Knockdown Of Fyn Research Articles

Chrysin alleviates lipopolysaccharide-induced neuron damage and behavioral deficits in mice through inhibition of Fyn

Fyn, a non-receptor tyrosine kinase, plays an important role in microglial-mediated neuroinflammation and may serve as a candidate therapeutic target for neuropsychiatric diseases. In this study, we discovered that chrysin, a natural flavonoid compound, suppressed the activation of Fyn kinase and further alleviated neuroinflammation-induced neuron damage and behavior deficits. Functionally, chrysin improved lipopolysaccharide (LPS)-induced memory impairment and depressive behaviors in mice, it also protected against LPS-induced neuronal degeneration and loss and synaptic defects in mice. Our study demonstrated that chrysin inhibited the activation of microglia and reduced the expression of NLRP3 and IL-1β. Furthermore, our data indicated that chrysin blocked phosphorylation of Fyn and activation of NF-κB. Transfection with siRNA-Fyn validated that knockdown of Fyn partly abolished the inhibitory effect of chrysin on the expression of the NLRP3 inflammasome and NF-κB activation. Taken together, our findings revealed that chrysin alleviated LPS-induced neuron damage and behavioral deficits by inhibiting the expression of the NLRP3 inflammasome and NF-κB pathway, which might be mediated by inhibition of Fyn.

Tyrosine kinase Fyn promotes apoptosis after intracerebral hemorrhage in rats by activating Drp1 signaling.

Tyrosine kinase Fyn is a member of the Src kinase family, which is involved in neuroinflammation, apoptosis, and oxidative stress. Its role in intracerebral hemorrhage (ICH) is not fully understood. In this study, we found that Fyn was significantly elevated in human brain tissue after ICH. Accordingly, we investigated the role of Fyn in a rat ICH model, which was constructed by injecting blood into the right basal ganglia. In this model, Fyn expression was significantly upregulated in brain tissue adjacent to the hematoma. SiRNA-induced Fyn knockdown was neuroprotective for secondary cerebral damage, as demonstrated by reduced brain edema, suppression of the modified neurological severity score, and mitigation of blood-brain barrier permeability and neuronal damage. Fyn downregulation reduced apoptosis following ICH, as indicated by downregulation of apoptosis-related proteins AIF, Cyt.c, caspase 3, and Bax; upregulation of anti-apoptosis-related protein Bcl-2; and decreased tunnel staining. Mdivi-1, a Drp1 inhibitor, reversed Fyn overexpression induced pro-apoptosis. However, Fyn did not significantly affect inflammation-related proteins NF-κB, TNF-α, caspase 1, MPO, IL-1β, or IL-18 after ICH. Fyn activated Drp1 signaling by phosphorylating Drp1 at serine 616, which increased apoptosis after ICH in rats. This study clarifies the relationship between Fyn, apoptosis, and inflammation following ICH and provides a new strategy for exploring the prevention and treatment of ICH. KEY MESSAGES: ICH induced an increase in Fyn expression in human and rat cerebral tissues. Knockdown of Fyn prevented cerebral damage following ICH. Inhibition of Fyn had no significant effects on inflammatory responses. However, the downregulation of Fyn exerted neuroprotective effects on apoptosis. Fyn perturbed ICH-induced cell apoptosis by interacting with and phosphorylating (Ser616) Drp1 in a rat ICH model.

Separate signaling events control TCR downregulation and T cell activation in primary human T cells.

IntroductionT‐cell antigen receptor (TCR) interaction with cognate peptide:MHC complexes trigger clustering of TCR:CD3 complexes and signal transduction. Triggered TCR:CD3 complexes are rapidly internalized and degraded in a process called ligand‐induced TCR downregulation. Classic studies in immortalized T‐cell lines have revealed a major role for the Src family kinase Lck in TCR downregulation. However, to what extent a similar mechanism operates in primary human T cells remains unclear.MethodsHere, we developed an anti‐CD3‐mediated TCR downregulation assay, in which T‐cell gene expression in primary human T cells can be knocked down by microRNA constructs. In parallel, we used CRISPR/Cas9‐mediated knockout in Jurkat cells for validation experiments.ResultsWe efficiently knocked down the expression of tyrosine kinases Lck, Fyn, and ZAP70, and found that, whereas this impaired T cell activation and effector function, TCR downregulation was not affected. Although TCR downregulation was marginally inhibited by the simultaneous knockdown of Lck and Fyn, its full abrogation required broad‐acting tyrosine kinase inhibitors.ConclusionsThese data suggest that there is substantial redundancy in the contribution of individual tyrosine kinases to TCR downregulation in primary human T cells. Our results highlight that TCR downregulation and T cell activation are controlled by different signaling events and illustrate the need for further research to untangle these processes.

