Identification and functional validation of SRC and RAPGEF1 as new direct targets of miR-203, involved in regulation of epidermal homeostasis.

The epidermis is mostly composed of keratinocytes and forms a protecting barrier against external aggressions and dehydration. Epidermal homeostasis is maintained by a fine-tuned balance between keratinocyte proliferation and differentiation. In the regulation of this process, the keratinocyte-specific miR-203 microRNA is of the outmost importance as it promotes differentiation, notably by directly targeting and down-regulating mRNA expression of genes involved in keratinocyte proliferation, such as ΔNp63, Skp2 and Msi2. We aimed at identifying new miR-203 targets involved in the regulation of keratinocyte proliferation/differentiation balance. To this end, a transcriptome analysis of human primary keratinocytes overexpressing miR-203 was performed and revealed that miR-203 overexpression inhibited functions like proliferation, mitosis and cell cycling, and activated differentiation, apoptosis and cell death. Among the down-regulated genes, 24 putative target mRNAs were identified and 8 of them were related to proliferation. We demonstrated that SRC and RAPGEF1 were direct targets of miR-203. Moreover, both were down-regulated during epidermal morphogenesis in a 3D reconstructed skin model, while miR-203 was up-regulated. Finally silencing experiments showed that SRC or RAPGEF1 contributed to keratinocyte proliferation and regulated their differentiation. Preliminary results suggest their involvement in skin carcinoma hyperproliferation. Altogether this data indicates that RAPGEF1 and SRC could be new mediators of miR-203 in epidermal homeostasis regulation.

Src42A is required for E-cadherin dynamics at cell junctions during Drosophila axis elongation.

Src kinases are important regulators of cell adhesion. Here, we have explored the function of Src42A in junction remodelling during Drosophila gastrulation. Src42A is required for tyrosine phosphorylation at bicellular (bAJ) and tricellular (tAJ) junctions in germband cells, and localizes to hotspots of mechanical tension. The role of Src42A was investigated using maternal RNAi and CRISPR-Cas9-induced germline mosaics. We find that, during cell intercalations, Src42A is required for the contraction of junctions at anterior-posterior cell interfaces. The planar polarity of E-cadherin is compromised and E-cadherin accumulates at tricellular junctions after Src42A knockdown. Furthermore, we show that Src42A acts in concert with Abl kinase, which has also been implicated in cell intercalations. Our data suggest that Src42A is involved in two related processes: in addition to establishing tension generated by the planar polarity of MyoII, it may also act as a signalling factor at tAJs to control E-cadherin residence time.

Hippo signaling suppresses tumor cell metastasis via a Yki-Src42A positive feedback loop.

Metastasis is an important cause of death from malignant tumors. It is of great significance to explore the molecular mechanism of metastasis for the development of anti-cancer drugs. Here, we find that the Hippo pathway hampers tumor cell metastasis in vivo. Silence of hpo or its downstream wts promotes tumor cell migration in a Yki-dependent manner. Furthermore, we identify that inhibition of the Hippo pathway promotes tumor cell migration through transcriptional activating src42A, a Drosophila homolog of the SRC oncogene. Yki activates src42A transcription through direct binding its intron region. Intriguingly, Src42A further increases Yki transcriptional activity to form a positive feedback loop. Finally, we show that SRC is also a target of YAP and important for YAP to promote the migration of human hepatocellular carcinoma cells. Together, our findings uncover a conserved Yki/YAP-Src42A/SRC positive feedback loop promoting tumor cell migration and provide SRC as a potential therapeutic target for YAP-driven metastatic tumors.

Genetic Biomarkers in Chronic Myeloid Leukemia: What Have We Learned So Far?

