Src Family Kinase Signaling Research Articles

Abstract 3404: Inhibiting EGF receptor or SRC family kinase signaling overcomes BRAF inhibitor resistance in melanoma.

Abstract We generated melanoma cell lines resistant to BRAF inhibitors and show that the EGF receptor (EGFR)-SRC family kinase (SFK)-STAT3 signaling pathway was upregulated in these cells. In addition to driving proliferation of resistant cells, this pathway also stimulated invasion and metastasis. EGFR inhibitors cooperated with BRAF inhibitors to block the growth of the resistant cells in vitro and in vivo, and monotherapy with the broad specificity tyrosine kinase inhibitor dasatinib blocked growth and metastasis in vivo. We analyzed a tumor from a BRAF mutant patient with intrinsic resistance to vemurafenib. Vemurafenib treatment induced SFK activation in this tumor and we show that dasatinib blocked its growth and metastasis in immunocompromised mice. Our data shows that BRAF inhibitor-mediated activation of EFGR/SFK/STAT3 signaling can mediate resistance in BRAF mutant melanoma patients. We describe two treatments that appear to overcome this resistance and could deliver therapeutic efficacy in drug-resistant BRAF mutant melanoma patients. Citation Format: Maria R. Girotti, Malin Pedersen, Berta Sanchez-Laorden, Amaya Viros, Samra Turajlic, Dan Niculescu-Duvaz, Alfonso Zambon, John Sinclair, Andrew Hayes, Martin Gore, Caroline Springer, James Larkin, Claus Jorgensen, Richard Marais. Inhibiting EGF receptor or SRC family kinase signaling overcomes BRAF inhibitor resistance in melanoma. [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 3404. doi:10.1158/1538-7445.AM2013-3404

The phosphatase CD148 promotes airway hyperresponsiveness through SRC family kinases

Increased airway smooth muscle (ASM) contractility and the development of airway hyperresponsiveness (AHR) are cardinal features of asthma, but the signaling pathways that promote these changes are poorly understood. Tyrosine phosphorylation is tightly regulated by the opposing actions of protein tyrosine kinases and phosphatases, but little is known about whether tyrosine phosphatases influence AHR. Here, we demonstrate that genetic inactivation of receptor-like protein tyrosine phosphatase J (Ptprj), which encodes CD148, protected mice from the development of increased AHR in two different asthma models. Surprisingly, CD148 deficiency minimally affected the inflammatory response to allergen, but significantly altered baseline pulmonary resistance. Mice specifically lacking CD148 in smooth muscle had decreased AHR, and the frequency of calcium oscillations in CD148-deficient ASM was substantially attenuated, suggesting that signaling pathway alterations may underlie ASM contractility. Biochemical analysis of CD148-deficient ASM revealed hyperphosphorylation of the C-terminal inhibitory tyrosine of SRC family kinases (SFKs), implicating CD148 as a critical positive regulator of SFK signaling in ASM. The effect of CD148 deficiency on ASM contractility could be mimicked by treatment of both mouse trachea and human bronchi with specific SFK inhibitors. Our studies identify CD148 and the SFKs it regulates in ASM as potential targets for the treatment of AHR.

Targeting Src Family Kinases Inhibits Bevacizumab-Induced Glioma Cell Invasion

Anti-VEGF antibody therapy with bevacizumab provides significant clinical benefit in patients with recurrent glioblastoma multiforme (GBM). Unfortunately, progression on bevacizumab therapy is often associated with a diffuse disease recurrence pattern, which limits subsequent therapeutic options. Therefore, there is an urgent need to understand bevacizumab's influence on glioma biology and block it's actions towards cell invasion.To explore the mechanism(s) of GBM cell invasion we have examined a panel of serially transplanted human GBM lines grown either in short-term culture, as xenografts in mouse flank, or injected orthotopically in mouse brain. Using an orthotopic xenograft model that exhibits increased invasiveness upon bevacizumab treatment, we also tested the effect of dasatinib, a broad spectrum SFK inhibitor, on bevacizumab-induced invasion.We show that 1) activation of Src family kinases (SFKs) is common in GBM, 2) the relative invasiveness of 17 serially transplanted GBM xenografts correlates strongly with p120 catenin phosphorylation at Y228, a Src kinase site, and 3) SFK activation assessed immunohistochemically in orthotopic xenografts, as well as the phosphorylation of downstream substrates occurs specifically at the invasive tumor edge. Further, we show that SFK signaling is markedly elevated at the invasive tumor front upon bevacizumab administration, and that dasatinib treatment effectively blocked the increased invasion induced by bevacizumab.Our data are consistent with the hypothesis that the increased invasiveness associated with anti-VEGF therapy is due to increased SFK signaling, and support testing the combination of dasatinib with bevacizumab in the clinic.