Fyn kinase mediates pro-inflammatory response in a mouse model of endotoxemia: Relevance to translational research

Systemic inflammation resulting from the release of pro-inflammatory cytokines and the chronic activation of the innate immune system remains a major cause of morbidity and mortality in the United States. After having demonstrated that Fyn, a Src family kinase, regulates microglial neuroinflammatory responses in cell culture and animal models of Parkinson's disease, we investigate here its role in modulating systemic inflammation using an endotoxic mouse model. Fyn knockout (KO) and their wild-type (WT) littermate mice were injected once intraperitoneally with either saline or 5 mg/kg lipopolysaccharide (LPS) and were killed 48 h later. LPS-induced mortality, endotoxic symptoms and hypothermia were significantly attenuated in Fyn KO, but not WT, mice. LPS reduced survival in Fyn WT mice to 49% compared to 84% in Fyn KO mice. Fyn KO mice were also protected from LPS-induced deficits in horizontal and vertical locomotor activities, total distance traveled and stereotypic movements. Surface body temperatures recorded at 24 h and 48 h post-LPS dropped significantly in Fyn WT, but not in KO, mice. Importantly, endotoxemia-associated changes to levels of the serum pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), splenocyte apoptosis and inducible nitric oxide synthase (iNOS) production in hepatocytes were also significantly attenuated in Fyn KO mice. Likewise, pharmacologically inhibiting Fyn with 10 mg/kg dasatinib (oral) significantly attenuated LPS-induced increases in plasma TNF-α and IL-6 protein levels and hepatic pro-IL-1β messenger ribonucleic acids (mRNAs). Collectively, these results indicate that genetic knockdown or pharmacological inhibition of Fyn dampens systemic inflammation, demonstrating for the first time that Fyn kinase plays a critical role in mediating the endotoxic inflammatory response.

Targeting Src family kinase member Fyn by Saracatinib attenuated liver fibrosis in vitro and in vivo

Recent studies suggest that Src family kinase (SFK) plays important roles in systemic sclerosis and pulmonary fibrosis. However, how SFKs contributed to the pathogenesis of liver fibrosis remains largely unknown. Here, we investigated the role of Fyn, a member of SFK, in hepatic stellate cell (HSC) activation and liver fibrosis, and evaluated the anti-fibrotic effects of Saracatinib, a clinically proven safe Fyn inhibitor. Fyn activation was examined in human normal and fibrotic liver tissues. The roles of Fyn in HSC activation and liver fibrosis were evaluated in HSC cell lines by using Fyn siRNA and in Fyn knockout mice. The effects of Saracatinib on HSC activation and liver fibrosis were determined in primary HSCs and CCl4 induced liver fibrosis model. We showed that the Fyn was activated in the liver of human fibrosis patients. TGF-β induced the activation of Fyn in HSC cell lines. Knockdown of Fyn significantly blocked HSC activation, proliferation, and migration. Fyn deficient mice were resistant to CCl4 induced liver fibrosis. Saracatinib treatment abolished the activation of Fyn, downregulated the Fyn/FAK/N-WASP signaling in HSCs, and subsequently prevented the activation of HSCs. Saracatinib treatment significantly reduced the severity liver fibrosis induced by CCl4 in mice. In conclusions, our findings supported the critical role of Fyn in HSC activation and development of liver fibrosis. Fyn could serve as a promising drug target for liver fibrosis treatment. Fyn inhibitor Saracatinib significantly inhibited HSC activation and attenuated liver fibrosis in mouse model.

497-P: Fyn-Dependent Tyrosine Phosphorylation of Tgm2 Modulates p53-Tgm2-p62 Complex in Renal Proximal Tubular Epithelial Cells

