A novel immuno-gold labeling protocol for nanobody-based detection of HER2 in breast cancer cells using immuno-electron microscopy.

Immuno-electron microscopy is commonly performed with the use of antibodies. In the last decade the antibody fragment indicated as nanobody (VHH or single domain antibody) has found its way to different applications previously done with conventional antibodies. Nanobodies can be selected to bind with high affinity and specificity to different antigens. They are small (molecular weight ca. 15kDa) and are usually easy to produce in microorganisms. Here we have evaluated the feasibility of a nanobody binding to HER2 for application in immuno-electron microscopy. To obtain highest labeling efficiency combined with optimal specificity, different labeling conditions were analysed, which included nanobody concentration, fixation and blocking conditions. The obtained optimal protocol was applied for post-embedment labeling of Tokuyasu cryosections and for pre-embedment labeling of HER2 for fluorescence microscopy and both transmission and scanning electron microscopy. We show that formaldehyde fixation after incubation with the anti-HER2 nanobody, improves labeling intensity. Among all tested blocking agents the best results were obtained with a mixture of cold water fish gelatine and acetylated bovine serum albumin, which prevented a-specific interactions causing background labeling while preserving specific interactions at the same time. In conclusion, we have developed a nanobody-based protocol for immuno-gold labeling of HER2 for Tokuyasu cryosections in TEM as well as for pre-embedment gold labeling of cells for both TEM and SEM.

Site-specific conjugation of single domain antibodies to liposomes enhances photosensitizer uptake and photodynamic therapy efficacy.

Photodynamic therapy for therapy-resistant cancers will greatly benefit from targeted delivery of tumor photosensitizing agents. In this study, a strategy for the site-specific conjugation of single domain antibodies onto liposomes containing the photosensitizer zinc phthalocyanine was developed and tested.

PEGylated and targeted extracellular vesicles display enhanced cell specificity and circulation time.

Extracellular vesicles (EVs) are increasingly being recognized as candidate drug delivery systems due to their ability to functionally transfer biological cargo between cells. However, the therapeutic applicability of EVs may be limited due to a lack of cell-targeting specificity and rapid clearance of exogenous EVs from the circulation. In order to improve EV characteristics for drug delivery to tumor cells, we have developed a novel method for decorating EVs with targeting ligands conjugated to polyethylene glycol (PEG). Nanobodies specific for the epidermal growth factor receptor (EGFR) were conjugated to phospholipid (DMPE)-PEG derivatives to prepare nanobody-PEG-micelles. When micelles were mixed with EVs derived from Neuro2A cells or platelets, a temperature-dependent transfer of nanobody-PEG-lipids to the EV membranes was observed, indicative of a 'post-insertion' mechanism. This process did not affect EV morphology, size distribution, or protein composition. After introduction of PEG-conjugated control nanobodies to EVs, cellular binding was compromised due to the shielding properties of PEG. However, specific binding to EGFR-overexpressing tumor cells was dramatically increased when EGFR-specific nanobodies were employed. Moreover, whereas unmodified EVs were rapidly cleared from the circulation within 10min after intravenous injection in mice, EVs modified with nanobody-PEG-lipids were still detectable in plasma for longer than 60min post-injection. In conclusion, we propose post-insertion as a novel technique to confer targeting capacity to isolated EVs, circumventing the requirement to modify EV-secreting cells. Importantly, insertion of ligand-conjugated PEG-derivatized phospholipids in EV membranes equips EVs with improved cell specificity and prolonged circulation times, potentially increasing EV accumulation in targeted tissues and improving cargo delivery.

Intraoperative fluorescence delineation of head and neck cancer with a fluorescent anti-epidermal growth factor receptor nanobody.