Loss-of-function screen in rhabdomyosarcoma identifies CRKL-YES as a critical signal for tumor growth

To identify novel signaling pathways necessary for rhabdomyosarcoma (RMS) survival, we performed a loss-of-function screen using an inducible small hairpin RNA (shRNA) library in an alveolar and an embryonal RMS cell line. This screen identified CRKL expression as necessary for growth of alveolar RMS and embryonal RMS both in vitro and in vivo. We also found that CRKL was uniformly highly expressed in both RMS cell lines and tumor tissue. As CRKL is a member of the CRK adapter protein family that contains an SH2 and two SH3 domains and is involved in signal transduction from multiple tyrosine kinase receptors, we evaluated CRKL interaction with multiple tyrosine kinase receptor signaling pathways in RMS cells. While we saw no interaction of CRKL with IGFIR, MET or PI3KAKT/mTOR pathways, we determined that CRKL signaling was associated with SRC family kinase (SFK) signaling, specifically with YES kinase. Inhibition of SFK signaling with dasatinib or another SFK inhibitor, sarcatinib, suppressed RMS cell growth in vitro and in vivo. These data identify CRKL as a novel critical component of RMS growth. This study also demonstrates the use of functional screening to identify a potentially novel therapeutic target and treatment approach for these highly aggressive pediatric cancers.

Early redox, Src family kinase, and calcium signaling integrate wound responses and tissue regeneration in zebrafish

Tissue injury can lead to scar formation or tissue regeneration. How regenerative animals sense initial tissue injury and transform wound signals into regenerative growth is an unresolved question. Previously, we found that the Src family kinase (SFK) Lyn functions as a redox sensor in leukocytes that detects H(2)O(2) at wounds in zebrafish larvae. In this paper, using zebrafish larval tail fins as a model, we find that wounding rapidly activated SFK and calcium signaling in epithelia. The immediate SFK and calcium signaling in epithelia was important for late epimorphic regeneration of amputated fins. Wound-induced activation of SFKs in epithelia was dependent on injury-generated H(2)O(2). A SFK member, Fynb, was responsible for fin regeneration. This work provides a new link between early wound responses and late regeneration and suggests that redox, SFK, and calcium signaling are immediate "wound signals" that integrate early wound responses and late epimorphic regeneration.

Fyn regeneration

A Src family kinase and several other signaling pathways help repair zebrafish fins after wounding.

Retracted: Ethanol disrupts axon outgrowth stimulated by netrin‐1, GDNF, and L1 by blocking their convergent activation of Src family kinase signaling

Pre-natal alcohol exposure causes fetal alcohol spectrum disorders (FASD), the most common, preventable cause of developmental disability. The developing cerebellum is particularly vulnerable to the effects of ethanol. We reported that ethanol inhibits the stimulation of axon outgrowth in cerebellar granule neurons (CGN) by NAP, an active motif of activity-dependent neuroprotective protein (ADNP), by blocking NAP activation of Fyn kinase and its downstream signaling molecule, the scaffolding protein Cas. Here, we asked whether ethanol inhibits the stimulation of axon outgrowth by diverse axon guidance molecules through a common action on the Src family kinases (SFK). We first demonstrated that netrin-1, glial cell line-derived neurotrophic factor (GDNF), and neural cell adhesion molecule L1 stimulate axon outgrowth in CGNs by activating SFK, Cas, and extracellular signal-regulated kinase 1 and 2 (ERK1/2). The specific SFK inhibitor, PP2, blocked the stimulation of axon outgrowth and the activation of the SFK-Cas-ERK1/2 signaling pathway by each of these axon-guidance molecules. In contrast, brain-derived neurotrophic factor (BDNF) stimulated axon outgrowth and activated ERK1/2 without first activating SFK or Cas. Clinically relevant concentrations of ethanol inhibited axon outgrowth and the activation of the SFK-Cas-ERK1/2 pathway by netrin-1, GDNF, and L1, but did not disrupt BDNF-induced axon outgrowth or ERK1/2 activation. These results indicate that SFK, but not ERK1/2, is a primary target for ethanol inhibition of axon outgrowth. The ability of ethanol to block the convergent activation of the SFK-Cas-ERK1/2 pathway by netrin-1, GDNF, L1, and ADNP could contribute significantly to the pathogenesis of FASD.

