Biomedical subjects

Margaret C Frame

Publications and source records attributed to Margaret C Frame.

At least 19 recordsLinked to original sources

PubMed2006

Identification of potential biomarkers for measuring inhibition of Src kinase activity in colon cancer cells following treatment with dasatinib.

Elevated levels of Src kinase expression have been found in a variety of human epithelial cancers. Most notably in colon cancer, elevated Src expression correlates with malignant potential and is also associated with metastatic disease. Dasatinib (BMS-354825) is a novel, orally active, multi-targeted kinase inhibitor that targets Src family kinases and is currently under clinical evaluation for the treatment of solid tumors. However, the effects of dasatinib on epithelial tumors are not fully understood. We show that concentrations of dasatinib that inhibit Src activity do not inhibit proliferation in 10 of 12 colon cancer cells lines. However, inhibition of integrin-dependent adhesion and migration by dasatinib correlated with inhibition of Src activity, suggesting that dasatinib may have anti-invasive or anti-metastatic activity and antiproliferative activity in epithelial tumors. Using phospho-specific antibodies, we show that inhibition of Src activity in colon cancer cell lines correlates with reduced phosphorylation of focal adhesion kinase and paxillin on specific Src-dependent phosphorylation sites. We have validated the use of phospho-specific antibodies against Src Tyr(419) and paxillin Tyr(118) as biomarkers of dasatinib activity in vivo. Colon carcinoma-bearing mice treated with dasatinib showed a decrease in both phospho-Src Tyr(419) and phospho-paxillin Tyr(118) in peripheral blood mononuclear cells, which correlated with inhibition of Src activity in the colon tumors. Thus, peripheral blood mononuclear cells may provide a useful surrogate tissue for biomarker studies with dasatinib using inhibition of Src Tyr(419) and paxillin Tyr(118) phosphorylation as read-outs of Src activity.

Animals↗

PubMed2006

Src-dependent phosphorylation of Scar1 promotes its association with the Arp2/3 complex.

The WAVE/Scar proteins regulate actin polymerisation at the leading edge of motile cells via activation of the Arp2/3 complex in response to extracellular cues. Within cells they form part of a pentameric complex that is thought to regulate their ability to interact and activate the Arp2/3 complex. However, the exact mechanism for this is not known. We set out to assess whether phosphorylation of Scar1 by the non-receptor tyrosine kinase Src may influence the function of Scar1 and its ability to regulate Arp2/3-mediated actin polymerisation. We show that Scar1 is phosphorylated by Src in vitro and in vivo and identify tyrosine 125 as the major site in Scar1 to be phosphorylated in cells. Src-dependent phosphorylation of Scar1 on tyrosine 125 enhances its ability to bind to the Arp2/3 complex and regulates its ability to control actin polymerisation in cells. Thus, Src may act as an intermediary to regulate the activity of the Arp2/3 complex in response to external stimuli, via modulation of its interaction with WAVE/Scar proteins.

Actin-Related Protein 2-3 Complex↗

PubMed2005

Identification of Src-specific phosphorylation site on focal adhesion kinase: dissection of the role of Src SH2 and catalytic functions and their consequences for tumor cell behavior.

Src tyrosine kinase expression and activity are elevated during colon cancer progression. How this contributes to the malignant phenotype is not fully understood. We show that in KM12C colon carcinoma cells, expression of kinase-deficient Src proteins (SrcMF and Src251) does not alter cell growth. Src kinase activity is required for turnover of cell-matrix adhesions and, in particular, the Src-dependent phosphorylation of focal adhesion kinase (FAK) is required for their disassembly. Surprisingly, we found that expression of SrcMF or Src251 resulted in increased tyrosine phosphorylation of FAK on Tyr(407), Tyr(576), Tyr(577), and Tyr(861), which are considered to be Src kinase substrates. This Src kinase-independent phosphorylation of FAK required an intact Src SH2 domain that mediates association of Src and FAK at peripheral adhesions. Use of a novel highly potent and selective Src kinase inhibitor AP23464 combined with experiments in Src/Fyn/Yes-deficient fibroblasts showed that increased phosphorylation of FAK in cells expressing SrcMF did not require Src-like kinases. However, specific phosphorylation on Tyr(925) of FAK was not evident in SrcMF- or Src251-expressing cells, and lack of Src kinase-dependent phosphorylation on this site was associated with impaired adhesion turnover. Our data show that Src kinase activity is required for adhesion turnover associated with cell migration in cancer cells and that, in addition to the catalytic activity, Src also acts as an adaptor to recruit other kinases that can phosphorylate key substrates including FAK. These studies have implications for tumor progression with respect to the use of Src kinase inhibitors.

