Activation Of Src Family Tyrosine Kinases Research Articles

Downregulation of Src-family tyrosine kinases by Srcasm and c-Cbl: A comparative analysis.

AIM:Elevated Src-Family tyrosine kinase (SFK) activity drives carcinogenesis in vivo and elevated SFK activity is found ubiquitously in human cancers. Although human squamous cell carcinomas (SCCs) demonstrate increased SFK activity, in silico analysis of SCCs demonstrates that only 0.4% of lesions contain mutations that could potentially increase SFK activity; similarly, a low frequency of activating SFK mutations is found in other major cancers. These findings indicate that SFK activation in cancers likely is not due to activating mutations but alternative mechanisms. To evaluate potential alternative mechanisms, we evaluated the selectivity of c-Cbl and Srcasm in downregulating native and activated mutant forms of SFKs.MATERIALS AND METHODS:We co-transfected native and activated forms of Src and Fyn with c-Cbl and Srcasm into HaCaT cells and monitored the ability of Srcasm and c-Cbl to downregulate native and activated forms of SFKs by Western blotting. The mechanism of downregulation was probed using mutant forms of Srcasm and c-Cbl and using proteosomal and lysosomal inhibition.RESULTS:The data indicate that Srcasm downregulates native Fyn and Src more effectively than c-Cbl, whereas c-Cbl preferentially downregulates activated SFK mutants, including Fyn Y528F, more effectively than Srcasm. Srcasm downregulates SFKs through a lysosomal-dependent mechanism while c-Cbl utilizes a proteosomal-dependent mechanism.CONCLUSION:Given the rarity of activating SFK mutations in human cancer, these data indicate that decreasing Srcasm level/function may represent a mechanism for increasing SFK activity in SCC and other human tumors.

Src Family Kinases as Therapeutic Targets in Advanced Solid Tumors: What We Have Learned So Far

Src is the prototypal member of Src Family tyrosine Kinases (SFKs), a large non-receptor kinase class that controls multiple signaling pathways in animal cells. SFKs activation is necessary for the mitogenic signal from many growth factors, but also for the acquisition of migratory and invasive phenotype. Indeed, oncogenic activation of SFKs has been demonstrated to play an important role in solid cancers; promoting tumor growth and formation of distant metastases. Several drugs targeting SFKs have been developed and tested in preclinical models and many of them have successfully reached clinical use in hematologic cancers. Although in solid tumors SFKs inhibitors have consistently confirmed their ability in blocking cancer cell progression in several experimental models; their utilization in clinical trials has unveiled unexpected complications against an effective utilization in patients. In this review, we summarize basic molecular mechanisms involving SFKs in cancer spreading and metastasization; and discuss preclinical and clinical data highlighting the main challenges for their future application as therapeutic targets in solid cancer progression

Depletion of Csk preferentially reduces the protein level of LynA in a Cbl-dependent manner in cancer cells

There are eight human Src-family tyrosine kinases (SFKs). SFK members c-Src, c-Yes, Fyn, and Lyn are expressed in various cancer cells. SFK kinase activity is negatively regulated by Csk tyrosine kinase. Reduced activity of Csk causes aberrant activation of SFKs, which can be degraded by a compensatory mechanism depending on Cbl-family ubiquitin ligases. We herein investigated whether all SFK members are similarly downregulated by Cbl-family ubiquitin ligases in cancer cells lacking Csk activity. We performed Western blotting of multiple cancer cells knocked down for Csk and found that the protein levels of the 56 kDa isoform of Lyn (LynA), 53 kDa isoform of Lyn (LynB), c-Src, and Fyn, but not of c-Yes, were reduced by Csk depletion. Induction of c-Cbl protein levels was also observed in Csk-depleted cells. The reduction of LynA accompanying the depletion of Csk was significantly reversed by the knockdown for Cbls, whereas such significant recovery of LynB, c-Src, and Fyn was not observed. These results suggested that LynA is selectively downregulated by Cbls in cancer cells lacking Csk activity.

A Role for Calcium-Activated Adenylate Cyclase and Protein Kinase A in the Lens Src Family Kinase and Na,K-ATPase Response to Hyposmotic Stress.