Chronic Myeloid Leukemia (CML) is a rare malignant proliferative disease of the hematopoietic system, whose molecular hallmark is the Philadelphia chromosome (Ph). The Ph chromosome originates an aberrant fusion gene with abnormal kinase activity, leading to the buildup of reactive oxygen species and genetic instability of relevance in disease progression. Several genetic abnormalities have been correlated with CML in the blast phase, including chromosomal aberrations and common altered genes. Some of these genes are involved in the regulation of cell apoptosis and proliferation, such as the epidermal growth factor receptor (EGFR), tumor protein p53 (TP53), or Schmidt-Ruppin A-2 proto-oncogene (SRC); cell adhesion, e.g., catenin beta 1 (CTNNB1); or genes associated to TGF-ß, such as SKI like proto-oncogene (SKIL), transforming growth factor beta 1 (TGFB1) or transforming growth factor beta 2 (TGFB2); and TNF-α pathways, such as Tumor necrosis factor (TNFA) or Nuclear factor kappa B subunit 1 (NFKB1). The involvement of miRNAs in CML is also gaining momentum, where dysregulation of some critical miRNAs, such as miRNA-451 and miRNA-21, which have been associated to the molecular modulation of pathogenesis, progression of disease states, and response to therapeutics. In this review, the most relevant genomic alterations found in CML will be addressed.

Qihuzha granule attenuated LPS-induced acute spleen injury in mice via Src/MAPK/Stat3 signal pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Qihuzha granule (QHZG), is one of traditional Chinese patent medicines composed of eleven edible medicinal plant, which has been used in the clinic for the treatment of indigestion and anorexia in children caused by deficiency of the spleen and stomach. Yet it is noteworthy that QHZG has therapeutic effect on recurrent respiratory tract infection (RRTI) in children. However, its potential molecular mechanisms remained unclear. AIM OF THE STUDY: The aim of this study was to investigate the therapeutic effect and potential mechanism of QHZG on lipopolysaccharide (LPS) induced acute spleen injury. MATERIALS AND METHODS: The acute spleen injury model was induced by intraperitoneal injection of LPS (10 mg/kg) and safe doses of QHZG was administered by gavage once a day for 23 days before LPS treatment. Serum inflammatory cytokines including interleukin-2 (IL-2), IL-1ß, IFN-γ, and tumor necrosis factor-α (TNF-α) were tested by ELISA. Related protein levels were detected by Western blotting. Hematoxylin-eosin (HE) staining was employed to observe the histological alterations. The distribution of macrophages and neutrophils in the mouse spleen was examined by immunofluorescence analysis. RESULTS: QHZG pretreatment significantly abolished the increased secretion of cytokines such as interleukin-2 (IL-2), IL-1ß, IFN-γ, and tumor necrosis factor-α (TNF-α), which were attributable to LPS treatment. Immunofluorescence staining and Histological analysis of spleen tissue revealed the protective effect of QHZG against LPS-induced acute spleen injury in mice. Further study indicated that pretreatment with QHZG significantly inhibited LPS-induced phosphorylation of Src. Accordingly, the increased phosphorylation of Src downstream components (JNK, ERK, P38 and STAT3) induced by LPS was remarkably diminished by QHZG, suggesting the involvement of Src/MAPK/STAT3 pathway in the inhibitory effects of QHZG on spleen injury in mice. CONCLUSION: Our study demonstrated that QHZG protected mice from LPS-induced acute spleen injury via inhibition of Src/MAPK/Stat3 signal pathway. These results suggested that QHZG might serve as a new drug for the treatment of LPS-stimulated spleen injury.

MiR-137 promotes cell growth and inhibits extracellular matrix protein expression in H2O2-induced human trabecular meshwork cells by targeting Src.

Glaucoma is a progressive optic neuropathy in more than 25 % of cases in patients with permanent blindness. The microRNA is implicated in modulating the cellular function of the trabecular meshwork (TM). The aim of this study is to investigate the role of miR-137 in glaucoma and illustrate the potential molecular mechanisms. We show that miR-137 was down-regulated in H2O2-induced human trabecular meshwork cells (HTMCs), and overexpression of miR-137 attenuated H2O2-induced cell growth inhibition, apoptosis and elevated extracellular matrix (ECM) protein expression. In addition, miR-137 blocked the activation of YAP/TAZ by directly targeting src. Overexpression of src or activation of the YAP/TAZ pathway partly abrogated the effects of miR-137 on H2O2-induced cell viability and apoptosis and dampened the inhibition effect on ECM protein expression. In conclusion, miR-137 promotes cell growth and inhibits extracellular matrix protein expression in H2O2-induced human trabecular meshwork cells via the YAP/TAZ pathway by targeting src. Hence, miR-137 might be used as a novel therapeutic target to treat glaucoma.