Intraoperative near-infrared (NIR) fluorescence imaging is a technology with high potential to provide the surgeon with real-time visualization of tumors during surgery. Our study explores the feasibility for clinical translation of an epidermal growth factor receptor (EGFR)-targeting nanobody for intraoperative imaging and resection of orthotopic tongue tumors and cervical lymph node metastases. The anti-EGFR nanobody 7D12 and the negative control nanobody R2 were conjugated to the NIR fluorophore IRDye800CW (7D12-800CW and R2-800CW). Orthotopic tongue tumors were induced in nude mice using the OSC-19-luc2-cGFP cell line. Tumor-bearing mice were injected with 25 µg 7D12-800CW, R2-800CW or 11 µg 800CW. Subsequently, other mice were injected with 50 or 75 µg of 7D12-800CW. The FLARE imaging system and the IVIS spectrum were used to identify, delineate and resect the primary tumor and cervical lymph node metastases. All tumors could be clearly identified using 7D12-800CW. A significantly higher tumor-to-background ratio (TBR) was observed in mice injected with 7D12-800CW compared to mice injected with R2-800CW and 800CW. The highest average TBR (2.00 ± 0.34 and 2.72 ± 0.17 for FLARE and IVIS spectrum, respectively) was observed 24 hr after administration of the EGFR-specific nanobody. After injection of 75 µg 7D12-800CW cervical lymph node metastases could be clearly detected. Orthotopic tongue tumors and cervical lymph node metastases in a mouse model were clearly identified intraoperatively using a recently developed fluorescent EGFR-targeting nanobody. Translation of this approach to the clinic would potentially improve the rate of radical surgical resections.

Crosstalk between epidermal growth factor receptor- and insulin-like growth factor-1 receptor signaling: implications for cancer therapy.

Both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) can contribute to tumor development and -progression through their effects on cell proliferation, inhibition of apoptosis, angiogenesis, anchorage-independent growth and tumor-associated inflammation. EGFR-targeting monoclonal antibodies and small molecule tyrosine kinase inhibitors are currently in clinical use for the treatment of several types of cancer. However, primary and acquired resistance to these agents often occurs and thereby limits the clinical efficacy of mono-specific targeted therapy. Results from both in vitro and in vivo studies indicate that cross-talk between EGFR and IGF-1R can lead to acquired resistance against EGFR-targeted drugs. This review describes the interface between the EGFR and IGF-1R signaling networks and the implications of the extensive cross-talk between these two receptor systems for cancer therapy. EGFR and IGF-1R interact on multiple levels, either through a direct association between the two receptors, by mediating the availability of each others ligands, or indirectly, via common interaction partners such as G protein coupled receptors (GPCR) or downstream signaling molecules. This multi-layered cross-talk and its involvement in the induction of resistance to targeted therapies provide a clear rationale for dual targeting of EGFR and IGF-1R. We discuss several (potential) strategies to simultaneously inhibit EGFR and IGF-1R signaling as promising novel therapeutic approaches.

The v-Crk oncogene enhances cell survival and induces activation of protein kinase B/Akt.

The v-Crk oncogene encodes an adaptor protein containing an SH2 domain and an SH3 domain. v-Crk-transformed fibroblast cells display enhanced tyrosine phosphorylation levels, and the v-Crk protein localizes in focal adhesions, suggesting that transformation may be due to enhanced focal complex signaling. Here we investigated the mechanism of transformation and found that v-Crk-transformed NIH 3T3 cells display growth rates and serum requirements similar to control cells. However, v-Crk enhanced survival in conditions of serum starvation. Both an intact SH2 and SH3 domain are required; moreover, SH2 mutants displayed dominant interfering properties, enhancing cell death. Using other cell death-inducing stimuli, it appeared that v-Crk in general inhibits apoptosis and enhances cell survival. In search of the signaling pathways involved, we found that v-Crk-transformed cells show constitutively higher levels of phospho-protein kinase B (PKB)/Akt and PKB/Akt activity, especially in conditions of serum starvation. These data strongly suggest involvement of the phosphatidylinositol 3-kinase/PKB survival pathway in the v-Crk-induced protection against apoptosis. In accordance, inhibition of this pathway by wortmannin or LY924002 reduced protection against starvation-induced apoptosis. In addition to the phosphatidylinositol 3-kinase/PKB pathway, a MEK-dependent pathway and an unknown additional pathway are also implicated in resistance against apoptosis. Activation of survival pathways may be the most important function of v-Crk in its oncogenic properties.