PurposeNa,K-ATPase activity in lens epithelium is subject to control by Src family tyrosine kinases (SFKs). Previously we showed hyposmotic solution causes an SFK-dependent increase in Na,K-ATPase activity in the epithelium. Here we explored the role of cAMP in the signaling mechanism responsible for the SFK and Na,K-ATPase response.MethodsIntact porcine lenses were exposed to hyposmotic Krebs solution (200 mOsm) then the epithelium was assayed for cAMP, SFK phosphorylation (activation) or Na,K-ATPase activity.ResultsAn increase of cAMP was observed in the epithelium of lenses exposed to hyposmotic solution. In lenses exposed to hyposmotic solution SFK phosphorylation in the epithelium approximately doubled as did Na,K-ATPase activity and both responses were prevented by H89, a protein kinase A inhibitor. The magnitude of the SFK response to hyposmotic solution was reduced by a TRPV4 antagonist HC067047 added to prevent TRPV4-mediated calcium entry, and by a cytoplasmic Ca2+ chelator BAPTA-AM. The Na,K-ATPase activity response in the epithelium of lenses exposed to hyposmotic solution was abolished by BAPTA-AM. As a direct test of cAMP-dependent SFK activation, intact lenses were exposed to 8-pCPT-cAMP, a cell-permeable cAMP analog. 8-pCPT-cAMP caused robust SFK activation. Using Western blot, two calcium-activated adenylyl cyclases, ADCY3 and ADCY8, were detected in lens epithelium.ConclusionsCalcium-activated adenylyl cyclases are expressed in the lens epithelium and SFK activation is linked to a rise of cAMP that occurs upon hyposmotic challenge. The findings point to cAMP as a link between TRPV4 channel-mediated calcium entry, SFK activation, and a subsequent increase of Na,K-ATPase activity.

Polymeric immunoglobulin receptor promotes tumor growth in hepatocellular carcinoma.

Our findings reveal the molecular mechanism by which pIgR promotes cancer malignancy, suggest the clinical potential of targeting this pathway in HCC, and provide new insight into the oncogenic role of immunoglobulin receptors. (Hepatology 2017;65:1948-1962).

Oncogenic signaling by Kit tyrosine kinase occurs selectively on the Golgi apparatus in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are caused by gain-of-function mutations in the Kit receptor tyrosine kinase. Most primary GIST patients respond to the Kit inhibitor imatinib, but this drug often becomes ineffective because of secondary mutations in the Kit kinase domain. The characteristic intracellular accumulation of imatinib-sensitive and -resistant Kit protein is well documented, but its relationship to oncogenic signaling remains unknown. Here, we show that in cancer tissue from primary GIST patients as well as in cell lines, mutant Kit accumulates on the Golgi apparatus, whereas normal Kit localizes to the plasma membrane (PM). In imatinib-resistant GIST with a secondary Kit mutation, Kit localizes predominantly on the Golgi apparatus. Both imatinib-sensitive and imatinib-resistant Kit (Kit(mut)) become fully auto-phosphorylated only on the Golgi and only if in a complex-glycosylated form. Kit(mut) accumulates on the Golgi during the early secretory pathway, but not after endocytosis. The aberrant kinase activity of Kit(mut) prevents its export from the Golgi to the PM. Furthermore, Kit(mut) on the Golgi signals and activates the phosphatidylinositol 3-kinase–Akt (PI3K–Akt) pathway, signal transducer and activator of transcription 5 (STAT5), and the Mek–Erk pathway. Blocking the biosynthetic transport of Kit(mut) to the Golgi from the endoplasmic reticulum inhibits oncogenic signaling. PM localization of Kit(mut) is not required for its signaling. Activation of Src-family tyrosine kinases on the Golgi is essential for oncogenic Kit signaling. These results suggest that the Golgi apparatus serves as a platform for oncogenic Kit signaling. Our study demonstrates that Kit(mut)’s pathogenicity is related to its mis-localization, and may offer a new strategy for treating imatinib-resistant GISTs.