Methionine restriction breaks obligatory coupling of cell proliferation and death by an oncogene Src in Drosophila.

Oncogenes often promote cell death as well as proliferation. How oncogenes drive these diametrically opposed phenomena remains to be solved. A key question is whether cell death occurs as a response to aberrant proliferation signals or through a proliferation-independent mechanism. Here, we reveal that Src, the first identified oncogene, simultaneously drives cell proliferation and death in an obligatorily coupled manner through parallel MAPK pathways. The two MAPK pathways diverge from a lynchpin protein Slpr. A MAPK p38 drives proliferation whereas another MAPK JNK drives apoptosis independently of proliferation signals. Src-p38-induced proliferation is regulated by methionine-mediated Tor signaling. Reduction of dietary methionine uncouples the obligatory coupling of cell proliferation and death, suppressing tumorigenesis and tumor-induced lethality. Our findings provide an insight into how cells evolved to have a fail-safe mechanism that thwarts tumorigenesis by the oncogene Src. We also exemplify a diet-based approach to circumvent oncogenesis by exploiting the fail-safe mechanism.

SLAP2 Adaptor Binding Disrupts c-CBL Autoinhibition to Activate Ubiquitin Ligase Function.

CBL is a RING type E3 ubiquitin ligase that functions as a negative regulator of tyrosine kinase signaling and loss of CBL E3 function is implicated in several forms of leukemia. The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL and are required for CBL-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling. Despite the established role of SLAP/SLAP2 in regulating CBL activity, the nature of the interaction and the mechanisms involved are not known. To understand the molecular basis of the interaction between SLAP/SLAP2 and CBL, we solved the crystal structure of CBL tyrosine kinase binding domain (TKBD) in complex with SLAP2. The carboxy-terminal region of SLAP2 adopts an α-helical structure which binds in a cleft between the 4H, EF-hand, and SH2 domains of the TKBD. This SLAP2 binding site is remote from the canonical TKBD phospho-tyrosine peptide binding site but overlaps with a region important for stabilizing CBL in its autoinhibited conformation. In addition, binding of SLAP2 to CBL in vitro activates the ubiquitin ligase function of autoinhibited CBL. Disruption of the CBL/SLAP2 interface through mutagenesis demonstrated a role for this protein-protein interaction in regulation of CBL E3 ligase activity in cells. Our results reveal that SLAP2 binding to a regulatory cleft of the TKBD provides an alternative mechanism for activation of CBL ubiquitin ligase function.

Characterization of the interactome of c-Src within the mitochondrial matrix by proximity-dependent biotin identification.

C-Src kinase is localized in several subcellular compartments, including mitochondria where it is involved in the regulation of organelle functions and overall metabolism. Surprisingly, the characterization of the intramitochondrial Src interactome has never been fully determined. Using in vitro proximity-dependent biotin identification (BioID) coupled to mass spectrometry, we identified 51 candidate proteins that may interact directly or indirectly with c-Src within the mitochondrial matrix. Pathway analysis suggests that these proteins are involved in a large array of mitochondrial functions such as protein folding and import, mitochondrial organization and transport, oxidative phosphorylation, tricarboxylic acid cycle and metabolism of amino and fatty acids. Among these proteins, we identified 24 tyrosine phosphorylation sites in 17 mitochondrial proteins (AKAP1, VDAC1, VDAC2, VDAC3, LonP1, Hsp90, SLP2, PHB2, MIC60, UBA1, EF-Tu, LRPPRC, ACO2, OAT, ACAT1, ETFß and ATP5ß) as potential substrates for intramitochondrial Src using in silico prediction of tyrosine phospho-sites. Interaction of c-Src with SLP2 and ATP5ß was confirmed using coimmunoprecipitation. This study suggests that the intramitochondrial Src could target several proteins and regulate different mitochondrial functions.

