Paxillin Is Required for Proper Spinal Motor Axon Growth into the Limb.

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.

CD99 Expression in Glioblastoma Molecular Subtypes and Role in Migration and Invasion.

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.

Activation of Focal Adhesion Kinase and Src Mediates Acquired Sorafenib Resistance in A549 Human Lung Adenocarcinoma Xenografts.

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.

Chemical Chaperone of Endoplasmic Reticulum Stress Inhibits Epithelial-Mesenchymal Transition Induced by TGF-ß1 in Airway Epithelium via the c-Src Pathway.

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-ß1-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-ß1. We found that E-cadherin, vimentin, fibronectin, and α-SMA expression was increased in nasal polyps compared to inferior turbinates. TGF-ß1 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-ß1 on migration of A549 cells and suppressed TGF-ß1-induced expression of EMT markers in PNECs and organ cultures of inferior turbinate. In conclusion, we demonstrated that 4PBA inhibits TGF-ß1-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-ß1 in upper airway chronic inflammatory disease such as CRS.

FAK, Src and p-Paxillin expression is decreased in liver metastasis of colorectal carcinoma patients.

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.

TC-PTP directly interacts with connexin43 to regulate gap junction intercellular communication.

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.

Karyotype analysis of the acute fibrosarcoma from chickens infected with subgroup J avian leukosis virus associated with v-src oncogene.

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.

SIRPα1-SHP2 Interaction Regulates Complete Freund Adjuvant-Induced Inflammatory Pain via Src-Dependent GluN2B Phosphorylation in Rats.

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.

RANK/OPG ratio of expression in primary clear-cell renal cell carcinoma is associated with bone metastasis and prognosis in patients treated with anti-VEGFR-TKIs.

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.

Phosphatidylinositol phosphate 5-kinase Iγi2 in association with Src controls anchorage-independent growth of tumor cells.

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.

Differential effects of c-Src and c-Yes on the endocytic vesicle-mediated trafficking events at the Sertoli cell blood-testis barrier: an in vitro study.

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.

Membrane androgen receptor down-regulates c-src-activity and beta-catenin transcription and triggers GSK-3beta-phosphorylation in colon tumor cells.

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.

Expression of a phosphorylated substrate domain of p130Cas promotes PyMT-induced c-Src-dependent murine breast cancer progression.

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.

Identification of signalling pathways triggered by changes in the mechanical environment in rat chondrocytes.

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, ß1 and ß3 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.

Enterovirus 71 modulates a COX-2/PGE2/cAMP-dependent viral replication in human neuroblastoma cells: role of the c-Src/EGFR/p42/p44 MAPK/CREB signaling pathway.

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.

[c-SRC knockdown decreases phosphorylated STAT3 expression and viability of HeLa cells].

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.

Novel modes of splicing repression by PTB.

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.

Multi-omics reveals novel prognostic implication of SRC protein expression in bladder cancer and its correlation with immunotherapy response.

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.

N-terminus-independent activation of c-Src via binding to a tetraspan(in) TM4SF5 in hepatocellular carcinoma is abolished by the TM4SF5 C-terminal peptide application.

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.

Binding of SH2 domains of phospholipase C gamma 1, GAP, and Src to activated growth factor receptors.

Phospholipase C gamma 1 (PLC gamma 1) and p21ras guanosine triphosphatase (GTPase) activating protein (GAP) bind to and are phosphorylated by activated growth factor receptors. Both PLC gamma 1 and GAP contain two adjacent copies of the noncatalytic Src homology 2 (SH2) domain. The SH2 domains of PLC gamma 1 synthesized individually in bacteria formed high affinity complexes with the epidermal growth factor (EGF)- or platelet derived growth factor (PDGF)-receptors in cell lysates, and bound synergistically to activated receptors when expressed together as one bacterial protein. In vitro complex formation was dependent on prior growth factor stimulation and was competed by intracellular PLC gamma 1. Similar results were obtained for binding of GAP SH2 domains to the PDGF-receptor. The isolated SH2 domains of other signaling proteins, such as p60src and Crk, also bound activated PDGF-receptors in vitro. SH2 domains, therefore, provide a common mechanism by which enzymatically diverse regulatory proteins can physically associate with the same activated receptors and thereby couple growth factor stimulation to intracellular signal transduction pathways.