A Computational Model for the AMPA Receptor Phosphorylation Master Switch Regulating Cerebellar Long-Term Depression

The expression of long-term depression (LTD) in cerebellar Purkinje cells results from the internalisation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) from the postsynaptic membrane. This process is regulated by a complex signalling pathway involving sustained protein kinase C (PKC) activation, inhibition of serine/threonine phosphatase, and an active protein tyrosine phosphatase, PTPMEG. In addition, two AMPAR-interacting proteins–glutamate receptor-interacting protein (GRIP) and protein interacting with C kinase 1 (PICK1)–regulate the availability of AMPARs for trafficking between the postsynaptic membrane and the endosome. Here we present a new computational model of these overlapping signalling pathways. The model reveals how PTPMEG cooperates with PKC to drive LTD expression by facilitating the effect of PKC on the dissociation of AMPARs from GRIP and thus their availability for trafficking. Model simulations show that LTD expression is increased by serine/threonine phosphatase inhibition, and negatively regulated by Src-family tyrosine kinase activity, which restricts the dissociation of AMPARs from GRIP under basal conditions. We use the model to expose the dynamic balance between AMPAR internalisation and reinsertion, and the phosphorylation switch responsible for the perturbation of this balance and for the rapid plasticity initiation and regulation. Our model advances the understanding of PF-PC LTD regulation and induction, and provides a validated extensible platform for more detailed studies of this fundamental synaptic process.

LynA regulates an inflammation-sensitive signaling checkpoint in macrophages.

Clustering of receptors associated with immunoreceptor tyrosine-based activation motifs (ITAMs) initiates the macrophage antimicrobial response. ITAM receptors engage Src-family tyrosine kinases (SFKs) to initiate phagocytosis and macrophage activation. Macrophages also encounter nonpathogenic molecules that cluster receptors weakly and must tune their sensitivity to avoid inappropriate responses. To investigate this response threshold, we compared signaling in the presence and absence of receptor clustering using a small-molecule inhibitor of Csk, which increased SFK activation and produced robust membrane-proximal signaling. Surprisingly, receptor-independent SFK activation led to a downstream signaling blockade associated with rapid degradation of the SFK LynA. Inflammatory priming of macrophages upregulated LynA and promoted receptor-independent signaling. In contrast, clustering the hemi-ITAM receptor Dectin-1 induced signaling that did not require LynA or inflammatory priming. Together, the basal-state signaling checkpoint regulated by LynA expression and degradation and the signaling reorganization initiated by receptor clustering allow cells to discriminate optimally between pathogens and nonpathogens.

Prolactin and growth hormone affect metaphase-II chromosomes in aging oocytes via cumulus cells using similar signaling pathways.

General senescence of the adult organism is closely connected with reproductive one. Meanwhile, the age-related reduction in the female fertility is primarily associated with a decline in the gamete quality. Molecular and cellular changes in oocytes of old mammalian females are very similar to those occurring during aging of matured ova of their young counterparts, suggesting similarities in underlying mechanisms. The aim of the present work was to study actions of two related pituitary hormones, prolactin (PRL) and growth hormone (GH), on age-associated modifications of metaphase-II (M-II) chromosomes in bovine oocytes using a model of the prolonged culture. We analyzed: (1) effects of PRL and GH on abnormal changes in the chromosome morphology in aging matured oocytes and the role of cumulus cells in these effects and (2) signaling pathways involved in the hormone actions. During the prolonged culture of oocytes, a gradual rise in the frequency of destructive modifications of M-II chromosomes was revealed. In the case of cumulus-enclosed oocytes (CEOs), PRL and GH exerted dose-dependent biphasic effects on the frequency of these modifications. Both PRL (50 ng/ml) and GH (10 ng/ml) decelerated the abnormal chromosome changes in CEOs, but did not affect the chromosome configuration in denuded oocytes. Concurrently, the presence of PRL and GH receptors in cumulus cells surrounding matured oocytes was demonstrated. Attenuating effects of both hormones on the chromosome modifications in aging CEOs were abolished by PP2 (an inhibitor of Src-family tyrosine kinases), triciribine (an inhibitor of Akt kinase), and calphostin C (a protein kinase C inhibitor). Our findings indicate that PRL and GH can exert the similar decelerating action on age-associated alterations in the M-II chromosome morphology in bovine ova, which is mediated by cumulus cells and may be related to activation of Src-family tyrosine kinases as well as Akt- and protein kinase C-dependent signal pathways.