Diabetic kidney disease (DKD) is one of the major diabetic complications, the leading cause of the end stage renal disease (ESRD). Recently autophagy, which is also implicated in diabetes and insulin resistance, was shown to regulate DKD. Previously, we reported that Fyn functions as a muscle mass regulator by suppressing autophagy but it is still not known whether Fyn regulates autophagy in kidney and functions as a pathogenesis of DKD. Recently, we demonstrated that over-expressing of Fyn in HK2 cells as an in vitro cell model of renal proximal tubular epithelial cells inhibits autophagy. SiRNA mediating knockdown of Fyn increased autophagy demonstrating Fyn has important role for autophagy in HK2 cells. Phospho-proteomic analysis revealed that Fyn phosphorylates Transglutaminase 2 (Tgm2), a known autophagic inhibitor through Beclin1, on Y369 and Y617. More recently, we demonstrated that Fyn dependent phosphorylation of Tgm2 could regulate autophagy in HK2 cells. While it has been reported that Tgm2 could cross-link Beclin1 to inhibits its activity, no cross-linking of Beclin1 was seen in Tgm2 expressing HK2 cells, indicating there could be another mechanism of Fyn-Tgm2 regulating autophagy in HK2 cells. Recently, Tgm2 was shown to make a complex with p53 and p62, known autophagy regulator/substrate to degrade p53 in autophagosome in cancer cells. Interestingly, we found that p53 expression was decreased in Tgm2 knock-downed HK2 cells and increased in Tgm2 expressing HK2 cells. More importantly co-immunoprecipitation assay showed that Tgm2-Y369,617F which could not be phosphorylated by Fyn bind to p53 more than WT and dissociated from p62. Taken together, these data suggest that Fyn phosphorylates Tgm2 on Y369, 617 to modulate p53-Tgm2-p62 complex in the renal tubulus cells leading to a pathogenesis of DKD. Disclosure E. Yamada: None. S. Okada: None. C.C. Bastie: None. M. Yamada: None.

Identifying a New Pathway to Regulate Autophagy in HK2 Cells

Diabetic nephropathy (DN) is one of the major diabetic complications, the leading cause of the end stage renal disease (ESRD). Recently autophagy, which is also implicated in diabetes and insulin resistance, was shown to regulate DN. Recently, we demonstrated that Fyn functions as a muscle mass regulator by suppressing macroautophagy through a STAT3-mediated inhibition of the Vps34/Beclin1/ATG14 complex 1 but it is still not known whether Fyn regulates autophagy in kidney and function as a pathogenesis of DN. To investigate this, siRNA mediating knockdown of Fyn in HK2 cells as an in vitro cell model of renal proximal tubular epithelial cells was performed and demonstrating not only decreased p62 expression but also increased LC3-1/2 ratio implicating increased autophagy. To further investigating autophagic flow we utilized ammonium chloride and leupeptin to inhibit the autophagic flow and consistently found that expression of LC3-2 was increased. Phospho-proteomic analysis were performed in vivo and revealed that Fyn phosphorylates Transglutaminase 2 (Tgm2), a known autophagic inhibitor through Beclin1, on Y369 and Y617. To demonstrate that Tgm2 is a direct Fyn substrate, we incubated human purified GST-Tgm2 protein with purified Fyn-CA. In the presence of ATP, Fyn was observed to robustly phosphorylate the Tgm2. Interestingly, Fyn-dependent phosphorylation of Tgm2 was abolished when Y369 and Y617 in Tgm2 was mutated (Tgm2-Y369/617F), suggesting that these sites were specific and unique for Fyn kinase. SiRNA mediating knockdown of Tgm2 in HK2 cells revealed decreased p62 expression and increased LC3-1/2 ratio while overexpressing Tgm2-WT decreased autophagic flow demonstrating Tgm2 could regulate autophagy in the renal tubulus. Taken together, these data suggest that Fyn phorphorylates Tgm2 on Y369 and Y617 and regulates autophagy in the renal tubulus implicating a pathogenesis of DN. Disclosure E. Yamada: None. S. Okada: None. M. Yamada: None.

Upregulation of Tyrosine Kinase FYN in Human Thyroid Carcinoma: Role in Modulating Tumor Cell Proliferation, Invasion, and Migration.

Tyrosine kinase FYN-a member of the SRC family of kinases-is associated with mediating mitogenic signals and regulating cell cycle entry, growth, and proliferation. It was hypothesized that FYN is upregulated in thyroid carcinoma, which plays a critical role in tumorigenesis. FYN expression level in thyroid carcinoma tissue and tumor cell lines was determined. Also, the effects of FYN on thyroid cancer (TC) growth, signaling, cell cycle, and apoptosis were evaluated in vitro. FYN was knocked down in thyroid cancer cells (TPC-1) by siRNA to investigate the effects on cell proliferation, invasion, and migration. First, FYN was upregulated both in thyroid carcinoma tissue and in tumor cell lines. Loss of FYN by siRNA weakened proliferation, invasion, and migration of PTC-1 cells. Furthermore, it was demonstrated that knockdown of FYN can inhibit the G0/G1 phase of cell cycle. It was also observed that reduced expression of FYN could increase the level of NF-κB/P65 and IκBα. This study was the first to demonstrate critical positive regulation of thyroid tumorigenesis by FYN, which could be a potential target gene for thyroid carcinoma treatment. In addition, findings from this study highlighted the potential role of FYN inhibitors in TC therapy.

Fyn Mediates High Glucose-Induced Actin Cytoskeleton Reorganization of Podocytes via Promoting ROCK Activation In Vitro.