UV radiation resistance-associated gene (UVRAG) promotes cell proliferation, migration, invasion by regulating cyclin-dependent kinases (CDK) and integrin-ß/Src signaling in breast cancer cells.

Breast cancer is a highly heterogeneous group of human cancer with distinct genetic, biological and clinicopathological features. Triple-negative breast cancer (TNBC) is the most aggressive and metastatic type of breast cancer and associated with poor patient survival. However, the role of UV Radiation Resistance-Associated Gene (UVRAG) in TNBC remains unknown. Here, we report that UVRAG is highly upregulated in all TNBC cells and its knockdown leads to the inhibition of cell proliferation, colony formation and progression of cell cycle, which is associated with and reduced expression of cell cycle related protein expression, including Cyclin A2, B1, D1, cdc2 and cdk6 in TNBC cells. Inhibition of UVRAG also suppressed cell motility, migration and invasion of TNBC cells by inhibition of Integrin ß1 and ß3 and Src activity. Our findings suggest for the first time that UVRAG expression contributes to proliferation, cell cycle progression, motility/migration and invasion of TNBC cells. Thus, targeting UVRAG could be a potential strategy in breast cancer especially against TNBC.

c-Src promotes the growth and tumorigenesis of hepatocellular carcinoma via the Hippo signaling pathway.

We investigated the association between c-Src and the progression of hepatocellular carcinoma (HCC) and its underlying mechanisms. The relationship between c-Src expression and the occurrence and development of HCC was explored using GEPIA and further confirmed by western blotting analysis and real-time quantitative PCR. CCK-8, flow cytometry, Transwell, and wound-healing assays were conducted to analyze the effects of c-Src on the growth, cell cycle, apoptosis, migration, and infiltration of HCC cells. Mouse models of transplanted xenogeneic human tumors were constructed to explore the effects of c-Src on HCC tumor growth. Compared with that in adjacent normal liver tissues, the expression level of c-Src in HCC tissues was significantly increased and was negatively correlated with patient survival. These findings are consistent with those in the GEPIA database. Downregulation of c-Src expression can inhibit the growth, infiltration, and migration of HCC cells. c-Src impeded the translocation of YAP from the nucleus to the cytoplasm and promoted Yes-associated protein transcriptional activity. In vivo experiments showed that c-Src inhibition suppressed tumor growth in mice. We found that c-Src can promote the growth and tumorigenesis of HCC cells by activating the Hippo signaling pathway.

Targeting the histone demethylase PHF8-mediated PKCα-Src-PTEN axis in HER2-negative gastric cancer.

Targeted treatments for advanced gastric cancer (GC) are needed, particularly for HER2-negative GC, which represents the majority of cases (80 to 88%). In this study, in silico analyses of the lysine histone demethylases (KDMs) involved in diverse biological processes and diseases revealed that PHD finger protein 8 (PHF8, KDM7B) was significantly associated with poor clinical outcome in HER2-negative GC. The depletion of PHF8 significantly reduced cancer progression in GC cells and in mouse xenografts. PHF8 regulated genes involved in cell migration/motility based on a microarray analysis. Of note, PHF8 interacted with c-Jun on the promoter of PRKCA which encodes PKCα. The depletion of PHF8 or PKCα greatly up-regulated PTEN expression, which could be rescued by ectopic expression of a PKCα expression vector or an active Src. These suggest that PTEN destabilization occurs mainly via the PKCα-Src axis. GC cells treated with midostaurin or bosutinib significantly suppressed migration in vitro and in zebrafish models. Immunohistochemical analyses of PHF8, PKCα, and PTEN showed a positive correlation between PHF8 and PKCα but negative correlations between PHF8 and PTEN and between PKCα and PTEN. Moreover, high PHF8-PKCα expression was significantly correlated with worse prognosis. Together, our results suggest that the PKCα-Src-PTEN pathway regulated by PHF8/c-Jun is a potential prognostic/therapeutic target in HER2-negative advanced GC.

Ligation of HLA Class I Molecules Induces YAP Activation through Src in Human Endothelial Cells.