Abstract 806: Modeling human UV-induced skin cancer through genetic manipulation of Src-kinases and Srcasm

Abstract UV-induced skin cancer is the most common form of cancer and its incidence continues to increase. Novel in vivo models that mimic human UV-induced skin cancer are needed to further studies and evaluate new therapies. Keratinocyte growth is regulated through oncogenic signals from EGFR and Src-family tyrosine kinases (SFKs); these tyrosine kinases are primary drivers of keratinocyte growth in UV-induced skin cancer. At the proteomic level, activated EGFR and SFKs are negatively regulated by Srcasm which targets activated tyrosine kinases for lysosomal degradation. Studies have shown that SFK activity is increased and Srcasm levels are decreased in human cutaneous squamous cell carcinomas and related precancerous lesions. We hypothesized that genetic downregulation of Srcasm by gene deletion or lentivirally mediated shRNA knockdown would promote oncogenic signaling and keratinocyte growth thereby promoting UV-induced skin cancer. To test this hypothesis Srcasm null mice and Srcasm lentiviral knockdown in human keratinocytes were generated and studied. Western blotting of epidermal lysates from Srcasm null mice demonstrates that loss of Srcasm promotes activation of EGFR and Src kinases and decreases levels of p53, NICD and p21. Activation of the canonical Erk1/2, STAT-3 and PDK-1 oncogenic pathways also was seen in Srcasm null keratinocytes. The epidermis of Srcasm null mice exhibits a higher Ki-67 index of 8.7 +/- 2.0 compared to 1.5 +/- 1.1 in controls (p<0.001). UVB irradiation (500 mJ/cm2 × 15 doses) produced precancerous lesions in Srcasm null mice resembling human actinic keratoses but not in controls; the precancerous lesions show an increased Ki-67 index and activation of Src kinases, STAT-3 and PDK-1. Acute UVB irradiation (1200 mJ/cm2 × 1 dose) of Srcasm null mice induced prominent erythema, epidermal necrosis and a prominent increase in the TUNEL-positive index (14.0 vs 1.3). Srcasm knockdown in human keratinocytes accelerated keratinocyte growth 130%; it also increased levels of EGFR and accelerated the kinetics of EGFR and MAPK activation in response to EGF stimulation. In human keratinocytes, Srcasm knockdown accelerated cell growth post-UV exposure which could promote neoplasia. These data show that loss of Srcasm is associated with increased oncogenic signaling, impaired p53 function and keratinocyte proliferation. Loss of Srcasm promotes keratinocyte proliferation post-UV exposure leading to the early stages of UVB-induced cutaneous neoplasia. Citation Format: Xiaoping Yang, Sheila Shankar, Hiroshi Maeno, Micheal Gober, Christine Marshall, Tzvete Dentchev, John Seykora. Modeling human UV-induced skin cancer through genetic manipulation of Src-kinases and Srcasm. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 806. doi:10.1158/1538-7445.AM2015-806

Effects of activation of Src-family tyrosine kinases on human LECs apoptosis and EMT under the high glucose environment