Fyn, a member of the Src family of tyrosine kinases, is a key regulator in cytoskeletal remodeling in a variety of cell types. Recent studies have demonstrated that Fyn is responsible for nephrin tyrosine phosphorylation, which will result in polymerization of actin filaments and podocyte damage. Thus detailed involvement of Fyn in podocytes is to be elucidated. In this study, we investigated the potential role of Fyn/ROCK signaling and its interactions with paxillin. Our results presented that high glucose led to filamentous actin (F-actin) rearrangement in podocytes, accompanied by paxillin phosphorylation and increased cell motility, during which Fyn and ROCK were markedly activated. Gene knockdown of Fyn by siRNA showed a reversal effect on high glucose-induced podocyte damage and ROCK activation; however, inhibition of ROCK had no significant effects on Fyn phosphorylation. These observations demonstrate that in vitro Fyn mediates high glucose-induced actin cytoskeleton remodeling of podocytes via promoting ROCK activation and paxillin phosphorylation.

Abstract 3459: SRC family kinase FYN promotes MET tyrosine kinase activation, epithelial to mesenchymal transition and metastasis in human prostate cancer

Abstract Introduction: FYN is a Src family kinase (SFK) member that has been shown to be upregulated in human prostate cancer tissues and cell lines (PCa). The majority of the studies on SRC kinases have focused on the classical c-SRC. We demonstrated previously that knockdown of FYN in PCa cell lines leads to reduced tumor growth in vitro and in vivo. We hypothesized that MET, an oncogenic tyrosine kinase receptor implicated in metastasis and therapeutic resistance of a variety of solid tumors is regulated by FYN expression in prostate cancer. Methods: We performed in vitro growth assays, migration and invasion assays and studied PCa cell response to HGF in wildtype, FYN-depleted and FYN-reconstituted cells. Additionally, we studied the role of FYN in promoting metastasis in vivo in xenograft mouse models. Summary: In this study, we demonstrate that knockdown of FYN leads to reduced migration, invasion and metastatic dissemination of PCa cells in vitro and in vivo. Conversely, ectopic expression of FYN promotes enhanced tumor growth and invasion. Hepatocyte growth factor (HGF) induced activation of MET is perturbed in FYN knockdown cells and overexpression of FYN leads to excessive MET activation suggesting a possible feedback loop between FYN and MET in prostate cancer. Consistent these findings, PC3 prostate cancer cells overexpressing FYN are resistant to treatment with a MET inhibitor upon ligand stimulation. More importantly, we show that FYN promotes epithelial to mesenchymal transition (EMT) of PCa cells and knockdown of FYN leads to reversal of EMT and alters radiation sensitivity of PCa cells. To further identify the molecular mechanism of FYN mediated regulation of tumor growth, we performed integrated RNA sequencing analysis of FYN knockdown cells and found MET and a network of molecular targets that appear to regulate FYN mediated tumor progression and metastasis. Conclusions: We demonstrate that FYN mediated MET activation promotes PCa invasion, EMT and metastasis. None of the SFK inhibitors currently in clinical trials are specific for SFKs alone. Therefore, it is imperative to develop and test novel inhibitors that selectively target FYN and other SFKs. Our findings have therapeutic implications since clinical trials are being performed for both SFK inhibitors and MET inhibitors in castration resistant PCa patients. Our studies reveal that FYN and MET interact in PCa cells and this interaction is clinically relevant given the success of XL-184 (a MET inhibitor) in phase III clinical trials in human prostate cancer. Clinical studies with translational endpoints are the need of the hour with agents that effectively inhibit FYN and MET in a variety of settings. Citation Format: Murali Gururajan, Margarit Sievert, Sheldon Mink, Chia-Yi (Gina) Chu, Matteo Morello, Jake Lichterman, Michael R. Freeman, Edwin M. Posadas. SRC family kinase FYN promotes MET tyrosine kinase activation, epithelial to mesenchymal transition and metastasis in human prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3459. doi:10.1158/1538-7445.AM2014-3459