Factor VIIa/tissue factor-induced signaling via activation of Src-like kinases, phosphatidylinositol 3-kinase, and Rac.

Tissue factor (TF), apart from activating the extrinsic pathway of the blood coagulation, is a principal regulator of embryonic angiogenesis and oncogenic neoangiogenesis, but also influences inflammation, leukocyte diapedesis and tumor progression. The intracellular domain of TF lacks homology to other classes of receptors and hence the signaling mechanism is poorly understood. Here we demonstrate that factor VIIa (the natural ligand for TF) induces the activation of the Src family members c-Src, Lyn, and Yes, and subsequently phosphatidylinositol 3-kinase (PI3K), followed by stimulation of c-Akt/protein kinase B as well as the small GTPases Rac and Cdc42. In turn Rac mediates p38 mitogen-activated protein (MAP) kinase activation and cytoskeletal reorganization, whereas factor VIIa-induced p42/p44 MAP kinase stimulation required PI3K enzymatic activity but was not inhibited by dominant negative Rac proteins. We propose that this Src family member/PI3K/Rac-dependent signaling pathway is a major mediator of factor VIIa/TF effects in pathophysiology.

The EH1 domain of Eps15 is structurally classified as a member of the S100 subclass of EF-hand-containing proteins.

The Eps15 homology (EH) domain is a protein-protein interaction module that binds to proteins containing the asparagine-proline-phenylalanine (NPF) or tryptophan/phenylalanine-tryptophan (W/FW) motif. EH domain-containing proteins serve important roles in signaling and processes connected to transport, protein sorting, and organization of subcellular structure. Here, we report the solution structure of the apo form of the EH1 domain of mouse Eps15, as determined by high-resolution multidimensional heteronuclear NMR spectroscopy. The polypeptide folds into six alpha-helices and a short antiparallel beta-sheet. Additionally, it contains a long, structured, topologically unique C-terminal loop. Helices 2-5 form two EF-hand motifs. Structural similarity and Ca(2+) binding properties lead to classification of the EH1 domain as a member of the S100 subclass of EF-hand-containing proteins, albeit with a unique set of interhelical angles. Binding studies using an eight-residue NPF-containing peptide derived from RAB, the cellular cofactor of the HIV Rev protein, show a hydrophobic peptide-binding pocket formed by conserved tryptophan and leucine residues.

Protein kinase Czeta is a negative regulator of protein kinase B activity.

Protein kinase B (PKB), also known as Akt or RAC-PK, is a serine/threonine kinase that can be activated by growth factors via phosphatidylinositol 3-kinase. In this article we show that PKCzeta but not PKCalpha and PKCdelta can co-immunoprecipitate PKB from CHO cell lysates. Association of PKB with PKCzeta was also found in COS-1 cells transiently expressing PKB and PKCzeta, and moreover we found that this association is mediated by the AH domain of PKB. Stimulation of COS-1 cells with platelet-derived growth factor (PDGF) resulted in a decrease in the PKB-PKCzeta interaction. The use of kinase-inactive mutants of both kinases revealed that dissociation of the complex depends upon PKB activity. Analysis of the activities of the interacting kinases showed that PDGF-induced activation of PKCzeta was not affected by co-expression of PKB. However, both PDGF- and p110-CAAX-induced activation of PKB were significantly abolished in cells co-expressing PKCzeta. In contrast, co-expression of a kinase-dead PKCzeta mutant showed an increased induction of PKB activity upon PDGF treatment. Downstream signaling of PKB, such as the inhibition of glycogen synthase kinase-3, was also reduced by co-expression of PKCzeta. A clear inhibitory effect of PKCzeta was found on the constitutively active double PKB mutant (T308D/S473D). In summary, our results demonstrate that PKB interacts with PKCzeta in vivo and that PKCzeta acts as a negative regulator of PKB.

