer cells. MDA-9/syntenin affects cancer cell motility and invasion through distinct biochemical and signaling pathways, including focal adhesion kinase and p38 mitogen-activated protein kinase (MAPK), resulting in activation of the nuclear factor (NF)-kappaB pathway. MDA-9/syntenin also promotes melanoma metastasis by activating c-Src, but how c-Src regulates NF-kappaB activation is unclear. Using a human melanoma model, we document that MDA-9/syntenin-c-Src interactions are positive regulators of NF-kappaB activation. Inhibition of c-Src by PP2 treatment, by blocking c-Src or mda-9/syntenin expression with small interfering RNA, or in c-Src (-/-) knockout cell lines, reduces NF-kappaB activation following overexpression of mda-9/syntenin or c-Src. Deletion or point mutations of the PDZ binding motif preventing MDA-9/syntenin association with c-Src reveals that both PDZ domains, with PDZ2 being the dominant module, are required for activating downstream signaling pathways, including p38 MAPK and NF-kappaB. We also document that MDA-9/syntenin-c-Src complexes functionally cooperate with NF-kappaB to promote anchorage-independent growth, motility and invasion of melanoma cells. These findings underscore PDZ domains of MDA-9/syntenin as promising potential therapeutic targets for intervening in a decisive component of cancer progression, namely, metastatic tumor spread.

A recent study from our laboratory indicated the cardioprotective ability of the tocotrienol-rich fraction (TRF) from red palm oil. The present study compared cardioprotective abilities of different isomers of tocotrienol against TRF as recently tocotrienol has been found to function as a potent neuroprotective agent against stroke. Rats were randomly assigned to one of the following groups: animals were given, by gavage, either 0.35%, 1%, or 3.5% TRF for two different periods of time (2 or 4 wk) or 0.03, 0.3, and 3 mg/kg body wt of one of the isomers of tocotrienol (alpha, gamma, or delta) for 4 wk; control animals were given, by gavage, vehicle only. After 2 or 4 wk, rats were killed, and their hearts were then subjected to 30 min of global ischemia followed by 2 h of reperfusion. Dose-response and time-response experiments revealed that the optimal concentration for TRF was 3.5% TRF and 0.3 mg/kg body wt of tocotrienol given for 4 wk. TRF as well as all the isomers of tocotrienol used in our study provided cardioprotection, as evidenced by their ability to improve postischemic ventricular function and reduce myocardial infarct size. The gamma-isoform of tocotrienol was the most cardioprotective of all the isomers followed by the alpha- and delta-isoforms. The molecular mechanisms of cardioprotection afforded by tocotrienol isoforms were probed by evaluating their respective abilities to stabilize the proteasome, allowing it to maintain a balance between prodeath and prosurvival signals. Our results demonstrated that tocotrienol isoforms reduced c-Src but increased the phosphorylation of Akt, thus generating a survival signal.

ANG II rapidly increases VEGF synthesis in proximal tubular epithelial cells through mRNA translation. The role of heterogeneous nuclear ribonucleoprotein K (hnRNP K) in ANG II regulation of VEGF mRNA translation initiation was examined. ANG II activated hnRNP K as judged by binding to poly(C)- and poly(U)-agarose. ANG II increased hnRNP K binding to VEGF mRNA at the same time as it stimulated its translation, suggesting that hnRNP K contributes to VEGF mRNA translation. Inhibition of hnRNP K expression by RNA interference significantly reduced ANG II stimulation of VEGF synthesis. ANG II increased hnRNP K phosphorylation on both tyrosine and serine residues with distinct time courses; only Ser302 phosphorylation paralleled binding to VEGF mRNA. Src inhibition using PP2 or RNA interference inhibited PKCdelta activity and prevented hnRNP K phosphorylation on both tyrosine and serine residues and its binding to VEGF mRNA. Under these conditions, ANG II-induced VEGF synthesis was inhibited. ANG II treatment induced redistribution of both VEGF mRNA and hnRNP K protein from light to heavy polysomal fractions, suggesting increased binding of hnRNP K to VEGF mRNA that is targeted for increased translation. This study shows that hnRNP K augments efficiency of VEGF mRNA translation stimulated by ANG II.