Background Diabetic cataract is one of the major ocular complications in diabetes mellitus, including cortical cataract, nuclear cataract, subcapsular cataract and mixed cataract, and different cataractogenesis may be associated with lens epithelial cells (LECs). Subcapsular cataract is one of diabetic cataracts.Studying the biological behavior of LECs in subcapsular cataract is crucial for prevention and treatment. Objective This study was to investigate the effects of Src-family tyrosine kinases (SFKs) on apoptosis and epithelial-mesenchymal transition (EMT) of human LECs cultured by high glucose. Methods Human LECs (HLE-B3) were cultured for 24 hours with DMEM containing 5.5 mmol/L glucose (normal control group), DMEM containing 35.5 mmol/L glucose (high glucose group) and DMEM containing 35.5 mmol/L glucose + 10 μmol/L PP1, a specific inhibitor of SFKs (PP1 group). In 3, 6, 12 and 24 hours after culture, the apoptosis of human LECs was detected by flow cytometry assay; morphological change of human LECs was observed under the inverted microscope, and the expressions of the markers of EMT, E-cadherin and α-smooth muscle actin (α-SMA), in the cells were detected by immnofluorescence staining.In addition, the alternations of p-Src418 (active c-Src), bcl-xl, survivin, caspase-3, E-cadherin and α-SMA proteins were assayed by Western blot analysis. Results An elevated expression level of p-Src418 was found in LECs in the high glucose group and peaked 6 hours after cultured.The expressions of p-Src418 (grey levels) were 0.125±0.036 in the high glucose group, and which was significantly higher than 0.042±0.011 in the normal control group and 0.035±0.018 in the PP1 group, respectively (both at P 0.05). The apoptotic rates of human LECs were(6.433±2.084)%, (10.333±2.610)% and (8.033±2.967)% in the PP1 group, which were higher than (3.233±1.320)%, (3.533±1.159)%, (5.733±0.230)% in the high glucose group and (3.133±1.170)%, (2.833±0.751)%, (3.333±1.201)% in the normal control group (all at P 0.05). In 6 hours and 12 hours after cell culture, the expression levels of bcl-xl and survivin (grey values) in human LECs were significantly declined, but the expression of caspase-3 was increased in the PP1 group compared with the the high glucose group and the normal control group (all at P<0.05). The LECs showed slender in shape 24 hours after culture in the high glucose group, but the cell shape was close to the normal in the PP1 group.Western blot and immunofluorescence assay revealed that the expression of E-cadherin in human LECs was significantly reduced and that of α-SMA was significantly increased 6 hours after culture in the high glucose group compared with the PP1 group and the normal control group (all at P<0.05). Conclusions High glucose activates c-Src kinase of LECs in high glucose environment and therefore induces EMT and inhibits apoptosis.However, PP1 impedes the biological process of EMT and apoptosis of LECs to maintain the epithelial characteristics even under the stress of high glucose. Key words: Glucose; Human; Epithelial cell, Lens; Apoptosis; Src-family tyrosine kinases; Epithelial-mesenchymal transition; Signaling pathway

Dasatinib enhances antitumor activity of paclitaxel in ovarian cancer through Src signaling.

Src family tyrosine kinase (SFK) activation is associated with ovarian cancer progression. Therefore, SFKs are targets for the development of potential treatments of ovarian cancer. Dasatinib is a tyrosine kinase inhibitor that targets SFK activity, and is used for the treatment of B cell and Abelson lymphomas. At the present time, the potential effect of dasatinib on ovarian cancer is not clear. The aim of the present study was to investigate the antitumor activity of dasatinib, alone and in combination with paclitaxel, in ovarian cancer in vitro and in vivo. In the present study, the expression of Src and phospho-Src-Y416 (p-Src) was measured in six ovarian cancer cell lines using western blotting and immunohistochemistry. In addition, cell viability and apoptosis were measured using an MTT assay and annexin V-fluorescein isothiocyanate staining. An ovarian cancer murine xenograft model was established, in order to evaluate the antitumor effect of dasatinib alone and in combination with paclitaxel in ovarian cancer. High levels of p-Src protein expression were observed in all cell lines, as compared with healthy cells, which indicated activation of the Src signaling pathway. p-Src expression increased in ovarian cancer cells following paclitaxel treatment. Dasatinib treatment demonstrated anti-ovarian cancer properties, by downregulating p-Src expression and by inducing cancer cell apoptosis. Combined treatment with dasatinib and paclitaxel markedly inhibited proliferation and promoted apoptosis of ovarian cancer cells, compared with control cells. Combined dasatinib and paclitaxel treatment exhibited antitumor activities in vivo and in vitro (combination indices, 0.25–0.93 and 0.31–0.75; and tumor growth inhibitory rates, 76.7% and 58.5%, in A2780 and HO8910 cell lines, respectively), compared with paclitaxel treatment alone. Dasatinib monotherapy demonstrated anti-ovarian cancer activities. The effects of dasatinib and paclitaxel treatments on ovarian cancer cells appeared to be mediated by the Src pathway.

TRPV4 Channels Enable Na,K‐ATPase Activity in the Lens Epithelium to Respond to a Remote Stimulus