Cyclooxygenase-dependent signalling: molecular events and consequences.

Non-steroidal anti-inflammatory drugs (NSAIDs) currently attract large interest. Next to pain relief, NSAIDs have important anti-thrombotic and anti-oncogenic effects. NSAIDs exert their action by inhibition of cyclooxygenase, the enzyme responsible for the production of prostanoids. Prostanoid signal transduction is still poorly understood, but it has become clear that these inflammatory lipids influence cellular physiology at three different levels: (1) activation of a 7 x transmembrane receptor coupled to heterotrimeric G proteins, (2) the inhibition of inflammation by activating corticosteroid-like receptors, (3) participation in receptor protein tyrosine kinase signal transduction. In this review prostanoid signalling at these three different levels will be reviewed and the relevance in (patho)physiological processes will be evaluated.

Activation of Rho-dependent cell spreading and focal adhesion biogenesis by the v-Crk adaptor protein.

The small GTPase RhoA plays a critical role in signaling pathways activated by serum-derived factors, such as lysophosphatidic acid (LPA), including the formation of stress fibers in fibroblasts and neurite retraction and rounding of soma in neuronal cells. Previously, we have shown that ectopic expression of v-Crk, an SH2/SH3 domain-containing adapter proteins, in PC12 cells potentiates nerve growth factor (NGF)-induced neurite outgrowth and promotes the survival of cells when NGF is withdrawn. In the present study we show that, when cultured in 15% serum or lysophosphatidic acid-containing medium, the majority of v-Crk-expressing PC12 cells (v-CrkPC12 cells) display a flattened phenotype with broad lamellipodia and are refractory to NGF-induced neurite outgrowth unless serum is withdrawn. v-Crk-mediated cell flattening is inhibited by treatment of cells with C3 toxin or by mutation in the Crk SH2 or SH3 domain. Transient cotransfection of 293T cells with expression plasmids for p160ROCK (Rho-associated coiled-coil-containing kinase) and v-Crk, but not SH2 or SH3 mutants of v-Crk, results in hyperactivation of p160ROCK. Moreover, the level of phosphatidylinositol-4,5-bisphosphate is increased in v-CrkPC12 cells compared to the levels in mutant v-Crk-expressing cells or wild-type cells, consistent with PI(4)P5 kinase being a downstream target for Rho. Expression of v-Crk in PC12 cells does not result in activation of Rac- or Cdc42-dependent kinases PAK and S6 kinase, demonstrating specificity for Rho. In contrast to native PC12 cells, in which focal adhesions and actin stress fibers are not observed, immunohistochemical analysis of v-CrkPC12 cells reveals focal adhesion complexes which are formed at the periphery of the cell and are connected to actin cables. The formation of focal adhesions correlates with a concomitant upregulation in the expression of focal adhesion proteins FAK, paxillin, alpha3-integrin, and a higher-molecular-weight form of beta1-integrin. Our results indicate that v-Crk activates the Rho-signaling pathway and serves as a scaffolding protein during the assembly of focal adhesions in PC12 cells.

The actin binding domain of the epidermal growth factor receptor is required for EGF-stimulated tissue invasion.

NIH-3T3 fibroblasts expressing epidermal growth factor receptors (EGFRs) lacking the actin binding domain (ABD) were analyzed for their EGF-induced capacity to invade a bone marrow stromal cell (BMSC) monolayer. The fibroblasts display a reduction in the percentage of cytoskeleton-associated EGFRs. Furthermore, EGF-induced tyrosine kinase activity is unaffected by the mutation. Cells expressing the mutant EGFRs hardly invade a BMSC monolayer upon EGF stimulation in contrast to cells expressing wild-type EGFRs. Using the same cells no difference was observed in PDGF-induced invasion, which ligand was as potent in both cell types as EGF was in wild-type cells. Inhibition of both the phosphatidyl inositol-3-kinase (PI-3-K) and lipoxygenase pathways in wild-type cells mimicked the effect of the ABD deletion. Our results point to an important role for the ABD of the EGFR in EGF-induced tissue invasion.