Src family tyrosine kinases are essential for neural development, but their in vivo functions remain elusive because of functional compensation among family members. To elucidate the roles of individual Src family members in vivo, we generated transgenic mice expressing the neuronal form of c-Src (n-Src), Fyn, and their constitutively active forms in cerebellar Purkinje cells using the L7 promoter. The expression of the constitutively active n-Src retarded the postnatal development of Purkinje cells and disrupted dendritic morphogenesis, whereas the wild-type n-Src had only moderate effects. Neither wild-type nor constitutively active Fyn over-expression significantly affected Purkinje-cell morphology. The aberrant Purkinje cells in n-Src transgenic mice retained multiple dendritic shafts extending in non-polarized directions and were located heterotopically in the molecular layer. Ultrastructural observation of the dendritic shafts revealed that the microtubules of n-Src transgenic mice were more densely and irregularly arranged, and had structural deformities. In primary culture, Purkinje cells from n-Src transgenic mice developed abnormally thick dendritic shafts and large growth-cone-like structures with poorly extended dendrites, which could be rescued by treatment with a selective inhibitor of Src family kinases, PP2. These results suggest that n-Src activity regulates the dendritic morphogenesis of Purkinje cells through affecting microtubule organization.

The developing neuroendocrine brain represents a potential target for endocrine active chemicals. The UV filter 4-methylbenzylidene camphor (4-MBC) exhibits estrogenic activity, but also interferes with the thyroid axis. We investigated effects of pre- and postnatal exposure to 4-MBC in the same rat offspring at brain and reproductive organ levels. 4-MBC (7, 24, 47 mg/kg/day) was administered in chow to the parent generation before mating, during gestation and lactation, and to the offspring until adulthood. mRNA of estrogen target genes involved in control of sexual behavior and gonadal functions was measured by real-time RT-PCR in ventromedial hypothalamic nucleus (VMH) and medial preoptic area (MPO) of adult offspring. 4-MBC exposure affected mRNA levels of ER alpha, progesterone receptor (PR), preproenkephalin (PPE) and insulin-like growth factor-I (IGF-I) in a sex- and region-specific manner. In order to assess possible changes in sensitivity of target genes to estrogens, offspring were gonadectomized on day 70, injected with estradiol (E2, 10 or 50 microg/kg s.c.) or vehicle on day 84, and sacrificed 6 h later. The acute induction of PR mRNA, and repression (at 6 h) of PPE mRNA by E2 was enhanced by 4-MBC in male and female VMH and female MPO, whereas male MPO exhibited reduced responsiveness of both genes. Steroid receptor coactivator SRC-1 mRNA levels were increased in female VMH and MPO. The data indicate profound sex- and region-specific alterations in the regulation of estrogen target genes at brain level. Effect patterns in baseline and E2-induced gene expression differ from those in uterus and prostate.