Neutrophil Extracellular Traps Exhibit Antibacterial Activity against Burkholderia pseudomallei and Are Influenced by Bacterial and Host Factors

Burkholderia pseudomallei is the causative pathogen of melioidosis, of which a major predisposing factor is diabetes mellitus. Polymorphonuclear neutrophils (PMNs) kill microbes extracellularly by the release of neutrophil extracellular traps (NETs). PMNs play a key role in the control of melioidosis, but the involvement of NETs in killing of B. pseudomallei remains obscure. Here, we showed that bactericidal NETs were released from human PMNs in response to B. pseudomallei in a dose- and time-dependent manner. B. pseudomallei-induced NET formation required NADPH oxidase activation but not phosphatidylinositol-3 kinase, mitogen-activated protein kinases, or Src family kinase signaling pathways. B. pseudomallei mutants defective in the virulence-associated Bsa type III protein secretion system (T3SS) or capsular polysaccharide I (CPS-I) induced elevated levels of NETs. NET induction by such mutants was associated with increased bacterial killing, phagocytosis, and oxidative burst by PMNs. Taken together the data imply that T3SS and the capsule may play a role in evading the induction of NETs. Importantly, PMNs from diabetic subjects released NETs at a lower level than PMNs from healthy subjects. Modulation of NET formation may therefore be associated with the pathogenesis and control of melioidosis.

A Src family kinase–Shp2 axis controls RUNX1 activity in megakaryocyte and T-lymphocyte differentiation

Hematopoietic development occurs in complex microenvironments and is influenced by key signaling events. Yet how these pathways communicate with master hematopoietic transcription factors to coordinate differentiation remains incompletely understood. The transcription factor RUNX1 plays essential roles in definitive hematopoietic stem cell (HSC) ontogeny, HSC maintenance, megakaryocyte (Mk) maturation, and lymphocyte differentiation. It is also the most frequent target of genetic alterations in human leukemia. Here, we report that RUNX1 is phosphorylated by Src family kinases (SFKs) and that this occurs on multiple tyrosine residues located within its negative regulatory DNA-binding and autoinhibitory domains. Retroviral transduction, chemical inhibitor, and genetic studies demonstrate a negative regulatory role of tyrosine phosphorylation on RUNX1 activity in Mk and CD8 T-cell differentiation. We also demonstrate that the nonreceptor tyrosine phosphatase Shp2 binds directly to RUNX1 and contributes to its dephosphorylation. Last, we show that RUNX1 tyrosine phosphorylation correlates with reduced GATA1 and enhanced SWI/SNF interactions. These findings link SFK and Shp2 signaling pathways to the regulation of RUNX1 activity in hematopoiesis via control of RUNX1 multiprotein complex assembly.