Identification of an intracellular domain of the EGF receptor required for high-affinity binding of EGF.

Although all EGF receptors in EGF receptor-expressing cells are molecularly identical, they can be subdivided in two different classes that have either a high or a low affinity for EGF. Specifically the high-affinity class is associated with filamentous actin. To determine whether the interaction of the EGF receptor with actin induces its high-affinity state, we studied EGF-binding properties of an EGF receptor mutant that lacks the actin-binding site. Interestingly, we found that cells expressing this mutant receptor still display both high- and low-affinity classes of EGF receptors, indicating that the actin-binding domain does not determine the high-affinity binding state. By further mutational analysis we identified a receptor domain, within the tyrosine kinase domain, that regulates the affinity for EGF.

Epidermal growth factor induces ubiquitination of Eps15.

Epidermal growth factor (EGF) receptor pathway substrate clone 15 (Eps15) has been described as a 142-kDa EGF receptor substrate. It has been shown to bind to the EGF receptor, adaptor protein-2, and clathrin and is present at clathrin-coated pits and vesicles. Upon stimulation of cells with EGF or transforming growth factor alpha, Eps15 becomes rapidly and transiently phosphorylated on tyrosine residues. This phosphorylation coincides with an increase of 8 kDa in molecular mass. Here we show that this increase in molecular mass is not due to tyrosine phosphorylation. Instead, we found both by Western blotting and protein sequencing that this EGF-induced increase in molecular mass is the result of monoubiquitination. Eps15 ubiquitination but not tyrosine phosphorylation was inhibited under conditions that blocked EGF-induced internalization of the EGF receptor. Our results establish ubiquitination as a second form of EGF-stimulated covalent modification of Eps15.

Association and colocalization of Eps15 with adaptor protein-2 and clathrin.

Eps15 has been identified as a substrate of the EGF receptor tyrosine kinase. In this report, we show that activation of the EGF receptor by either EGF or TGF-alpha results in phosphorylation of Eps15. Stimulation of cells with PDGF or insulin did not lead to Eps15 phosphorylation, suggesting that phosphorylation of Eps15 is a receptor-specific process. We demonstrate that Eps15 is constitutively associated with both alpha-adaptin and clathrin. Upon EGF stimulation, Eps15 and alpha-adaptin are recruited to the EGF receptor. Using a truncated EGF receptor mutant, we demonstrate that the regulatory domain of the cytoplasmic tail of the EGF receptor is essential for the binding of Eps15. Fractionation studies reveal that Eps15 is present in cell fractions enriched for plasma membrane and endosomal membranes. Immunofluorescence studies show that Eps15 colocalizes with adaptor protein-2 (AP-2) and partially with clathrin. No colocalization of Eps15 was observed with the early endosomal markers rab4 and rab5. These observations indicate that Eps15 is present in coated pits and coated vesicles of the clathrin-mediated endocytic pathway, but not in early endosomes. Neither AP-2 nor clathrin are required for the binding of Eps15 to coated pits or coated vesicles, since in membranes lacking AP-2 and clathrin, Eps15 still shows the same staining pattern. These findings suggest that Eps15 may play a critical role in the recruitment of active EGF receptors into coated pit regions before endocytosis of ligand-occupied EGF receptors.

v-Crk-induced cell transformation: changes in focal adhesion composition and signaling.

