Expression Of C-terminal Src Kinase Research Articles

CSK may be a potential prognostic biomarker reflecting the immune status of gastric cancer.

C-terminal Src kinase (CSK), a sarcoma (Src) homologous family kinase, is one of the most important negative regulators. It acts as a tumor suppressor by inhibiting the activity of Src family tyrosine kinases. Paradoxically, CSK is highly expressed in a variety of common tumors. Therefore, we report the expression profile of CSK in pan-cancer patients, focusing on the prognostic value, immune infiltration pattern, and biological function of CSK in gastric cancer. We used the TCGA database to analyze CSK expression, clinical relevance, prognostic significance, assessment of the tumor immune microenvironment, and GO and Kegg enrichment analysis based on co-expressed genes using a bioinformatics approach. CSK is a protective factor in gastric cancer, and its expression correlates with the level of immune cell infiltration and immune checkpoint molecules. Our findings suggest that CSK is an independent prognostic factor in gastric cancer and may predict molecular targeting and immunotherapy and provide ideas for its therapeutic strategy.

Abstract 18390: Ibrutinib Increases Ventricular Arrhythmia Burden Independently of Atrial Arrhythmia

Introduction: Use of Bruton Tyrosine Kinase inhibitors (BTKi) such as ibrutinib is frequently limited by atrial arrhythmia, possibly through off-target inhibition of C-Terminal-Src-Kinase (CSK). Whether BTKi can directly trigger ventricular arrhythmia is less clear. Case reports and retrospective analysis have suggested a link, but causal effect is not established. The association is further put into doubt by absent expression of CSK or BTK in ventricular cardiomyocytes, and absence of ventricular arrhythmia signals in meta-analyses of BTKi RCTs. Further study is critically needed. Hypothesis: Ibrutinib induces ventricular arrhythmia through a unique mechanism. Methods: We performed a retrospective crossover study of ibrutinib-treated patients with serial cardiac monitoring both on and off ibrutinib (pacemakers, loop recorders, or multiple holters) across a 10-year span in a large academic center to compare burden of atrial/ventricular ectopy on and off ibrutinib. A separate group of Chronic Lymphocytic Leukemia (CLL) patients with serial cardiac monitoring not on BTKi was used as additional controls. To determine biological correlation, we tested the ability of ibrutinib to induce arrhythmia in cultured human Induced Pluripotent Stem Cell derived ventricular cardiomyocytes (iPSC-vCMs). Results: 88% (15/17) of patients on ibrutinib with serial cardiac monitoring had increased burden of Premature Ventricular Contractions (PVCs), with an average increase of 155 PVCs/hour (p<0.0001). An increase was not observed in the control cohort. Ventricular arrhythmia burden was not associated with atrial arrhythmia burden. Removing patient-related confounders, in vitro analysis of iPSC-vCMs demonstrated that ibrutinib induced arrhythmia following three days of treatment in a dose-dependent manner. Conclusions: In patients with continuous/serial cardiac monitoring, ibrutinib is associated with a significant increase in ventricular ectopy (average of 155 PVCs/hr). Lack of ventricular and atrial arrhythmia concordance suggests an alternative mechanism of arrhythmia induction. Increased arrhythmogenicity of human ventricular cardiomyocytes treated with ibrutinib indicates a direct myocardial mechanism.

O092 SRC intervention for treating breast cancer

Abstract Introduction The non-receptor tyrosine kinase SRC interacts with multiple signalling pathways and has important roles in tumourigenesis. Thus far, SRC intervention for treating breast cancer remains inconclusive. To investigate the underlying molecular mechanisms affecting responses we generated breast cancer cells resistant to the SRC-inhibitor, Bosutinib (BOS). Methods BOS-Resistant (BOS-R) cell lines: ER+ (MCF-7, ZR-75-30) and ER- (Hs578T, MDA-MB-231) were developed by cultivation in increasing concentrations of BOS (0.1-10uM); protein expression changes were determined by Western Blot and densitometry. Dose-responses to [BOS] were assessed by IC50, mitochondrial and LDH assays. Results BOS sensitivity did not correlate with sub-type: MCF-7, ZR-75-30, Hs578T were inhibited significantly (p<0.05) by 5uM BOS and above (IC50=6uM, 6.4uM, 5.5uM respectively) while MDA-MB-231 tolerated >8.5uM BOS (IC50 7.6uM). Sustained growth of ZR-75-30BOS-R and Hs578TBOS-R was achieved in 5uM, MCF-7BOS-R in 7.5uM and MDA-MB-231BOS-R in 10uM BOS (proliferation rates similar to MDA-MB-231). Densitometry comparisons showed increased levels of SRC/SFK (SRC Family Kinases) in all BOS-R cells (range: 12%-57%). In contrast, expression of CSK (C-terminal SRC Kinase) was reduced by >34% (MCF-7BOS-R, MDA-MB-231BOS-R) or lost (ZR-75-30BOS-R, Hs578TBOS-R). Expression of FAK (Focal Adhesion Kinase) increased in all BOS-R cells (range 25%-50%). Furthermore, differential expression of SRC/SFK and alternative SRC-transcripts distinguished five ER+ from four ER- cell lines. Conclusion Regulation of SRC/SFK and their interactions are highly complex. We show resistance to BOS is, in part, associated with increased expressions of SRC/SFK and FAK.