Animals↗

PubMed2005

Src and FAK signalling controls adhesion fate and the epithelial-to-mesenchymal transition.

Src kinase controls cellular adhesions, including cadherin-based intercellular adhesions and integrin-mediated cell-matrix adhesions. In epithelial cells, Src activation, or increased signalling from migratory growth factor receptors via Src, induces an adhesion switch that enhances dynamic cell-matrix adhesions and migratory capacity while suppressing intercellular contact. Moreover, Src and the associated tyrosine kinase FAK are at the heart of the recently identified crosstalk between integrin- and cadherin-mediated adhesions of epithelial cells, particularly during the epithelial-to-mesenchymal transition.

Animals↗

PubMed2005

N-cadherin differentially determines Schwann cell and olfactory ensheathing cell adhesion and migration responses upon contact with astrocytes.

Olfactory ensheathing cells (OECs) and Schwann cells provide a cellular environment that promotes axonal outgrowth in several models of CNS injury. However, they exhibit different properties when in contact with astrocytes. Schwann cells, but not OECs, induce characteristics that typify hypertrophy in astrocytes and exhibit a poor capacity to migrate within astrocyte-rich areas, making them less favourable for transplant-mediated repair. N-cadherin has been implicated in the adhesion of Schwann cells to astrocytes. Despite indistinguishable expression of N-cadherin, Schwann cells adhered more strongly to an astrocyte monolayer and migrated more slowly on astrocytes when compared to OECs. We have examined the role of N-cadherin in mediating these cellular interactions using RNA interference and found differing effects. In Schwann cells, suppression of N-cadherin reduced heterotypic and homotypic adhesion and they gained adhesion properties more akin to OECs. In contrast, suppression of N-cadherin in OECs had no effect. These findings imply that N-cadherin is differentially regulated in OECs and Schwann cells.

Animals↗

PubMed2005

The role of focal-adhesion kinase in cancer - a new therapeutic opportunity.

Focal-adhesion kinase (FAK) is an important mediator of growth-factor signalling, cell proliferation, cell survival and cell migration. Given that the development of malignancy is often associated with perturbations in these processes, it is not surprising that FAK activity is altered in cancer cells. Mouse models have shown that FAK is involved in tumour formation and progression, and other studies showing that FAK expression is increased in human tumours make FAK a potentially important new therapeutic target.

Animals↗

PubMed2005

The SRC-induced mesenchymal state in late-stage colon cancer cells.

One major function of elevated Src kinase in epithelial cancer cells is to drive adhesion changes that are associated with the mesenchymal transition and metastasis. Here we review recent work that describes Src-induced shape changes, and the mechanisms involved, in cells derived from a model of colon cancer metastasis. Src activity in these cells is associated with formation and dynamic regulation of integrin adhesions and disorganization of E-cadherin-dependent cell-cell contacts. Furthermore, Src-induced deregulation of E-cadherin requires integrin signalling, demonstrating a complex interdependence between integrin- and cadherin-associated adhesion changes induced by Src. The integrin-induced signals that co-operate with Src to cause deregulation of cadherin-dependent cell-cell contacts include activation of the MEK/ERK and MLCK/myosin activities. Inhibition of this pathway suppresses integrin complexes formed on fibronectin, while promoting E-cadherin redistribution to sites of cell-cell contacts. Also, in embryonic fibroblasts that express N-cadherin (which is normally diffusely cytoplasmic as these cells maintain a fibroblastic morphology) suppressing integrin signalling and inhibiting the MEK/ERK/MLCK/myosin pathway relocalizes N-cadherin to cell-cell contacts. Our recent data therefore imply an important, and perhaps general, role for spatially controlled contractility in suppressing normal cadherin localization and inducing a mesenchymal-like phenotype.