Abstract 511: EGFR-initated NADPH oxidase activity regulates Fyn expression in glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) constitute the most aggressive and common form of primary brain tumors, conferring the worst clinical prognosis. Epidermal growth factor receptor (EGFR) amplification, mutation and re-arrangement are commonly observed genetic lesions in GBM. The most frequently occurring EGFR variant in GBM, EGFRvIII, is characterized by a truncated extracellular domain, leading to a receptor which is unable to bind ligand yet rendered constitutively active. In addition to increasing proliferation and survival signaling, EGFRvIII increases the production of reactive oxygen species (ROS); redox signaling in GBM, however, remains an understudied oncogenic feature. Consistent with published findings, our studies indicate that EGFRvIII elevates ROS levels in comparison to wild-type EGFR or vector-transfected control in U87-MG and the observed increase in ROS levels is alleviated by pharmacological and genetic means of EGFR inhibition. ROS increases were determined to occur independent of augmented spare mitochondrial respiratory capacity, but did coincide with elevated basal levels of non-mitochondrial oxygen consumption as measured by Seahorse extracellular flux analysis. Given this finding, our studies focused on the NADPH oxidase (NOX), where chemical inhibition of NOX by apocynin effectively reduced ROS by greater than 50% in EGFRvIII expressing cells. Likewise, EGFRvIII increased the consumption of NADPH greater than 2.0-fold relative to vector control, as measured by cellular NADP/NADPH ratios, and the resulting increases in NADPH consumption were diminished by chemical inhibition of EGFRvIII as well as apocynin inhibition of NOX. These findings indicated that EGFRvIII-induced ROS increases might be related to increased activity of the NOX complex. To this end, siRNA knockdown of p47phox, an essential component of the NOX-2 complex, markedly reduced ROS content as well as cellular proliferation in these cells, thus reinforcing a possible link between aberrant EGFR signaling and ROS production through NOX. Our studies additionally indicate that EGFRvIII-mediated elevations in ROS up-regulate mRNA and protein level expression (>3.0-fold) of the SRC family kinase member Fyn, as Fyn expression is up-regulated in an EGFRvIII-dependent manner and concordantly down-regulated via chemical and genetic means of NOX inhibition. Moreover, siRNA knockdown of Fyn effectively lowers cellular proliferation, survival and increases sensitivity to EGFR kinase inhibition in EGFRvIII, thus indicating that redox-initiated signaling through Fyn, in part, mediates GBM proliferation. Taken together, our results suggest that EGFRvIII increases NOX-2 generated ROS through p47phox, enhancing the proliferative capacity of GBM, in part, via increased Fyn expression, suggesting that targeting redox alterations may prove beneficial in improving clinical outcomes in GBM. Citation Format: Blake Johnson, Joya Chandra. EGFR-initated NADPH oxidase activity regulates Fyn expression in glioblastoma multiforme. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 511. doi:10.1158/1538-7445.AM2014-511

The inhibition of SRC family kinase suppresses pancreatic cancer cell proliferation, migration, and invasion.

Src is considered a rising therapeutic target for the treatment of solid tumors, and Src family kinases (SFKs) participate in cancer cell proliferation and survival. The role of SFK suppression was investigated in the proliferation, migration, and invasion of pancreatic cancer cells. Knockdown of the SFKs in pancreatic cancer cells was achieved by transfecting small interfering RNAs, and its effects were investigated using proliferation, wound, and invasion assays. The SFK inhibitors suppressed proliferation and induced cell cycle arrest in pancreatic cancer cells. The SFK messenger RNA profiles showed that Yes1, Lyn, Fyn, Frk, Hck, and Src were expressed. Specific small interfering RNA transfection suppressed the messenger RNA expressions of Yes1, Lyn, Fyn, Frk, and Src, and the knockdown suppressed cell proliferation by 16.7% to 47.3% in PANC-1 cells. Knockdown of any of these 5 SFKs suppressed proliferation in other pancreatic cancer cell lines by 3.0% to 40.5%. The knockdowns significantly reduced pancreatic cancer cell migration by 24.9% to 66.7% and completely inhibited invasion. These results suggest that the knockdown of Yes1, Lyn, Fyn, Frk, or Src reduce human pancreatic cancer cell proliferation, migration, and invasion, and that SFKs should be viewed as critical therapeutic targets of pancreatic cancer.

Gene expression profiling identifies FYN as an important molecule in tamoxifen resistance and a predictor of early recurrence in patients treated with endocrine therapy.

To elucidate the molecular mechanisms of tamoxifen resistance in breast cancer, we performed gene array analyses and identified 366 genes with altered expression in four unique tamoxifen-resistant (TamR) cell lines vs the parental tamoxifen-sensitive MCF-7/S0.5 cell line. Most of these genes were functionally linked to cell proliferation, death and control of gene expression, and include FYN, PRKCA, ITPR1, DPYD, DACH1, LYN, GBP1 and PRLR. Treatment with FYN-specific small interfering RNA or a SRC family kinase inhibitor reduced cell growth of TamR cell lines while exerting no significant effect on MCF-7/S0.5 cells. Moreover, overexpression of FYN in parental tamoxifen-sensitive MCF-7/S0.5 cells resulted in reduced sensitivity to tamoxifen treatment, whereas knockdown of FYN in the FYN-overexpressing MCF-7/S0.5 cells restored sensitivity to tamoxifen, demonstrating growth- and survival-promoting function of FYN in MCF-7 cells. FYN knockdown in TamR cells led to reduced phosphorylation of 14-3-3 and Cdc25A, suggesting that FYN, by activation of important cell cycle-associated proteins, may overcome the anti-proliferative effects of tamoxifen. Evaluation of the subcellular localization of FYN in primary breast tumors from two cohorts of endocrine-treated ER+ breast cancer patients, one with advanced disease (N=47) and the other with early disease (N=76), showed that in the former, plasma membrane-associated FYN expression strongly correlated with longer progression-free survival (P<0.0002). Similarly, in early breast cancer patients, membrane-associated expression of FYN in the primary breast tumor was significantly associated with increased metastasis-free (P<0.04) and overall (P<0.004) survival independent of tumor size, grade or lymph node status. Our results indicate that FYN has an important role in tamoxifen resistance, and its subcellular localization in breast tumor cells may be an important novel biomarker of response to endocrine therapy in breast cancer.