Na,K-ATPase activity is low or absent in the fiber cells that make up most of the eye lens. Ion and water homeostasis of the entire structure is made possible by Na,K-ATPase activity in a single epithelial cell layer at the anterior lens surface. Previously we have shown Src family tyrosine kinase-dependent changes of Na,K-ATPase activity in the epithelium. Here we provide evidence that the epithelium is able to increase Na,K-ATPase activity in response to a remote stimulus in the fiber mass. The posterior pole of intact pig lenses was subjected to localized damage either by a 2.5 mm diameter freeze-thaw wound or by injection of 5 µl of hyperosmotic saline just beneath the surface. After 2-30 min the epithelium was removed from the lens, homogenized and the rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was determined. Src family tyrosine kinase (SFK) activation (phosphorylation) was assessed by Western blot. Localized damage to lens fibers at posterior pole caused SFK activation and a significant increase in Na,K-ATPase activity in the epithelium that occurred within minutes. Na,K-ATPase activity almost doubled. The increase in Na,K-ATPase activity was prevented by the SFK inhibitor PP2 (10 µM). Importantly, the SFK phosphorylation response as well as the increase in Na,K-ATPase activity were suppressed by a TRPV4 channel antagonist, HC067947 (10 µM). By Western blot, TRPV4 was detected in the epithelium but not fiber cells. The findings point to a TRPV4-dependent mechanism in the anterior epithelium that enables the cells to sense remote damage in the fiber mass and respond by activating SFK and increasing Na,K-ATPase activity.

Csk-homologous kinase (Chk/Matk): a molecular policeman suppressing cancer formation and progression

Aberrant activation of Src-family tyrosine kinases (SFKs) directs initiation of metastasis and development of drug resistance in multiple solid tumors and hematological cancers. Since oncogenic mutations of SFKs are rare events, aberrant activation of SFKs in cancer is likely due to dysregulation of the two major upstream inhibitors: C-terminal Src kinase (Csk) and its homolog Csk-homologous kinase (Chk/Matk). Csk and Chk/Matk inhibit SFKs by selectively phosphorylating the inhibitory tyrosine residue at their C-terminal tail. Additionally, Chk/Matk can also employ a noncatalytic inhibitory mechanism to inhibit multiple active forms of SFKs, suggesting that Chk/Matk is a versatile inhibitor capable of constraining the activity of multiple active forms of SFKs. Mounting evidence suggests that Chk/Matk is a potential tumor suppressor downregulated by epigenetic silencing and/or missense mutations in several cancers such as colorectal and lung carcinoma. In spite of the potential significance of Chk/Matk in cancer, little is known about its structure and regulation. This review focuses on the mechanisms by which Chk/Matk expression and activity is downregulated in cancers. Specifically, we assessed the evidence demonstrating downregulation of Chk/Matk by epigenetic silencing and missense mutations in cancers. The other focus is the tumor suppressive mechanism of Chk/Matk. The final focus of the review is on the clinical applications of the investigations into the mechanism of epigenetic silencing of Chk/Matk expression and the tumor suppressive mechanism of Chk/Matk; specifically we discussed how they can benefit the development of biomarkers for early diagnosis of cancers and specific SFK inhibitors for use as cancer therapeutics.

Abstract B73: Integration of a personalized molecular targeted therapy into the multimodal treatment of refractory early childhood sPNET