Cadherins↗

PubMed2004

Specific deletion of focal adhesion kinase suppresses tumor formation and blocks malignant progression.

We have generated mice with a floxed fak allele under the control of keratin-14-driven Cre fused to a modified estrogen receptor (CreER(T2)). 4-Hydroxy-tamoxifen treatment induced fak deletion in the epidermis, and suppressed chemically induced skin tumor formation. Loss of fak induced once benign tumors had formed inhibited malignant progression. Although fak deletion was associated with reduced migration of keratinocytes in vitro, we found no effect on wound re-epithelialization in vivo. However, increased keratinocyte cell death was observed after fak deletion in vitro and in vivo. Our work provides the first experimental proof implicating FAK in tumorigenesis, and this is associated with enhanced apoptosis.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2004

Recruitment of phosphoinositide 3-kinase defines a positive contribution of tyrosine kinase signaling to E-cadherin function.

Classical cadherin adhesion molecules can function as adhesion-activated cell-signaling receptors. One key target for cadherin signaling is the lipid kinase phosphoinositide (PI) 3-kinase, which is recruited to cell-cell contacts and activated by E-cadherin. In this study, we sought to identify upstream factors necessary for E-cadherin to activate PI 3-kinase signaling. We found that inhibition of tyrosine kinase signaling blocked recruitment of PI 3-kinase to E-cadherin contacts and abolished the ability of E-cadherin to activate PI 3-kinase signaling. Tyrosine kinase inhibitors further perturbed several parameters of cadherin function, including cell adhesion and the ability of cells to productively extend nascent cadherin-adhesive contacts. Notably, the functional effects of tyrosine kinase blockade were rescued by expression of a constitutively active form of PI 3-kinase that restores PI 3-kinase signaling. Finally, using dominant negative Src mutants and Src-null cells, we identified Src as one key upstream kinase in the E-cadherin/PI 3-kinase-signaling pathway. Taken together, our findings indicate that tyrosine kinase activity, notably Src signaling, can contribute positively to cadherin function by supporting E-cadherin signaling to PI 3-kinase.

Animals↗

PubMed2004

Expression of kinase-defective mutants of c-Src in human metastatic colon cancer cells decreases Bcl-xL and increases oxaliplatin- and Fas-induced apoptosis.

Tumor resistance to current drugs prevents curative treatment of human colon cancer. A pressing need for effective, tumor-specific chemotherapies exists. The non-receptor-tyrosine kinase c-Src is overexpressed in >70% of human colon cancers and represents a tractable drug target. KM12L4A human metastatic colon cancer cells were stably transfected with two distinct kinase-defective mutants of c-src. Their response to oxaliplatin, to SN38, the active metabolite of irinotecan (drugs active in colon cancer), and to activation of the death receptor Fas was compared with vector control cells in terms of cell cycle arrest and apoptosis. Both kinase-defective forms of c-Src co-sensitized cells to apoptosis induced by oxaliplatin and Fas activation but not by SN38. Cells harboring kinase-defective forms of c-Src carrying function blocking point mutations in SH3 or SH2 domains were similarly sensitive to oxaliplatin, suggesting that reduction in kinase activity and not a Src SH2-SH3 scaffold function was responsible for the observed altered sensitivity. Oxaliplatin-induced apoptosis, potentiated by kinase-defective c-Src mutants, was dependent on activation of caspase 8 and associated with Bid cleavage. Each of the stable cell lines in which kinase-defective mutants of c-Src were expressed had reduced levels of Bcl-x(L.) However, inhibition of c-Src kinase activity by PP2 in vector control cells did not alter the oxaliplatin response over 72 h nor did it reduce Bcl-x(L) levels. The data suggest that longer term suppression of Src kinase activity may be required to lower Bcl-x(L) levels and sensitize colon cancer cells to oxaliplatin-induced apoptosis.

Apoptosis↗

PubMed2004

PDE4-regulated cAMP degradation controls the assembly of integrin-dependent actin adhesion structures and REF52 cell migration.