OP0026 Gene expression profiling identifies FYN as important in tamoxifen resistance and a predictor of early recurrence in patients treated with endocrine therapy

Background We aimed to elucidate the molecular mechanisms of tamoxifen resistance in breast cancer. Methods We did gene array analyses and identified 366 genes with altered expression in four unique tamoxifen-resistant cell lines versus the parental tamoxifen-sensitive MCF-7/S0.5 cell line. Most of these genes were functionally linked to cell proliferation, death, and control of gene expression, and include FYN, PRKCA, ITPR1, DPYD, DACH1, LYN, GBP1, and PRLR. We also evaluated the subcellular localisation of FYN in primary breast tumours from two cohorts of endocrine-treated ER+ breast cancer patients, one with advanced disease (n = 47) and the other with early disease (n = 76). Findings Treatment with FYN-specific small interfering RNA or a SRC family kinase inhibitor reduced cell growth of tamoxifen-resistant cell lines, while exerting no significant effect on MCF-7/S0.5 cells. Moreover, overexpression of FYN in parental tamoxifen-sensitive MCF-7/S0.5 cells resulted in reduced sensitivity to tamoxifen treatment, while knockdown of FYN in the FYN-overexpressing MCF-7/S0.5 cells restored sensitivity to tamoxifen, demonstrating growth-promoting and survival-promoting function of FYN in MCF-7 cells. FYN knockdown in tamoxifen-resistant cells led to reduced phosphorylation of 14-3-3 and Cdc25A, suggesting that FYN, by activation of important cell cycle-associated proteins, could overcome the antiproliferative effects of tamoxifen. Evaluation of the subcellular localisation of FYN in breast cancer patients showed that in those with advanced disease, plasma membrane-associated FYN expression strongly correlated with longer progression-free survival (p Interpretation Our results indicate that FYN plays an important part in tamoxifen resistance, and its subcellular localisation in breast tumour cells could be an important novel biomarker of response to endocrine therapy in breast cancer.

Glial cell line-derived neurotrophic factor promotes increased phenotypic marker expression in femoral sensory and motor-derived Schwann cell cultures

Schwann cells (SCs) secrete growth factors and extracellular matrix molecules that promote neuronal survival and help guide axons during regeneration. Transplantation of SCs is a promising strategy for enhancing peripheral nerve regeneration. However, we and others have shown that after long-term in vitro expansion, SCs revert to a de-differentiated state similar to the phenotype observed after injury. In vivo, glial cell-line derived neurotrophic factor (GDNF) may guide the differentiation of SCs to remyelinate regenerating axons. Therefore, we hypothesized that exogenous GDNF may guide the differentiation of SCs into their native phenotypes in vitro through stimulation of GDNF family receptor (GFR)α-1. When activated in SCs, GFRα-1 promotes phosphorylation of Fyn, a Src family tyrosine kinase responsible for mediating downstream signaling for differentiation and proliferation. In this study, SCs harvested from the sensory and motor branches of rat femoral nerve were expanded in vitro and then cultured with 50 or 100ng/mL of GDNF. The exogenous GDNF promoted differentiation of sensory and motor-derived SCs back to their native phenotypes, as demonstrated by decreased proliferation after 7days and increased expression of S100Ββ and phenotype-specific markers. Furthermore, inhibiting Fyn with Src family kinase inhibitors, PP2 and SU6656, and siRNA-mediated knockdown of Fyn reduced GDNF-stimulated differentiation of sensory and motor-derived SCs. These results demonstrate that activating Fyn is necessary for GDNF-stimulated differentiation of femoral nerve-derived SCs into their native phenotypes in vitro. Therefore GDNF could be incorporated into SC-based therapies to promote differentiation of SCs into their native phenotype to improve functional nerve regeneration.