Ab cross-linking of HLA class I (HLA I) molecules on the surface of endothelial cells (EC) triggers proliferative and prosurvival intracellular signaling, which is implicated in the process of chronic allograft rejection, also known as transplant vasculopathy. Despite the importance of Ab-mediated rejection in transplantation, the mechanisms involved remain incompletely understood. In this study, we examined the regulation of yes-associated protein (YAP) localization, phosphorylation, and transcriptional activity in human ECs challenged with Abs that bind HLA I. In unstimulated ECs, YAP localized mainly in the cytoplasm. Stimulation of these cells with Ab W6/32 induced marked translocation of YAP to the nucleus. The nuclear import of YAP was associated with a rapid decrease in YAP phosphorylation at Ser127 and Ser397, sites targeted by LATS1/2 and with the expression of YAP-regulated genes, including connective tissue growth factor (CTGF), and cysteine-rich angiogenic inducer 61 (CYR61). Transfection of small interfering RNAs targeting YAP/TAZ blocked the migration of ECs stimulated by ligation of HLA I, indicating that YAP mediates the increase in EC migration induced by HLA I ligation. Treatment of intact ECs with Src family inhibitors induced cytoplasmic localization of YAP in unstimulated ECs and, strikingly, blocked the nuclear import of YAP induced by Ab-induced HLA I activation in these cells and the increase in the expression of the YAP-regulated genes CTGF and CYR61 induced by HLA I stimulation. Our results identify the Src/YAP axis as a key player in promoting the proliferation and migration of ECs that are critical in the pathogenesis of transplant vasculopathy.

The 14-3-3ζ-c-Src-integrin-ß3 complex is vital for platelet activation.

Several adaptor molecules bind to cytoplasmic tails of ß-integrins and facilitate bidirectional signaling, which is critical in thrombosis and hemostasis. Interfering with integrin-adaptor interactions spatially or temporally to inhibit thrombosis without affecting hemostasis is an attractive strategy for the development of safe antithrombotic drugs. We show for the first time that the 14-3-3ζ-c-Src-integrin-ß3 complex is formed during platelet activation. 14-3-3ζ-c-Src interaction is mediated by the -PIRLGLALNFSVFYYE- fragment (PE16) on the 14-3-3ζ and SH2-domain on c-Src, whereas the 14-3-3ζ-integrin-ß3 interaction is mediated by the -ESKVFYLKMKGDYYRYL- fragment (EL17) on the 14-3-3ζ and -KEATSTF- fragment (KF7) on the ß3-integrin cytoplasmic tail. The EL17-motif inhibitor, or KF7 peptide, interferes with the formation of the 14-3-3ζ-c-Src-integrin-ß3 complex and selectively inhibits ß3 outside-in signaling without affecting the integrin-fibrinogen interaction, which suppresses thrombosis without causing significant bleeding. This study characterized a previously unidentified 14-3-3ζ-c-Src-integrin-ß3 complex in platelets and provided a novel strategy for the development of safe and effective antithrombotic treatments.

Anti-Cancer Potential of Oxialis obtriangulata in Pancreatic Cancer Cell through Regulation of the ERK/Src/STAT3-Mediated Pathway.

As a plant medicine, Oxalidaceae has been used to treat various diseases in Korea. However, there is little data on the anti-cancer efficacy of Oxalidaceae, particularly O. obtriangulata. This study aimed to investigate the anti-cancer effect of O. obtriangulata methanol extract (OOE) and its regulatory actions on pancreatic carcinoma. OOE showed anti-proliferative effects and induced cell death in the colony formation and cell viability assays, respectively. The Fluorescence-activated cell sorting (FACS) data confirmed that OOE significantly induced cell cycle accumulation at the G2/M phase and apoptotic effects. Additionally, OOE inhibited the activated ERK (extracellular-signal-regulated kinase)/Src (Proto-oncogene tyrosine-protein kinase Src)/STAT3 (signal transducers and activators of transcription 3) pathways including nuclear translocation of STAT3. Furthermore, suppression of Ki67, PARP(Poly ADP-ribose polymerase), caspase-3, P27(Cyclin-dependent kinase inhibitor 1B), and c-Myc as well as the STAT3 target genes CDK(cyclin-dependent kinase)1, CDK2, Cyclin B1, VEGF-1(vascular endothelial growth factor-1), MMP-9(Matrix metallopeptidase 9), and Survivin by OOE was observed in BxPC3. We speculate that these molecular actions might support an anti-cancer effect of OOE. In this study, we demonstrated that OOE may be a promising anti-cancer material and may serve as a natural therapy and alternative remedy for pancreatic cancer treatment.