Abstract Background: Supratentorial primitive neuroectodermal tumors (sPNET) are highly-malignant and rare brain tumors that primarily affect young children. Despite multimodal treatment that includes surgery, radiotherapy and chemotherapy, less than half of affected patients will survive 5 years post-diagnosis. Moreover, neurocognitive impairment associated with current treatment protocols is a significant complication in survivors. Therefore, new molecular targeted therapies that are effective and less toxic are needed to further improve outcomes in sPNET patients. Tyrosine kinase inhibitors (TKI) have emerged as an important class of molecular targeted anti-cancer agents with demonstrated clinical efficacy and generally favorable toxicity profiles. Dasatinib, an orally-administered TKI that is FDA approved for Ph+ CML/ALL, has been reported in off-label use for cancers of the head and neck, breast, colon, prostate and lung. Dasatinib targets multiple tyrosine kinases that are involved in numerous cancers, including ABL, SRC family tyrosine kinases (SFKs), and receptor tyrosine kinases c-KIT and PDGFR. Notably, SFKs have been implicated in driving the development and progression of numerous solid tumors, including PNET and medulloblastoma. Methods: Here we report the successful use of dasatinib with informed consent in multimodal treatment of a 32 month old, ex-29 week twin B female patient. She was diagnosed with sPNET that demonstrated high tumor SFK-activity, had progressed on standard chemotherapy, and for whom irradiation was avoided due to the unwanted side effects of radiation on the developing brain. Multiplex kinase-activity profiling, and electron microscopy analysis was performed on pre- and post-dasatinib treatment tumor tissue after partial resections. Additionally, pre-and post-treatment MRI was done to quantitate the clinical response with dasatinib. Results: Ex vivo multiplex analysis revealed aberrant activation of SFKs SRC, LYN and FYN in the sPNET tumor, which was shown to be significantly reduced following dasatinib therapy. In addition, blockade of SFK activity with dasatinib correlated with greatly reduced activation of downstream proliferative transcription factors STAT1 and STAT3. Electron microscopy analysis of the tumor upon surgical resection revealed phenotypic changes with significant presence of autophagocytic vacuoles, indicative of cellular sequestration, and increased prominence of nuclear heterochromatin. Dasatinib therapy over a 4-month period was very well-tolerated by the patient and resulted in significant tumor reduction as determined by radiographic detection. Conclusions: We have demonstrated how tyrosine kinase activity-based tumor profiling can be utilized to devise a successful personalized molecular targeted multimodal treatment strategy in a patient for whom standard therapy had failed. Dasatinib was clinically and molecularly effective in the treatment of a progressive refractory sPNET displaying high-SFK activity. These results could aid future patient stratification schemes that direct targeted therapies based upon an aberrant molecular pathway strategy, therefore larger clinical studies using this approach are warranted. Citation Format: Lisa LR Hartman, Diba Farah, Arlynn F. Mulne, Bruce B. Storrs, Harry L. Wilson, Staci Bryson, Derrick M. Oaxaca, Robert A. Kirken, Jeremy A. Ross. Integration of a personalized molecular targeted therapy into the multimodal treatment of refractory early childhood sPNET. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B73.

Activation of Src family tyrosine kinases by ferric ions

The Src-family tyrosine kinases (SFKs) are oncogenic enzymes that contribute to the initiation and progression of many types of cancer. In normal cells, SFKs are kept in an inactive state mainly by phosphorylation of a consensus regulatory tyrosine near the C-terminus (Tyr530 in the SFK c-Src). As recent data indicate that tyrosine modification enhances binding of metal ions, the hypothesis that SFKs might be regulated by metal ions was investigated. The c-Src C-terminal peptide bound two Fe3+ ions with affinities at pH4.0 of 33 and 252μM, and phosphorylation increased the affinities at least 10-fold to 1.4 and 23μM, as measured by absorbance spectroscopy. The corresponding phosphorylated peptide from the SFK Lyn bound two Fe3+ ions with much higher affinities (1.2pM and 160nM) than the Src C-terminal peptide. Furthermore, when Lyn or Hck kinases, which had been stabilised in the inactive state by phosphorylation of the C-terminal regulatory tyrosine, were incubated with Fe3+ ions, a significant enhancement of kinase activity was observed. In contrast Lyn or Hck kinases in the unphosphorylated active state were significantly inhibited by Fe3+ ions. These results suggest that Fe3+ ions can regulate SFK activity by binding to the phosphorylated C-terminal regulatory tyrosine.

Distinct roles for N‐Cadherin linked c‐Src and fyn kinases in lens development

Src family tyrosine kinases (SFKs) are often coincidently expressed but few studies have dissected their individual functions in the same cell during development. Using the classical embryonic lens as our model, we investigated SFK signaling in the regulation of both differentiation initiation and morphogenesis, and the distinct functions of c-Src and Fyn in these processes. Blocking SFK activity with the highly specific inhibitor PP1 induced initiation of the lens differentiation program but blocked lens fiber cell elongation and organization into mini lens-like structures called lentoids. These dichotomous roles for SFK signaling were discovered to reflect distinct functions of c-Src and Fyn and their differentiation-state-specific recruitment to and action at N-cadherin junctions. c-Src was highly associated with the nascent N-cadherin junctions of undifferentiated lens epithelial cells. Its siRNA knockdown promoted N-cadherin junctional maturation, blocked proliferation, and induced lens cell differentiation. In contrast, Fyn was recruited to mature N-cadherin junctions of differentiating lens cells and siRNA knockdown suppressed differentiation-specific gene expression and blocked morphogenesis. Through inhibition of N-cadherin junction maturation, c-Src promotes lens epithelial cell proliferation and the maintenance of the lens epithelial cell undifferentiated state, while Fyn, signaling downstream of mature N-cadherin junctions, promotes lens fiber cell morphogenesis.