The Src family tyrosine kinase, fyn, is a negative regulator of inflammation‐induced apoptosis in the lung (1176.6)

Endothelial cell apoptosis is an early event in the development of acute respiratory distress syndrome (ARDS). We hypothesized that the Src‐family tyrosine kinases (STK) would differentially regulate endothelial cell apoptosis. Human pulmonary microvascular endothelial cells (hPMVEC) were transfected with siRNA against fyn or yes and then treated with cytomix (LPS, TNF‐α, IL‐1β and INF‐γ) or vehicle. Following cytomix treatment knock‐down of fyn resulted in greater levels of caspase 3 activity and lower viable cell numbers, while knock‐down of yes resulted in lower levels of caspase 3 activity and greater viable cell numbers, than in scramble treated control hPMVEC. We then tested the hypothesis that mice deficient in fyn will have greater lung injury following exposure to LPS. Wild‐type and fyn‐/‐ mice were given LPS 10 mg/kg or saline before harvesting tissues or measuring static lung compliance. The fyn‐/‐ mice had lower static lung compliance 24 hours after LPS compared to the WT mice. LPS exposure also caused more cleaved‐caspase 3 and p21 expression in the lungs of fyn‐/‐ mice than in WT mice. Real time‐PCR results showed that following LPS treatment the mRNA levels for arginase I, iNOS, and COX‐2 were greater in the fyn knockout mice than in WT mice. Our results demonstrate that fyn is an important negative regulator of LPS‐induced iNOS expression and apoptosis in cultured endothelial cell and in a mouse model of inflammatory lung disease.

Mechanically activated fyn utilizes mTORC2 to regulate RhoA and adipogenesis in mesenchymal stem cells

Mechanical strain provides an anti-adipogenic, pro-osteogenic stimulus to mesenchymal stem cells (MSC) through generating intracellular signals and via cytoskeletal restructuring. Recently, mTORC2 has been shown to be a novel mechanical target critical for the anti-adipogenic signal leading to preservation of β-catenin. As mechanical activation of mTORC2 requires focal adhesions (FAs), we asked whether proximal signaling involved Src and FAK, which are early responders to integrin-FA engagement. Application of mechanical strain to marrow-derived MSCs was unable to activate mTORC2 when Src family kinases were inhibited. Fyn, but not Src, was specifically required for mechanical activation of mTORC2 and was recruited to FAs after strain. Activation of mTORC2 was further diminished following FAK inhibition, and as FAK phosphorylation (Tyr-397) required Fyn activity, provided evidence of Fyn/FAK cooperativity. Inhibition of Fyn also prevented mechanical activation of RhoA as well as mechanically induced actin stress fiber formation. We thus asked whether RhoA activation by strain was dependent on mTORC2 downstream of Fyn. Inhibition of mTORC2 or its downstream substrate, Akt, both prevented mechanical RhoA activation, indicating that Fyn/FAK affects cytoskeletal structure via mTORC2. We then sought to ascertain whether this Fyn-initiated signal pathway modulated MSC lineage decisions. siRNA knockdown of Fyn, but not Src, led to rapid attainment of adipogenic phenotype with significant increases in adipocyte protein 2, peroxisome proliferator-activated receptor gamma, adiponectin, and perilipin. As such, Fyn expression in mdMSCs contributes to basal cytoskeletal architecture and, when associated with FAs, functions as a proximal mechanical effector for environmental signals that influence MSC lineage allocation.

Abstract 3392: New findings of kinase switch in gastrointestinal stromal tumor under imatinib using phosphoproteomic analysis.