Interplay between the tyrosine kinases Chk and Csk and phosphatase PTPRJ is critical for regulating platelets in mice.

The Src family kinases (SFKs) Src, Lyn, and Fyn are essential for platelet activation and also involved in megakaryocyte (MK) development and platelet production. Platelet SFKs are inhibited by C-terminal Src kinase (Csk), which phosphorylates a conserved tyrosine in their C-terminal tail, and are activated by the receptor-type tyrosine phosphatase PTPRJ (CD148, DEP-1), which dephosphorylates the same residue. Deletion of Csk and PTPRJ in the MK lineage in mice results in increased SFK activity, but paradoxically hypoactive platelets resulting from negative feedback mechanisms, including upregulation of Csk homologous kinase (Chk) expression. Here, we investigate the role of Chk in platelets, functional redundancy with Csk, and the physiological consequences of ablating Chk, Csk, and PTPRJ in mice. Platelet count was normal in Chk knockout (KO) mice, reduced by 92% in Chk;Csk double KO (DKO) mice, and partially rescued in Chk;Csk;Ptprj triple KO (TKO) mice. Megakaryocyte numbers were significantly increased in both DKO and TKO mice. Phosphorylation of the inhibitory tyrosine of SFKs was almost completely abolished in DKO platelets, which was partially rescued in Src and Fyn in TKO platelets. This residual phosphorylation was abolished by Src inhibitors, revealing an unexpected mechanism in which SFKs autoinhibit their activity by phosphorylating their C-terminal tyrosine residues. We demonstrate that reduced inhibitory phosphorylation of SFKs leads to thrombocytopenia, with Csk being the dominant inhibitor in platelets and Chk having an auxiliary role. PTPRJ deletion in addition to Chk and Csk ameliorates the extent of thrombocytopenia, suggesting targeting it may have therapeutic benefits in such conditions.

Activation of Src mediates acquired cisplatin resistance in human lung carcinoma cells.

Cisplatin (CDDP) is the most effective chemotherapeutic drug against lung carcinoma. However, the emergence of resistant clones has severely limited its clinical application. We found that the cisplatin-resistant lung carcinoma cell line A549/CDDP had increased levels of the phosphorylated gap junction protein Cx43 and SRC tyrosine kinase, and low levels of total Cx43 protein and reduced gap junction formation. The SRC kinase inhibitor PP2 increased the expression of total Cx43 protein and enhanced cisplatin sensitivity, indicating that activated SRC kinase induces chemoresistance by decrease total Cx43 level. Furthermore, Cx43 gene silencing in the drug-resistant cell lines abrogated the sensitizing effect of PP2. Taken together, targeting SRC kinase by PP2 reverses cisplatin resistance by upregulating Cx43 protein levels, indicating a novel pathway of cisplatin resistance that may be amenable to therapeutic intervention.

Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves ß1-integrin signalling.