Activation of Src, Fyn and Yes non-receptor tyrosine kinases in keratinocytes expressing human papillomavirus (HPV) type 16 E7 oncoprotein

BackgroundThe Src family tyrosine kinases (SFK) are cellular regulatory proteins that influence cell adhesion, proliferation, invasion and survival during tumor development. Elevated activity of Src was associated with increased cell proliferation and invasivity in human papillomavirus (HPV)-associated malignancies; therefore, transduced human foreskin keratinocytes (HFK) were used to investigate whether SFK activation is a downstream effect of papillomaviral oncoproteins. Activation of ubiquitously expressed SFKs, namely Src, Yes and Fyn, was investigated in both proliferating and differentiating keratinocytes.ResultsIn proliferating keratinocytes, Src, Yes and Fyn mRNA levels were not affected by HPV 16 E6 or E7 oncoproteins, while at the protein level as detected by western blot, the presence of both E6 and E7 resulted in substantial increase in Src and Yes expression, but did not alter the high constitutive level of Fyn. Phospo-kinase array revealed that all ubiquitously expressed SFKs are activated by phosphorylation in the presence of HPV 16 E7 oncoprotein. Keratinocyte differentiation led to increased Yes mRNA and protein levels in all transduced cell lines, while it did not influence the Src transcription but resulted in elevated Src protein level in HPV16 E7 expressing lines.ConclusionsThis study revealed that HPV 16 oncoproteins upregulate Src family kinases Src and Yes via posttranscriptional mechanisms. A further effect of HPV 16 E7 oncoprotein is to enhance the activating phosphorylation of SFKs expressed in keratinocytes.

Phosphatidylinositol‐3 kinase activity in B cells is negatively regulated by Lyn tyrosine kinase

Phosphatidylinositol-3 kinase (PI3K) activity is essential for normal B-cell receptor (BCR)-mediated responses. To understand the mechanisms of PI3K regulation during B-cell activation, we performed a series of biochemical analysis on primary B cells, and found that activity of Src family tyrosine kinases (SFK) is crucial for the activation of PI3K following BCR ligation and this is regulated by the SFK Lyn. We show that the hyperresponsive phenotype of B cells lacking Lyn is predicated on significantly increased basal and inducible PI3K activity that correlates with the constitutive hypophosphorylation of PAG/Cbp (phosphoprotein associated with glycosphingolipid-enriched microdomains/Csk-binding protein), a concomitant reduction in bound Csk in Lyn(-/-) B cells and elevated levels of active Fyn. Regulating SFK activity may thus be a central mechanism by which Lyn regulates PI3K activity in B cells. This study defines the molecular connection between the BCR and PI3K and reveals this to be a point of Lyn-mediated regulation.

Src family tyrosine kinase regulates acrosome reaction but not motility in porcine spermatozoa

During the capacitation process, spermatozoa acquire the ability to fertilize an oocyte, and upregulation of cAMP-dependent protein tyrosine phosphorylation occurs. Recently, Src family tyrosine kinase (SFK) has been involved in spermatozoa capacitation as a key PKA-dependent tyrosine kinase in several species. This work investigates the expression and role of SFK in porcine spermatozoa. SFK members Lyn and Yes are identified in porcine spermatozoa by western blotting as well as two proteins named SFK1 and SFK2 were also detected by their tyrosine 416 phosphorylation, a key residue for SFK activation. Spermatozoa with SFK1 and SFK2 increase their Y416 phosphorylation time-dependently under capacitating conditions compared with noncapacitating conditions. The specific SFK inhibitor SU6656 unaffected porcine spermatozoa motility or viability. Moreover, SFK inhibition in spermatozoa under capacitating conditions leads to a twofold increase in both nonstimulated and calcium-induced acrosome reaction. Our data show that capacitating conditions lead to a time-dependent increase in actin polymerization in boar spermatozoa and that long-term incubation with SFK inhibitor causes a reduction in the F-actin content. In summary, this work shows that the SFK members Lyn and Yes are expressed in porcine spermatozoa and that SFK1 and SFK2 are phosphorylated (activated) during capacitation. Our results point out the important role exerted by SFK in the acrosome reaction, likely mediated in part by its involvement in the actin polymerization process that accompanies capacitation, and rule out its involvement in porcine spermatozoa motility.