Identifying bioactive phytoconstituents as C-terminal Src kinase inhibitors: a virtual screening and molecular simulation approach

Tyrosine-protein kinase CSK otherwise known as C-terminal Src kinase (CSK), is involved in multiple pathways and processes, including regulating cell growth, differentiation, migration, and immune responses. Altered expression of CSK has been associated with various complexities, including cancer, CD45 deficiency, Osteopetrosis and lupus erythematosus. Important auxiliary roles of CSK in cancer progression make it a crucial target in developing novel anticancer therapy. Thus, CSK inhibitors are of concern as potent immuno-oncology agents. In this perspective, phytochemicals can be a significant source for unraveling novel CSK inhibitors. In this study, we carried out a systematic structure-based virtual screening of bioactive phytoconstituents against CSK to identify its potential inhibitors. After a multi-step screening process, two hits (Shinpterocarpin and Justicidin B) were selected based on their druglike properties and binding affinity towards CSK. The selected hits were further analyzed for their stability and interaction via all-atom molecular dynamics (MD) simulations. The selected hits indicated their potential as selective binding partners of CSK, which can further be used for therapeutic development against CSK-associated malignancies. Communicated by Ramaswamy H. Sarma

Evaluating the in vitro therapeutic effects of human amniotic mesenchymal stromal cells on MiaPaca2 pancreatic cancer cells using 2D and 3D cell culture model

Human amniotic mesenchymal stromal cells (hAMSCs) are considered as a population of multipotent cells. The molecular events associated with mesenchymal stromal cell (MSC)/tumor cell interactions should be studied to identify the role of MSCs on suppressing or inducing the key signaling pathways of tumor cells. Thus, designing therapeutic approaches is considered as important. In the present study, hAMSCs and MiaPaca2 cells were first cultured separately. In addition, both cell lines were co-cultured by using 0.4 μm pore sized transwell membranes in different times. Further, the RNA of the cells was extracted, and Bcl2, Bax, epidermal growth factor receptor (EGFR), c-Src, C-terminal Src Kinase (CSK), and SGK223 expression were analyzed through quantitative real time PCR. Furthermore, the total cell lysates of the cells were prepared and analyzed by using western blot. Based on the results, the expression of EGFR, c-Src, SGK223, and CSK in MiaPaca2 cells reduced after treating with hAMSCs. Notably, the cellular apoptosis of MiaPaca2 cells was induced in 2D cell culture system. Further, the anti-cancer activity of conditioned medium from hAMSCs was confirmed in a 3D cell culture model by using hanging drop technique. Finally, hAMSCs have inhibitory effects on pancreatic cancer cells and can be considered as a therapeutic way to suppress EGFR, c-Src, and SGK223, as the potent targets in cancer cell signaling.

Positive and Negative Regulatory Roles of C-Terminal Src Kinase (CSK) in FcεRI-Mediated Mast Cell Activation, Independent of the Transmembrane Adaptor PAG/CSK-Binding Protein.

C-terminal Src kinase (CSK) is a major negative regulator of Src family tyrosine kinases (SFKs) that play critical roles in immunoreceptor signaling. CSK is brought in contiguity to the plasma membrane-bound SFKs via binding to transmembrane adaptor PAG, also known as CSK-binding protein. The recent finding that PAG can function as a positive regulator of the high-affinity IgE receptor (FcεRI)-mediated mast cell signaling suggested that PAG and CSK have some non-overlapping regulatory functions in mast cell activation. To determine the regulatory roles of CSK in FcεRI signaling, we derived bone marrow-derived mast cells (BMMCs) with reduced or enhanced expression of CSK from wild-type (WT) or PAG knockout (KO) mice and analyzed their FcεRI-mediated activation events. We found that in contrast to PAG-KO cells, antigen-activated BMMCs with CSK knockdown (KD) exhibited significantly higher degranulation, calcium response, and tyrosine phosphorylation of FcεRI, SYK, and phospholipase C. Interestingly, FcεRI-mediated events in BMMCs with PAG-KO were restored upon CSK silencing. BMMCs with CSK-KD/PAG-KO resembled BMMCs with CSK-KD alone. Unexpectedly, cells with CSK-KD showed reduced kinase activity of LYN and decreased phosphorylation of transcription factor STAT5. This was accompanied by impaired production of proinflammatory cytokines and chemokines in antigen-activated cells. In line with this, BMMCs with CSK-KD exhibited enhanced phosphorylation of protein phosphatase SHP-1, which provides a negative feedback loop for regulating phosphorylation of STAT5 and LYN kinase activity. Furthermore, we found that in WT BMMCs SHP-1 forms complexes containing LYN, CSK, and STAT5. Altogether, our data demonstrate that in FcεRI-activated mast cells CSK is a negative regulator of degranulation and chemotaxis, but a positive regulator of adhesion to fibronectin and production of proinflammatory cytokines. Some of these pathways are not dependent on the presence of PAG.