Accumulation of tissue factor (TF) within cells leads to cellular apoptosis mediated through p38 and p53 pathways. In this study, the involvement of Src1 in the induction of TF-mediated cell apoptosis, and the mechanisms of Src1 activation were investigated. Human coronary artery endothelial cell (HCAEC) were transfected with plasmids to express the wild-type TF (TFWt-tGFP), or a mutant (Ser253 → Ala) which is incapable of being released from cells (TFAla253-tGFP). The cells were then activated with PAR2-agonist peptide (SLIGKV-NH) and the phosphorylation of Src and Rac, and also the kinase activity of Src were assessed. Transfected cells were also pre-incubated with pp60c Src inhibitor, FAK inhibitor-14, or a blocking anti-ß1-integrin antibody prior to activation and the phosphorylation of p38 as well as cellular apoptosis was examined. Finally, cells were co-transfected with the plasmids, together with a Src1-specific siRNA, activated as above and the cellular apoptosis measured. Activation of PAR2 lead to the phosphorylation of Src1 and Rac1 proteins at 60 min regardless of TF expression. Moreover, Src phosphorylation and kinase activity was prolonged up to 100 min in the presence of TF, with a significantly higher magnitude when the non-releasable TFAla253-tGFP was expressed in HCAEC. Inhibition of Src with pp60c, or suppression of Src1 expression in cells, reduced p38 phosphorylation and prevented cellular apoptosis. In contrast, inhibition of FAK had no significant influence on Src kinase activity or cellular apoptosis. Finally, pre-incubation of cells with an inhibitory anti-ß1-integrin antibody reduced both Src1 activation and cellular apoptosis. Our data show for the first time that the over-activation of Src1 is a mediator of TF-induced cellular apoptosis in endothelial cells through a mechanism that is dependent on its interaction with ß1-integrin.

Epigenome-Wide Association Study for All-Cause Mortality in a Cardiovascular Cohort Identifies Differential Methylation in Castor Zinc Finger 1 (CASZ1).

Background DNA methylation is implicated in many chronic diseases and may contribute to mortality. Therefore, we conducted an epigenome-wide association study (EWAS) for all-cause mortality with whole-transcriptome data in a cardiovascular cohort (CATHGEN [Catheterization Genetics]). Methods and Results Cases were participants with mortality≥7 days postcatheterization whereas controls were alive with≥2 years of follow-up. The Illumina Human Methylation 450K and EPIC arrays (Illumina, San Diego, CA) were used for the discovery and validation sets, respectively. A linear model approach with empirical Bayes estimators adjusted for confounders was used to assess difference in methylation (Δß). In the discovery set (55 cases, 49 controls), 25 629 (6.5%) probes were differently methylated (P<0.05). In the validation set (108 cases, 108 controls), 3 probes were differentially methylated with a false discovery rate-adjusted P<0.10: cg08215811 (SLC4A9; log2 fold change=-0.14); cg17845532 (MATK; fold change=-0.26); and cg17944110 (castor zinc finger 1 [CASZ1]; FC=0.26; P<0.0001; false discovery rate-adjusted P=0.046-0.080). Meta-analysis identified 6 probes (false discovery rate-adjusted P<0.05): the 3 above, cg20428720 (intergenic), cg17647904 (NCOR2), and cg23198793 (CAPN3). Messenger RNA expression of 2 MATK isoforms was lower in cases (fold change=-0.24 [P=0.007] and fold change=-0.61 [P=0.009]). The CASZ1, NCOR2, and CAPN3 transcripts did not show differential expression (P>0.05); the SLC4A9 transcript did not pass quality control. The cg17944110 probe is located within a potential regulatory element; expression of predicted targets (using GeneHancer) of the regulatory element, UBIAD1 (P=0.01) and CLSTN1 (P=0.03), were lower in cases. Conclusions We identified 6 novel methylation sites associated with all-cause mortality. Methylation in CASZ1 may serve as a regulatory element associated with mortality in cardiovascular patients. Larger studies are necessary to confirm these observations.

Src inhibition attenuates polyglutamine-mediated neuromuscular degeneration in spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by an expanded CAG repeat in the androgen receptor (AR) gene. Here, we perform a comprehensive analysis of signaling pathways in a mouse model of SBMA (AR-97Q mice) utilizing a phosphoprotein assay. We measure the levels of 17 phosphorylated proteins in spinal cord and skeletal muscle of AR-97Q mice at three stages. The level of phosphorylated Src (p-Src) is markedly increased in the spinal cords and skeletal muscles of AR-97Q mice prior to the onset. Intraperitoneal administration of a Src kinase inhibitor improves the behavioral and histopathological phenotypes of the transgenic mice. We identify p130Cas as an effector molecule of Src and show that the phosphorylated p130Cas is elevated in murine and cellular models of SBMA. These results suggest that Src kinase inhibition is a potential therapy for SBMA.