Plating of REF52 cells onto extracellular matrix components leads to the formation of integrin-dependent actin adhesion microspikes. We show that the formation of these structures is sensitive to chemical agents that regulate cAMP levels, such as forskolin and IBMX. In particular, by using the specific inhibitor rolipram, we identify the PDE4 family of cAMP-specific phosphodiesterases as critical regulators of this process. The effect of PDE4 on microspike formation is mediated by actions exerted through the activation of PKA - rather than through the alternative cAMP effector, Epac. We provide evidence that peripheral microspikes are RhoA-, ROCK- and myosin-dependent, and that this pathway is suppressed by PDE4 inhibition. In addition, PDE4 inhibition impairs cell locomotion that requires dynamic protrusion and retraction of peripheral spike structures. Our data demonstrate that PDE4 activity is a key modulator of integrin-induced actin assembly at the cell periphery which, in turn, controls cell migration.

1-Methyl-3-isobutylxanthine↗

PubMed2004

Src SH3/2 domain-mediated peripheral accumulation of Src and phospho-myosin is linked to deregulation of E-cadherin and the epithelial-mesenchymal transition.

Elevated Src kinase in epithelial cancer cells induces adhesion changes that are associated with a mesenchymal-like state. We recently showed that Src induces dynamic integrin adhesions in KM12C colon cancer cells, whereas E-cadherin-dependent cell-cell contacts become disorganized. This promotes a fibroblastic-like morphology and expression of the mesenchymal marker vimentin. Furthermore, Src-induced deregulation of E-cadherin, and the associated mesenchymal transition, is dependent on integrin signaling (Avizienyte et al., Nat. Cell Biol. 2002, 4, 632-638), although the nature of downstream signals that mediate these Src- and integrin-dependent effects are unknown. Here we show that the SH2 and SH3 domains of Src mediate peripheral accumulation of phospho-myosin, leading to integrin adhesion complex assembly, whereas loss of SH2 or SH3 function restores normal regulation of E-cadherin and inhibits vimentin expression. Inhibitors of MEK, ROCK, or MLCK also suppress peripheral accumulation of phospho-myosin and Src-induced formation of integrin-dependent adhesions, whereas at the same time restoring E-cadherin redistribution to regions of cell-cell contact. Our data therefore implicate peripheral phospho-myosin activity as a point of convergence for upstream signals that regulate integrin- and E-cadherin-mediated adhesions. This further implicates spatially regulated contractile force as a determinant of epithelial cell plasticity, particularly in cancer cells that can switch between epithelial and mesenchymal-like states.

Cadherins↗

PubMed2004

Newest findings on the oldest oncogene; how activated src does it.

Oncogenic forms of the non-receptor tyrosine kinase Src alter cell structure, in particular the actin cytoskeleton and the adhesion networks that control cell migration, and also transmit signals that regulate proliferation and cell survival. Recent work indicates that they do so by influencing the RhoA-ROCK pathway that controls contractile actin filament assembly, the STAT family of transcription factors needed for transformation, and the Cbl ubiquitin ligase that controls Src protein levels. These studies also shed light on the role of focal adhesion kinase (FAK) downstream of v-Src and other signalling pathways in controlling migration, invasion and survival of transformed cells. Src directly phosphorylates integrins and can also modulate R-Ras activity. Moreover, it stimulates the E-cadherin regulator Hakai, interacts with and phosphorylates the novel podosome-linked adaptor protein Fish, and progressively phosphorylates the gap junction component connexion 43. A recurring theme is the identification of novel and important Src substrates that mediate key biological events associated with transformation.

Animals↗

PubMed2004

RhoB and actin polymerization coordinate Src activation with endosome-mediated delivery to the membrane.

We have used a c-Src-GFP fusion protein to address the spatial control of Src activation and the nature of Src-associated intracellular structures during stimulus-induced transit to the membrane. Src is activated during transit, particularly in RhoB-containing cytoplasmic endosomes associated with the perinuclear recycling compartment. Knocking out RhoB or expressing a dominant-interfering Rab11 mutant suppresses both catalytic activation of Src and translocation of active kinase to peripheral membrane structures. In addition, the Src- and RhoB-containing endosomes harbor proteins involved in actin polymerization and filament assembly, for example Scar1, and newly polymerized actin can associate with these endosomes in a Src-dependent manner. This implies that Src may regulate an endosome-associated actin nucleation activity. In keeping with this, Src controls the actin dependence of RhoB endosome movement toward the plasma membrane. This work identifies RhoB as a component of "outside-in" signaling pathways that coordinate Src activation with translocation to transmembrane receptors.