Estrogen-regulated feedback loop limits the efficacy of estrogen receptor–targeted breast cancer therapy

Endocrine therapy resistance invariably develops in advanced estrogen receptor-positive (ER+) breast cancer, but the underlying mechanisms are largely unknown. We have identified C-terminal SRC kinase (CSK) as a critical node in a previously unappreciated negative feedback loop that limits the efficacy of current ER-targeted therapies. Estrogen directly drives CSK expression in ER+ breast cancer. At low CSK levels, as is the case in patients with ER+ breast cancer resistant to endocrine therapy and with the poorest outcomes, the p21 protein-activated kinase 2 (PAK2) becomes activated and drives estrogen-independent growth. PAK2 overexpression is also associated with endocrine therapy resistance and worse clinical outcome, and the combination of a PAK2 inhibitor with an ER antagonist synergistically suppressed breast tumor growth. Clinical approaches to endocrine therapy-resistant breast cancer must overcome the loss of this estrogen-induced negative feedback loop that normally constrains the growth of ER+ tumors.

Genome-wide methylation profiling of ovarian cancer patient-derived xenografts treated with the demethylating agent decitabine identifies novel epigenetically regulated genes and pathways.

BackgroundIn high-grade serous ovarian cancer (HGSOC), intrinsic and/or acquired resistance against platinum-containing chemotherapy is a major obstacle for successful treatment. A low frequency of somatic mutations but frequent epigenetic alterations, including DNA methylation in HGSOC tumors, presents the cancer epigenome as a relevant target for innovative therapy. Patient-derived xenografts (PDXs) supposedly are good preclinical models for identifying novel drug targets. However, the representativeness of global methylation status of HGSOC PDXs compared to their original tumors has not been evaluated so far. Aims of this study were to explore how representative HGSOC PDXs are of their corresponding patient tumor methylome and to evaluate the effect of epigenetic therapy and cisplatin on putative epigenetically regulated genes and their related pathways in PDXs.MethodsGenome-wide analysis of the DNA methylome of HGSOC patients with their corresponding PDXs, from different generations, was performed using Infinium 450 K methylation arrays. Furthermore, we analyzed global methylome changes after treatment of HGSOC PDXs with the FDA approved demethylating agent decitabine and cisplatin. Findings were validated by bisulfite pyrosequencing with subsequent pathway analysis. Publicly available datasets comprising HGSOC patients were used to analyze the prognostic value of the identified genes.ResultsOnly 0.6–1.0 % of all analyzed CpGs (388,696 CpGs) changed significantly (p < 0.01) during propagation, showing that HGSOC PDXs were epigenetically stable. Treatment of F3 PDXs with decitabine caused a significant reduction in methylation in 10.6 % of CpG sites in comparison to untreated PDXs (p < 0.01, false discovery rate <10 %). Cisplatin treatment had a marginal effect on the PDX methylome. Pathway analysis of decitabine-treated PDX tumors revealed several putative epigenetically regulated pathways (e.g., the Src family kinase pathway). In particular, the C-terminal Src kinase (CSK) gene was successfully validated for epigenetic regulation in different PDX models and ovarian cancer cell lines. Low CSK methylation and high CSK expression were both significantly associated (p < 0.05) with improved progression-free survival and overall survival in HGSOC patients.ConclusionsHGSOC PDXs resemble the global epigenome of patients over many generations and can be modulated by epigenetic drugs. Novel epigenetically regulated genes such as CSK and related pathways were identified in HGSOC. Our observations encourage future application of PDXs for cancer epigenome studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0361-5) contains supplementary material, which is available to authorized users.