Bone disorders with increased osteoclastic bone resorption are frequently associated with bone pain and inhibitors of osteoclasts reduce bone pain. Osteoclasts degrade bone minerals by secreting protons through the vacuolar H+-ATPase, creating acidic microenvironments. Because acidosis is a well-known cause of pain, we reasoned that osteoclasts cause pain through proton secretion. We explored this using an animal model in which a single subcutaneous injection of the complete Freund's adjuvant (CFA) in the hind-paw caused inflammatory hyperalgesia (hyper-responsiveness to noxious stimuli). Osteoclastic bone resorption was increased in the metatarsal bones in the CFA-injected hind-paws. CFA-induced hyperalgesia was significantly suppressed by the bisphosphonates, zoledronic acid (ZOL) and alendronate and osteoprotegerin. c-src-deficient mice in which osteoclasts are inherently dysfunctional exhibited reduced CFA-induced hyperalgesia. Repeated subcutaneous injections of parathyroid hormone-related protein into the hind-paw also induced hyperalgesia with increased osteoclastic bone resorption. The hyperalgesia was associated with increased mRNA expression of acid-sensing ion channel (ASIC) 1a, 1b and 3 in the ipsi-lateral dorsal root ganglions (DRGs) by RT-PCR and c-Fos in the ipsi-lateral spinal dorsal horn by immunohistochemistry. Of note, ZOL decreased the ASIC1a mRNA expression and c-Fos. Treatment of the DRG cell line F-11 with acid (pH5.5) increased ASIC1a, 1b and 3 mRNA expression and nuclear c-Fos expression. The ASIC blocker amiloride inhibited acid-induced c-Fos expression in F-11 cells. Moreover, F-11 cells transfected with the transient receptor potential channel vanilloid subfamily member 1 (TRPV1) showed increased acid-induced nuclear c-Fos expression compared with parental F-11 cells. Finally, bafilomycin A1, an inhibitor of the vacuolar H+-ATPase, reversed the hyperalgesia and down-regulated ASIC1a mRNA expression in the DRGs. These results led us to propose that osteoclasts play a part in CFA-induced inflammatory pain through an activation of the acid-sensing receptors including ASICs and TRPV1 by creating acidosis.

Background: One of the outcomes positively associated with dioxin exposure in humans is type 2 diabetes., Objectives: This study was conducted in order to find the molecular biological evidence for the diabetogenic action of dioxin in adipose samples from Vietnam veterans., Methods: We obtained 313 adipose tissue samples both from Vietnam veterans who were exposed to dioxin (Operation Ranch Hand) and from comparison veterans who served in Southeast Asia with no record of dioxin exposure. We conducted quantitative reverse-transcribed polymerase chain reaction studies on selected marker mRNAs from these samples., Results: We found the most sensitive and reliable molecular indicator of dioxin-induced diabetes to be the ratio of mRNA of glucose transporter 4 (GLUT4) and nuclear transcription factor kappa B (NFkappaB), a marker of inflammation. This ratio showed significant correlations to serum dioxin residues and to fasting glucose among those in the Ranch Hand group and, surprisingly, even in the comparison group, who have low levels of dioxin comparable to the general public. Such a correlation in the comparison group was particularly significant among those with known risk factors such as obesity and family history of diabetes., Conclusions: These results show that the GLUT4:NFkappaB ratio is a reliable marker for the diabetogenic action of dioxin, particularly at very low exposure levels that are not much higher than those found in the general public, implying a need to address current exposure levels.

Mandibular condylar cartilage is sensitive to masticatory force, while mice lacking the c-src gene (c-src-deficient mice) have osteopetrosis and tooth eruption failure. The purpose of this study was to investigate the morphology of the mandibular condyle in these mice, which were maintained with a soft-food diet for 240 days after birth. The condylar head in the c-src-deficient mice showed slight deformity in shape before weaning, but showed remarkable undergrowth after weaning. No significant morphological or histological differences were detected between the mandibular condyle in wild-type mice fed soft food and those fed hard food, indicating that osteopetrosis, as well as abnormal masticatory force, influences the morphology of the mouse mandibular condyle, and that malocclusion rather than dietary consistency may have greater influence. After 70 days, incremental line-like striations consisting of cartilaginous and non-cartilaginous layers were detected in the mandibular condyle of the c-src-deficient mice, but not in the tibial growth plate. Immunostaining of aggrecan, collagen types II and X, and osteopontin was detected in the cartilaginous layers, but not in the non-cartilaginous layers showing collagen type I immunostaining. Chondrocyte lacunae were not eroded in the cartilaginous layers, and complete circumferential mineralisation around the lacunae and impaired osteoclast (chondroclast) function can account for this phenomenon. However, repeated cessation of chondrocyte differentiation may be required to completely explain the formation of the striations. These results indicate that the mandibular condyle in the c-src-deficient mice has unique structural features, adding to its deformity.