3T3 Cells↗

PubMed2004

Focal adhesion and actin dynamics: a place where kinases and proteases meet to promote invasion.

Integrin-linked focal adhesion complexes provide the main sites of cell adhesion to extracellular matrix and associate with the actin cytoskeleton to control cell movement. Dynamic regulation of focal adhesions and reorganization of the associated actin cytoskeleton are crucial determinants of cell migration. There are important roles for tyrosine kinases, extracellular signal-regulated protein kinase/mitogen-activated protein kinase signalling, and intracellular and extracellular proteases during actin and adhesion modulation. Dysregulation of these is associated with tumour cell invasion. In this article, we discuss established roles for these signalling pathways, as well as the functional interplay between them in controlling the migratory phenotype.

Actins↗

PubMed2003

A novel role for FAK as a protease-targeting adaptor protein: regulation by p42 ERK and Src.

Cell migration on extracellular matrix requires the turnover of integrin-dependent adhesions. The nonreceptor tyrosine kinases Src and FAK regulate focal-adhesion turnover by poorly understood mechanisms. ERK/MAP kinase-mediated activation of the protease Calpain 2 also promotes focal-adhesion turnover; however, it is not known if this is linked to the activities of Src and FAK. Calpain 2 has previously been demonstrated to colocalize with focal-adhesion structures and can cleave several focal-adhesion complex components, including FAK. Studies utilizing Calpain inhibitors or Calpain-deficient cells confirm that Calpain's role in regulating focal-adhesion turnover is necessary for cell migration. We have identified a novel and kinase-independent function for FAK as an adaptor molecule that mediates the assembly of a complex consisting of at least Calpain 2 and p42ERK. Mutation of proline residues (Pro2) in the amino-terminal region of FAK blocks direct binding with Calpain 2 and also prevents formation of the Calpain 2/p42ERK complex in cells. We show that both complex formation and MEK/ERK activity are associated with Calpain-mediated proteolysis of FAK and focal adhesion turnover during transformation and migration. Furthermore, FAK is necessary for recruiting both Calpain 2 and p42ERK/MAPK to peripheral adhesion sites facilitating maximal Calpain activity.

Adaptor Proteins, Vesicular Transport↗

PubMed2003

Focal adhesion kinase as a potential target in oncology.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays a pivotal role in signal transduction at integrin-linked cellular adhesions, which mediate cell contact with the extracellular matrix. It has been shown to play a role in the survival of anchorage-dependent cells and to be essential for integrin-linked cell migration - processes that are likely to play important roles in the development of malignancies. FAK is upregulated in a wide variety of human epithelial cancers, with expression being closely correlated to invasive potential. Recently, evidence has emerged directly linking FAK expression to tumour development in vivo, raising the possibility that intervention strategies to block FAK function may potentially provide an opportunity for the development of anticancer therapeutics.

Animals↗

PubMed2002

Src in cancer: deregulation and consequences for cell behaviour.

Considerable evidence now implicates elevated expression and/or activity of Src in cancer development. In cells, endogenous Src is switched from an inactive to an active state by a variety of mechanisms that simultaneously relieve constraints on the kinase and protein-interacting Src homology (SH) domains. As a result, Src is translocated to the cell periphery, often to sites of cell adhesion, where myristylation mediates attachment to the inner surface of the plasma membrane. From these peripheral sites, Src's catalytic activity initiates intracellular signal transduction pathways that influence cell growth and adhesion strength, the latter contributing to control of cell migration. De-regulation in cancer cells may therefore enhance tumour growth and/or stimulate migratory or invasive potential in cells that would normally be relatively non-motile. Evidence now exists to suggest that Src may also influence the life or death decisions that cells make during many biological processes. Thus, Src modulation in cancer cells can alter cell responses that are often perturbed in cancer. Consequently, there is optimism that drugs which inhibit Src's kinase activity, or the activity of its downstream effectors, might have profound effects on cancer cell behaviour and be useful therapeutic agents.

Animals↗