Striatal-enriched Protein-tyrosine Phosphatase (STEP) Regulates Pyk2 Kinase Activity

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family and is highly expressed in brain and hematopoietic cells. Pyk2 plays diverse functions in cells, including the regulation of cell adhesion, migration, and cytoskeletal reorganization. In the brain, it is involved in the induction of long term potentiation through regulation of N-methyl-d-aspartate receptor trafficking. This occurs through the phosphorylation and activation of Src family tyrosine kinase members, such as Fyn, that phosphorylate GluN2B at Tyr(1472). Phosphorylation at this site leads to exocytosis of GluN1-GluN2B receptors to synaptic membranes. Pyk2 activity is modulated by phosphorylation at several critical tyrosine sites, including Tyr(402). In this study, we report that Pyk2 is a substrate of striatal-enriched protein-tyrosine phosphatase (STEP). STEP binds to and dephosphorylates Pyk2 at Tyr(402). STEP KO mice showed enhanced phosphorylation of Pyk2 at Tyr(402) and of the Pyk2 substrates paxillin and ASAP1. Functional studies indicated that STEP opposes Pyk2 activation after KCl depolarization of cortical slices and blocks Pyk2 translocation to postsynaptic densities, a key step required for Pyk2 activation and function. This is the first study to identify Pyk2 as a substrate for STEP.

Transactivation of EGFR by LPS Induces COX-2 Expression in Enterocytes

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal morbidity and mortality in preterm infants. NEC is characterized by an exaggerated inflammatory response to bacterial flora leading to bowel necrosis. Bacterial lipopolysaccharide (LPS) mediates inflammation through TLR4 activation and is a key molecule in the pathogenesis of NEC. However, LPS also induces cyclooxygenase-2 (COX-2), which promotes intestinal barrier restitution through stimulation of intestinal cell survival, proliferation, and migration. Epidermal growth factor receptor (EGFR) activation prevents experimental NEC and may play a critical role in LPS-stimulated COX-2 production. We hypothesized that EGFR is required for LPS induction of COX-2 expression. Our data show that inhibiting EGFR kinase activity blocks LPS-induced COX-2 expression in small intestinal epithelial cells. LPS induction of COX-2 requires Src-family kinase signaling while LPS transactivation of EGFR requires matrix metalloprotease (MMP) activity. EGFR tyrosine kinase inhibitors block LPS stimulation of mitogen-activated protein kinase ERK, suggesting an important role of the MAPK/ERK pathway in EGFR-mediated COX-2 expression. LPS stimulates proliferation of IEC-6 cells, but this stimulation is inhibited with either the EGFR kinase inhibitor AG1478, or the selective COX-2 inhibitor Celecoxib. Taken together, these data show that EGFR plays an important role in LPS-induction of COX-2 expression in enterocytes, which may be one mechanism for EGF in inhibition of NEC.

Phosphoproteomics Identifies Driver Tyrosine Kinases in Sarcoma Cell Lines and Tumors

Driver tyrosine kinase mutations are rare in sarcomas, and patterns of tyrosine phosphorylation are poorly understood. To better understand the signaling pathways active in sarcoma, we examined global tyrosine phosphorylation in sarcoma cell lines and human tumor samples. Anti-phosphotyrosine antibodies were used to purify tyrosine phosphorylated peptides, which were then identified by liquid chromatography and tandem mass spectrometry. The findings were validated with RNA interference, rescue, and small-molecule tyrosine kinase inhibitors. We identified 1,936 unique tyrosine phosphorylated peptides, corresponding to 844 unique phosphotyrosine proteins. In sarcoma cells alone, peptides corresponding to 39 tyrosine kinases were found. Four of 10 cell lines showed dependence on tyrosine kinases for growth and/or survival, including platelet-derived growth factor receptor (PDGFR)α, MET, insulin receptor/insulin-like growth factor receptor signaling, and SRC family kinase signaling. Rhabdomyosarcoma samples showed overexpression of PDGFRα in 13% of examined cases, and sarcomas showed abundant tyrosine phosphorylation and expression of a number of tyrosine phosphorylated tyrosine kinases, including DDR2, EphB4, TYR2, AXL, SRC, LYN, and FAK. Together, our findings suggest that integrating global phosphoproteomics with functional analyses with kinase inhibitors can identify drivers of sarcoma growth and survival.