Abstract Despite the initial effectiveness of imatinib for gastrointestinal stromal tumors (GISTs), patients must continue lifelong imatinib therapy, and may eventually relapse owing to acquired drug resistance. Pathology reveals the persistence of viable cells during imatinib therapy, even in the disappearance region upon radiological examination. These cells are thought to cause acquired drug resistance and relapse. To uncover the mechanisms underlying these clinical issues, we used GIST-T1 cells to investigate the phosphoproteomic alterations induced by imatinib treatment. Because some tyrosine kinases might be activated after imatinib exposure, it is important to reveal the increased phosphorylation of proteins on a proteomic scale, and elucidate drug-resistant proteins. Proteins extracted from imatinib-treated or untreated GIST-T1 cells were digested with trypsin. The resulting tyrosine-phosphorylated peptides were purified using an immunoaffinity-enriched and iTRAQ-labeled reagent, and analyzed using mass spectroscopy. Using this phosphoproteomic approach, we identified 171 tyrosine phosphorylation sites spanning 134 proteins. We also detected increased tyrosine phosphorylation of Fyn (Y420) and focal adhesion kinase (FAK) (Y576). Increased phosphorylation was also confirmed by Western blot analysis, and knockdown of Fyn or FAK increased cells’ sensitivity to imatinib. Furthermore, the anti-proliferative effect of imatinib significantly enhanced imatinib-resistant GIST-T1 cells, which exhibited constitutive phosphorylation of FAK when treated with a FAK-selective kinase inhibitor. Moreover, FYN was proved to be a signal of FAK of upper reaches. These results indicate that an iTRAQ-based quantitative phospho-tyrosine-focused phosphoproteomic approach is a powerful method for screening phosphoproteins associated with drug resistance. Fyn or FAK might be potential therapeutic targets to overcome acquired resistance to imatinib in GISTs. Citation Format: Maiko Ako, Tsuyoshi Takahashi, Satoshi Serada, Minoru Fujimoto, Yasuaki Miyazaki, Kazuki Shimada, Takayuki Ikezoe, Takahiro Taguchi, Yukinori Kurokawa, Makoto Yamasaki, Hiroshi Miyata, Kiyokazu Nakajima, Shuji Takiguchi, Masaki Mori, Yuichirou Doki, Tetsuji Naka, Toshirou Nishida. New findings of kinase switch in gastrointestinal stromal tumor under imatinib using phosphoproteomic analysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3392. doi:10.1158/1538-7445.AM2013-3392

AMP-activated protein kinase negatively regulates FcεRI-mediated mast cell signaling and anaphylaxis in mice

Aggregation of FcεRI activates a cascade of signaling events leading to mast cell activation, followed by inhibitory signals that turn off the activating signals. However, the overall view of negative signals in mast cells is still incomplete. Although AMP-activated protein kinase (AMPK), which is generally known as a regulator of energy metabolism, is also associated with anti-inflammation, little is known about the role of AMPK in mast cells. We investigated the role of AMPK and its regulatory mechanism in mast cells. The roles of AMPK in FcεRI-dependent activation of bone marrow-derived mast cells (BMMCs) were evaluated by using chemical agents, small interfering RNAs (siRNAs), or adenovirus that modulated the activity or expression of AMPK signaling components. In addition, AMPKα2(-/-) mice were used to verify the role of AMPK in anaphylactic models. FcεRI signaling and associated effector functions in BMMCs were suppressed by the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) and were conversely augmented by siRNA knockdown of AMPKα2 or liver kinase B1 (LKB1), an upstream kinase of AMPK. Furthermore, AMPKα2 deficiency led to increased FcεRI-mediated BMMC activation and anaphylaxis that were insensitive to AICAR, whereas enforced expression of AMPKα2 in AMPKα2(-/-) BMMCs reversed the hypersensitive FcεRI signaling to normal levels. Pharmacologic inhibition or siRNA knockdown of Fyn mimicked AMPK activation, suggesting that Fyn counterregulates the LKB1-AMPK axis. Mechanistically, Fyn controlled AMPK activity by regulating LKB1 localization. The Fyn-regulated LKB1-AMPK axis acts as a novel inhibitory module for mast cell activation, which points to AMPK activators as therapeutic drugs for allergic diseases.

Src kinase-induced phosphorylation of annexin A2 mediates glucocorticoid resistance in MLL-rearranged infant acute lymphoblastic leukemia

MLL-rearranged infant acute lymphoblastic leukemia (ALL) (<1 year of age) are frequently resistant to glucocorticoids, like prednisone and dexamethasone. As poor glucocorticoid responses are strongly associated with therapy failure, overcoming glucocorticoid resistance may be a crucial step towards improving prognosis. Unfortunately, the mechanisms underlying glucocorticoid resistance in MLL-rearranged ALL largely remain obscure. We here defined a gene signature that accurately discriminates between prednisolone-resistant and prednisolone-sensitive MLL-rearranged infant ALL patient samples, demonstrating that, among other genes, high-level ANXA2 is associated with prednisolone resistance in this type of leukemia. Further investigation demonstrated that the underlying factor of this association was the presence of Src kinase-induced phosphorylation (activation) of annexin A2, a process requiring the adapter protein p11 (encoded by human S100A10). shRNA-mediated knockdown of either ANXA2, FYN, LCK or S100A10, all led to inhibition of annexin A2 phosphorylation and resulted in marked sensitization to prednisolone. Likewise, exposure of prednisolone-resistant MLL-rearranged ALL cells to different Src kinase inhibitors exerting high specificity towards FYN and/or LCK had similar effects. In conclusion, we here present a novel mechanism of prednisolone resistance in MLL-rearranged leukemias, and propose that inhibition of annexin A2 phosphorylation embodies a therapeutic strategy for overcoming resistance to glucocorticoids in this highly aggressive type of leukemia.