v-Crk is an oncogene product in which a viral Gag sequence is fused to a cellular Crk sequence. It contains one SH2 and one SH3 domain. To gain insight into the molecular mechanisms underlying v-Crk-induced cell transformation, we studied the subcellular localization and molecular interactions of v-Crk in v-Crk-transformed NIH-3T3 cells. Our results show that v-Crk specifically localizes to focal adhesions where it induces protein tyrosine phosphorylation. Subcellular fractionation studies indicated that a significant amount of v-Crk is present in the cytoskeletal cell fraction, a fraction that includes focal adhesions. Tyrosine phosphorylated proteins, including p130CAS, were also predominantly found in the cytoskeletal fraction. We show that v-Crk induces a translocation of p130CAS to the cytoskeleton, which is accompanied by hyperphosphorylation of this protein. Mutational analyses showed that functional v-Crk SH2 domain is required for the localization of v-Crk in focal adhesions. Functional v-Crk SH2 and SH3 domains were both found to be required for the observed increase in tyrosine phosphorylation of focal adhesion proteins and for the translocation and hyperphosphorylation of p130CAS. v-Crk immunoprecipitation studies revealed that cytoskeleton-associated v-Crk interacts with both p130CAS and an unidentified tyrosine kinase. These findings suggest that formation of a focal adhesion-located complex consisting of v-Crk, a tyrosine kinase and p130CAS, which may lead to the hyperphosphorylation of p130CAS. These specific and localized signaling events may represent initial steps in the process of v-Crk-induced cell transformation.

Epidermal growth factor-induced activation and translocation of c-Src to the cytoskeleton depends on the actin binding domain of the EGF-receptor.

In the epidermal growth factor (EGF)-receptor signal transduction cascade, the non-receptor tyrosine kinase c-Src has been demonstrated to become activated upon EGF stimulation. In this paper we show that c-Src associates with the cytoskeleton and co-isolates with actin filaments upon EGF treatment of NIH-3T3 cells transfected with the EGF receptor. Immunofluorescence studies using CLSM show colocalization of F-actin and endogenous c-Src predominantly around endosomes and not on stress fibers and cell-cell contacts. Stimulation of EGF receptor-transfected NIH-3T3 cells with EGF induces an activation and translocation of c-Src to the cytoskeleton. These processes depend upon the presence of the actin binding domain of the EGF-receptor since in cells that express EGF-receptors lacking this domain, EGF fails to induce an activation and translocation to the cytoskeleton of c-Src. These data suggest a role for the actin binding domain of the EGF-receptor in the translocation of c-Src.

Maximal epidermal growth-factor-induced cytosolic phospholipase A2 activation in vivo requires phosphorylation followed by an increased intracellular calcium concentration.

The 85 kDa cytosolic phospholipase A2 (cPLA2) preferentially catalyses the hydrolysis of arachidonic acid from the sn-2 position of phospholipids. cPLA2 can be activated by extracellular stimuli such as thrombin, platelet-derived growth factor and epidermal growth factor (EGF): A full activation of cPLA2 requires an increase of intracellular Ca2+ concentration and phosphorylation on Ser-505 by mitogen-activated protein (MAP) kinase. Because EGF can provoke an increase in intracellular [Ca2+] ([Ca2+]i) and activation of MAP kinase, we investigated the role of these pathways in EGF-induced activation of cPLA2. Characterization of two cell lines expressing different numbers of EGF receptors (HERc13 and HER14) revealed that both were activating MAP kinase in response to EGF, but only HER14 responded with an increase in [Ca2+]i. In this study we used both cell lines as a tool to clarify the role of each pathway in cPLA2 activation. We show that EGF stimulates cPLA2 activity in both cell lines in vitro as measured in cytosolic fractions, but only in HER14 in vivo as measured by 3H release from cells prelabelled with [3H]arachidonic acid. This latter activation can be restored in HERc13 cells by the addition of the ionophore A23187. Interestingly, this effect is only observed when EGF stimulation precedes A23187 addition. The phosphorylation of MAP kinase, however, was identical under identical conditions. We conclude that a maximal cPLA2 activation by EGF requires both, and in this order: MAP kinase activation followed by a rise in [Ca2+]i concentration.