Activating Mutations In Fyn Kinase In Peripheral T-Cell Lymphomas

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of aggressive non Hodgkin lymphomas with poor prognosis. Molecular and cytogenetic studies have shown a prominent role for kinase fusion oncogenes, mostly NPM-ALK, in ALK+ anaplastic large cell lymphomas (ALCLs) and ITK-SYK kinase in unspecified PTCLs. To gain further insight on the genetics and pathogenic mechanisms of aggressive PTCLs we performed an integrated mutation analysis using whole exome sequencing (n=12) and RNAseq (n=35) data. This analysis identified 288 candidate coding somatic mutations in 268 genes including known recurrent mutations in the TET2, DNMT3A and IDH2 epigenetic factor genes and pointed to the FYN kinase gene as a new recurrently mutated oncogene in T-cell transformation. The FYN tyrosine kinase is, with LCK, the predominant SRC family kinase found in T lymphocytes and plays an important role in T-cell activation upon T-cell receptor (TCR) stimulation. FYN mutations in PTCL included a FYN L174R mutation detected in one AITL patient sample, a FYN R176C allele recurrently found in two PTCL NOS cases and a FYN Y531H mutation present in a PTCL NOS sample. Notably, each of these alleles are predicted to specifically disrupt the inhibition of FYN kinase activity by the C terminal SRC kinase (CSK). Thus, structure analysis of FYN and FYN mutant proteins predicted that FYN L174R and, most prominently, FYN R176C and FYN Y531H can disrupt the inhibitory interaction of the FYN SH2 domain with the CSK-phosphorylated Y531. Consistently, pull down assays using GST-FYN-SH2 recombinant proteins and biotinylated C-terminal FYN peptides encompassing Y531 showed abrogation of the interaction between FYN-SH2 and P-Y531 in each of these mutants. In agreement with these results, expression of FYN L174R, FYN R176C and FYN Y531H resulted in increased levels of FYN activation. Moreover, CSK expression effectively inhibited wild type FYN, but failed to abrogate FYN L174R, FYN R176C or FYN Y531H activation. In contrast, pharmacologic kinase inhibition with dasatinib, a multikinase inhibitor which blocks ABL1 and SRC kinases, effectively abrogated the activity of FYN L174R, FYN R176C and FYN Y531H mutant proteins and suppressed the growth of cells transformed via expression of activated FYN mutant alleles. Overall these results support an oncogenic role for FYN activating mutations in the pathogenesis of PTCL and support a role for SRC kinase inhibitors for the treatment of this disease. Disclosures:No relevant conflicts of interest to declare.

Chemopreventive activity of plant flavonoid isorhamnetin in colorectal cancer is mediated by oncogenic Src and β-catenin.

Analysis of the Polyp Prevention Trial showed an association between an isorhamnetin-rich diet and a reduced risk of advanced adenoma recurrence; however, the mechanism behind the chemoprotective effects of isorhamnetin remains unclear. Here, we show that isorhamnetin prevents colorectal tumorigenesis of FVB/N mice treated with the chemical carcinogen azoxymethane and subsequently exposed to colonic irritant dextran sodium sulfate (DSS). Dietary isorhamnetin decreased mortality, tumor number, and tumor burden by 62%, 35%, and 59%, respectively. MRI, histopathology, and immunohistochemical analysis revealed that dietary isorhamnetin resolved the DSS-induced inflammatory response faster than the control diet. Isorhamnetin inhibited AOM/DSS-induced oncogenic c-Src activation and β-catenin nuclear translocation, while promoting the expression of C-terminal Src kinase (CSK), a negative regulator of Src family of tyrosine kinases. Similarly, in HT-29 colon cancer cells, isorhamnetin inhibited oncogenic Src activity and β-catenin nuclear translocation by inducing expression of csk, as verified by RNA interference knockdown of csk. Our observations suggest the chemoprotective effects of isorhamnetin in colon cancer are linked to its anti-inflammatory activities and its inhibition of oncogenic Src activity and consequential loss of nuclear β-catenin, activities that are dependent on CSK expression.

Expression of C-terminal &lt;italic&gt;src&lt;/italic&gt; Kinase in Human Colorectal Cancer Cell Lines

C-terminal src kinase (CSK) is a cytoplasmic protein which decreases activities of the Src family protein-tyrosine kinases. We produced a polyclonal antibody specific for CSK and analyzed the expression of CSK by immunoblotting in two human colorectal normal cell lines and eighteen cancer cell lines. CSK was detected in both the two normal and all the eighteen cancer cell lines. The expression of CSK obtained from human colorectal cancer cell lines was greater than that from human colorectal normal cell lines in most cases. The elevated expression of CSK in human colorectal cancer cell lines appeared to correspond to the elevated p60c-src (c-Src) and p62c-yes (c-Yes) protein-tyrosine kinase activities found in other studies. Thus, CSK may not have an anti-oncogenic role to play through the negative regulation of Src family kinases in colorectal carcinogenesis.