Abstract 2154: CDCP1 links RAS and Src signaling pathways as a common effector for the promotion of tumor metastasis

Abstract The oncogenic RAS is one of the most intensively studied proteins which are well recognized as a master regulator of cancer initiation and progression, however rather limited information is available as for their contribution to the cell properties associated with tumor metastasis such as anoikis resistance, cell migration and invasion. We have revealed that CUB domain-containing protein 1 (CDCP1) protein, a substrate of Src family kinase (SFK), in solid cancers is involved in the regulation of anoikis resistance, cell migration and invasion. CDCP1 contributes significantly to the metastatic potential of various cancers including lung, stomach, and pancreatic cancers in a tyrosine phosphorylation dependent manner. It was recently observed that human lung cancer cell lines with K-RAS mutations show significantly higher expression of CDCP1 than those without, while the other common mutations such as EGFR, LKB1 and p53 did not show such relationship. The introduction of the activating mutation of K-RAS clearly induced the expression of CDCP1 and that this enhancement is also affected by the expression levels of Myc protein. Treatment of PD98059, an inhibitor of MEK downstream of Ras, as well as expression of dominant-negative Ras, reduced the expression of CDCP1, and at the same time suppressed cell survival in suspension culture, cell migration and invasion. However, these properties were rescued only when both CDCP1 and Fyn, a member of SFKs were expressed. On the contrary, metastatic potentials caused by expression of activated K-Ras in lung cancer cells was blocked by knockdown of in CDCP1 expression induced by Ras. Using three dimensional organotypic culture assay of human cervical keratinocytes (HCKs) which mimics in vivo tissue invasion of cervical cancers, marked expression of CDCP1 was detected at the site of cell invasion into the collagen raft. The treatment of CDCP1 siRNA or SFKs inhibitor PP2 repressed cell invasion into the collagen raft induced by Ras and Myc. As a summary, it was revealed that expression of CDCP1 is induced by oncogenic Ras, and upregulated CDCP1 triggers cancer invasion and metastasis when phosphorylated by SFKs. Metastatic potential induced by Ras is dependent on the CDCP1 induction, while CDCP1 is not responsive for proliferative change also induced by Ras. These findings demonstrated the central role of CDCP1 as the functional link between RAS and SFKs signaling during the progression of human malignant tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2154. doi:1538-7445.AM2012-2154

CBL mutation-related patterns of phosphorylation and sensitivity to tyrosine kinase inhibitors

Recurrent homozygous CBL-inactivating mutations in myeloid malignancies decrease ubiquitin ligase activity that inactivates SRC family kinases (SFK) and receptor tyrosine kinases (RTK). However, the most important SFK and RTK affected by these mutations, and hence, the most important therapeutic targets, have not been clearly characterized. We compared SFK and RTK pathway activity and inhibitors in acute myeloid leukemia cell lines containing homozygous R420Q mutation (GDM-1), heterozygous deletion (MOLM13) and wild-type (WT) CBL (THP1, U937). As expected with CBL loss, GDM-1 displayed high KIT expression and granulocyte-macrophage colony-stimulating factor (GM-CSF) hypersensitivity. Ectopic expression of WT CBL decreased GDM-1 proliferation but not cell lines with WT CBL. GDM-1, but not the other cell lines, was highly sensitive to growth inhibition by dasatinib (dual SFK and RTK inhibitor, LD50 50 nM); there was less or no selective inhibition of GDM-1 growth by sunitinib (RTK inhibitor), imatinib (ABL, KIT inhibitor), or PP2 (SFK inhibitor). Phosphoprotein analysis identified phosphorylation targets uniquely inhibited by dasatinib treatment of GDM-1, including a number of proteins in the KIT and GM-CSF receptor pathways (for example, KIT Tyr721, STAT3 Tyr705). In conclusion, the promiscuous effects of CBL loss on SFK and RTK signaling appear to be best targeted by dual SFK and RTK inhibition.