Background: Genetic alterations of the SRC1 gene have not been thoroughly studied in prostate cancer., Materials and Methods: Five prostate cancer cell lines and 32 xenografts were screened for mutations and gene copy number alterations. Subsequently, frequencies of detected sequence variations were further analyzed in 44 clinical prostate cancers, 6 benign prostate hyperplasias, and 48 normal controls. Finally, the protein expression of SRC1 in 254 clinical prostate tumors was investigated., Results: Three non-recurrent sequence variations, and one single nucleotide polymorphism in the coding region of SRC1, as well as one case of SRC1 gene amplification were found. The protein expression of SRC1 was higher in androgen ablation resistant than untreated prostate carcinomas, but the difference was not statistically significant (P = 0.0796)., Conclusions: Genetic alterations of SRC1 are rare in prostate cancer. The nuclear protein accumulation of SRC1 seems to be mildly increased in androgen ablation resistant prostate cancers. .

Context: A natural missense mutation in the signal transducer and activator of transcription (STAT) 5b gene was recently identified in association with a female patient presenting with severe growth failure and immune dysfunction. The mutation results in an alanine to proline substitution at residue 630 (A630P) in the src-homology-2 domain, a region essential for docking of STATs to phospho-tyrosines on activated receptors, STAT dimerization, and stabilization of phospho-STAT-DNA interactions., Objective: The purpose of this study was to explore the molecular mechanisms underlying the GH insensitivity and IGF-I deficiency caused by the A630P-mutated STAT5b., Results: In reconstitution experiments using HEK293 cells, both GH and interferon-gamma were unable to activate mutant STAT5b (A630P), as demonstrated by lack of immunodetectable phospho-tyrosyl-STAT5b (A630P) and inability to drive luciferase reporter activity. However, the Src family of nonreceptor kinases [constitutively active v-src and epithelial growth factor-induced c-src] tyrosine-phosphorylated STAT5b(A630P). The v-src-induced phospho-STAT5b(A630P) translocated to the nucleus but, unlike wild-type Stat5b, was unable to bind DNA., Conclusions: The A630P mutation disrupts the src-homology-2 architecture such that: 1) mutant STAT5b most likely cannot dock to phospho-tyrosines on ligand-activated receptors; and 2) stable interactions with DNA are prevented. Because STAT5b (A630P) is an inefficient signal transducer and transcription factor, the detrimental impact on signaling pathways important for normal growth and immunity explains, in part, the complex clinical phenotype of GH insensitivity and immune dysfunction.

Polypyrimidine-tract-binding protein (PTB) is a repressive regulator of alternative splicing. Models for PTB activity have ranged from simple binding competition with splicing factor U2AF(65) at regulated polypyrimidine tracts to looping out of repressed exons by binding of PTB to flanking sites. Structural analysis of PTB bound to RNA suggests how PTB monomers can induce loops, but two recent publications indicate that repression by PTB involves more than just binding to RNA.

Nonreceptor protein tyrosine kinases of the Src family regulate the survival, proliferation, differentiation, and motility of many cell types, but their roles in brain development are unclear. Biochemical and in vitro experiments implicate Src and Fyn in the Reelin-dependent tyrosine phosphorylation of Dab1, which controls the positioning of radially migrating neurons in many brain regions. However, genetic evidence that either Src or Fyn mediates Reelin-dependent migrations in vivo has been lacking. Here, we report that, although Src is dispensable and although the absence of Fyn causes an intermediate phenotype, the combined absence of Src and Fyn almost abolishes tyrosine phosphorylation of Dab1 and causes defects in the fetal cortex and cerebellum very similar to those of dab1 mutants of the same age. Neurogenesis is not detectably affected, but the layering of neurons in the cortex is inverted, and the formation of the Purkinje plate is impaired. This implies that Src and Fyn are needed for Reelin-dependent events during brain development.