The regulation of N‐methyl‐d‐aspartate receptors by Src kinase

Src family kinases (SFKs) play critical roles in the regulation of many cellular functions by growth factors, G-protein-coupled receptors and ligand-gated ion channels. Recent data have shown that SFKs serve as a convergent point of multiple signaling pathways regulating N-methyl-d-aspartate (NMDA) receptors in the central nervous system. Multiple SFK molecules, such as Src and Fyn, closely associate with their substrate, NMDA receptors, via indirect and direct binding mechanisms. The NMDA receptor is associated with an SFK signaling complex consisting of SFKs; the SFK-activating phosphatase, protein tyrosine phosphatase α; and the SFK-inactivating kinase, C-terminal Src kinase. Early studies have demonstrated that intramolecular interactions with the SH2 or SH3 domain lock SFKs in a closed conformation. Disruption of the interdomain interactions can induce the activation of SFKs with multiple signaling pathways involved in regulation of this process. The enzyme activity of SFKs appears 'graded', exhibiting different levels coinciding with activation states. It has also been proposed that the SH2 and SH3 domains may stimulate catalytic activity of protein tyrosine kinases, such as Abl. Recently, it has been found that the enzyme activity of neuronal Src protein is associated with its stability, and that the SH2 and SH3 domain interactions may act not only to constrain the activation of neuronal Src, but also to regulate the enzyme activity of active neuronal Src. Collectively, these findings demonstrate novel mechanisms underlying the regulation of SFKs.

Abstract LB-25: Global phosphoproteomics reveals crosstalk between Bcr-Abl and negative feedback mechanisms controlling Src signaling

Abstract In subtypes and late stages of leukemias driven by the tyrosine kinase fusion protein Bcr-Abl, signaling by the Src family kinases (SFKs) critically contributes to the leukemic phenotype. We performed global tyrosine phosphoprofiling using quantitative mass spectrometry of Bcr-Abl transformed cells in which the activities of the Src family kinases were perturbed to build a detailed context-dependent network of cancer signaling. Perturbation of the SFKs Lyn and Hck with genetics or inhibitors revealed Bcr-Abl downstream phosphorylation events either mediated by or independent of SFKs. We identified multiple negative feedback mechanisms within the network of signaling events affected by Bcr-Abl and SFKs, and found that Bcr-Abl attenuated these inhibitory mechanisms. The Csk binding protein Pag1 (also known as Cbp) and the tyrosine phosphatase Ptpn18 both mediated negative feedback to SFKs. We observed Bcr-Abl-mediated phosphorylation of the phosphatase Shp2 (Ptpn11) and this may contribute to the suppression of these negative feedback mechanisms to promote Bcr-Abl-activated SFK signaling. Csk and a kinase-deficient Csk mutant both produced similar globally repressive signaling consequences, suggesting a critical role for the adaptor protein function of Csk in its inhibition of Bcr-Abl and SFK signaling. The identified Bcr-Abl-activated SFK regulatory mechanisms are candidates for dysregulation during leukemia progression and acquisition of SFK-mediated drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-25. doi:10.1158/1538-7445.AM2011-LB-25

Abstract 3131: A novel tyrosine kinase signaling pathway in human basal breast cancer

Abstract Basal-type breast cancers account for up to 25 % of breast cancer cases, depending on the patient population. These cancers usually lack estrogen, progesterone and erbB2 receptors and are therefore resistant to endocrine or trastuzumab therapy, and markers that stratify this subset according to clinical behaviour have yet to be identified. Consequently, basal breast cancers present a major treatment dilemma. In order to identify potential therapeutic targets and prognostic markers for this disease subgroup, we are currently undertaking a detailed characterization of tyrosine kinase signaling networks in basal breast cancer cells. Mass spectrometry (MS)-based global profiling of the tyrosine phosphoproteome of basal breast cancer cells identified a prominent Src family kinase (SFK) signaling network that featured high expression of the SFK Lyn (Hochgräfe et al, Cancer Res 2010 doi: 10.1158/0008-5472.CAN-10-0911). In order to identify Lyn substrates, we used stable isotope labelling with amino acids in cell culture (SILAC) in combination with immunoaffinity-based phosphotyrosine enrichment and LC-MS/MS to profile cellular tyrosine phosphorylation following transient siRNA-mediated Lyn knockdown in MDA-MB-231 and BT-549 basal breast cancer cells. This identified several Lyn substrates characteristic of both cell lines, which included annexin A2 (Y42), 40S ribosomal protein S10 (Y12) and SgK269 (Y635). The latter is a relatively understudied kinase that contains several tyrosine phosphorylation sites that are likely to act as recruitment sites for SH2/PTB domain-containing signalling proteins, and a C-terminal kinase-like domain that lacks the consensus DFG motif usually regarded as essential for kinase activity. Protein expression of SgK269 was markedly higher in basal, versus luminal, breast cancer cells, while mRNA transcript levels in the two cell types were similar, indicating post-transcriptional regulation of SgK269 expression. Phosphorylation of SgK269 on Y635 could be detected readily in basal breast cancer cells. Overexpression of SgK269 in MCF-10A immortalized mammary epithelial cells led to a more elongated morphology in monolayer and perturbed acinar morphogenesis in Matrigel, resulting in large, multilobular acini that failed to undergo proliferative suppression in long-term culture. SgK269-overexpressing cells also exhibited enhanced tyrosine phosphorylation of Stat3. In addition, we undertook stable shRNA-mediated knockdown of SgK269 in BT-549 cells. This reduced soft agar growth and Stat3 activation. These studies identify a novel oncogenic signalling pathway in basal breast cancer involving the kinases Lyn and SgK269, which may represent a target for therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3131. doi:10.1158/1538-7445.AM2011-3131

Imatinib Upregulates Compensatory Integrin Signaling in a Mouse Model of Gastrointestinal Stromal Tumor and Is More Effective When Combined with Dasatinib

Activating mutations in the Kit receptor tyrosine kinase are associated with gastrointestinal stromal tumor (GIST). Imatinib inhibits Kit and is front-line therapy for GIST. However, imatinib most often elicits a partial response or stable disease, and most GIST patients who initially respond to imatinib eventually acquire resistance. Thus, improved treatment strategies for GIST are needed. We investigated the role of Src family kinases (SFK) in tumorigenesis in a mouse model of human GIST. The SFKs Src and Lyn were active in GIST, and surprisingly, imatinib treatment stimulated their phosphorylation/activation. We show that integrin signaling activates focal adhesion kinase and, consequently, SFKs in GIST and that imatinib enhances integrin signaling, implying a role for the extracellular matrix and integrin signaling in tumor maintenance and imatinib resistance. Dasatinib, an inhibitor of SFKs and Kit, inhibited SFK and focal adhesion kinase activation in GIST but also inhibited Kit and Kit-dependent downstream signaling pathways including phosphoinositide 3-kinase and mitogen-activated protein kinase, but not signal transducer and activator of transcription (STAT) signaling. Whereas dasatinib and imatinib alone both produced a minimal histopathologic response, combination therapy improved their efficacy, leading to increased necrosis in GIST. These results highlight the importance of SFK and STAT signaling in GIST and suggest that the clinical efficacy of imatinib may be limited by the stimulation of integrin signaling.

Multiplex Analysis of Src Family Kinase Signaling by Microbead Suspension Arrays

There is renewed interest in the Src family of protein tyrosine kinases (SFKs) as a result of their potential utility as molecular targets for cancer therapy. This protein family consists of 9 nonreceptor tyrosine kinases that, although implicated in a diverse array of cellular functions, possess a similar modular structure. Here we describe a simple and efficient multiplex microbead immunoassay (MMIA), based on Luminex xMAP technology, which allows for the simultaneous detection of 8 phosphorylated SFKs in a single assay. Microbead sets identifiable by unique fluorescence were individually coated with antibodies specific for an individual SFK member. Detection of phosphorylated SFKs was accomplished using a secondary antibody directed against phosphotyrosine. The assay requires < or = 10 microg of cell lysate or nanogram amounts of purified SFK. The use of a generic secondary antibody allows for the expansion of the assay to include any other tyrosine kinase for which a specific antibody exists. Using either mammalian cell lines or purified, recombinant kinases as the SFK source, we demonstrate the utility of the assay by evaluating the phosphorylation status of SFK members following several in vitro manipulations designed to modulate the phosphotyrosine content of the kinases. These results show that the SFK multiplex assay is a robust tool to investigate the function of SFKs in basic and potentially in clinical research.

Src family kinase signaling during egg maturation and fertilization in a marine protostome worm

Src family kinase signaling during egg maturation and fertilization in a marine protostome worm