http://informahealthcare.com/grf ISSN: 0897-7194 (print), 1029-2292 (electronic) Growth Factors, 2014; 32(2): 74–81 ! 2014 Informa UK Ltd. DOI: 10.3109/08977194.2014.896355

RESEARCH PAPER

EGF receptor family: twisting targets for improved cancer therapies Antony W. Burgess1, Yoav I. Henis2, Nancy E. Hynes3, Thomas Jovin4, Alexander Levitzki5, Ronit Pinkas-Kramarski2, and Yosef Yarden6 The Walter & Eliza Hall Institute of Medical Research, Burgess Lab Structural Biology, Parkville, Australia, 2Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel, 3Department of Mechanisms of Cancer, Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse, Basel, Switzerland, 4Laboratory of Cellular Dynamics, Max Planck Institute for Biophysical Chemistry, Go¨ttingen, Germany, 5Department of Biological Chemistry, Unit of Cellular Signaling, Alexander Siberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel, and 6Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel

Abstract

Keywords

The epidermal growth factor receptor (EGFR) undergoes a conformational change in response to ligand binding. The ligand-induced changes in cell surface aggregation and mobility have a profound effect on the function of all the family members. Ligand also activates the EGFR intracellular kinase, stimulating proliferation and cell survival. The EGFR family are often activated, overexpressed or mutated in cancer cells and therapeutic drugs (including antibodies) can slow the progress of some cancers. This article provides a brief, annotated summary of the presentations and discussion which occurred at the Epidermal Growth Factor Receptor – Future Directions Conference held in Jerusalem in November 2013.

Cell surface, erbB2, erbB3, FLIM, FRET, receptor dynamics, therapeutic antibodies

Introduction A summary of Epidermal Growth Factor Receptor – Future Directions, a Joint International Research Conference of the Israel Institute for Advanced Studies and the Israel Science Foundation held at the Israel Institute for Advanced Studies, Hebrew University, Jerusalem from 17 November to 20 November 2013 (see poster). The epidermal growth factor receptor (EGFR) family members have been targeted for cancer therapeutics. Recent advances in the use of genomics in cancer medicine are leading to the identification of patients suitable for therapeutic targeting EGFR family members. Biophysical techniques including X-ray crystallography, fluorescence lifetime imaging microscopy (FLIM) and fluorescence resonance energy transfer (FRET) have defined extracellular and intracellular interactions between EGFR family members which regulate the activity of the receptor. Interactions between HER2 and the cell membrane can also change the curvature of the cell membrane. Antibody, nanobody and Designed Ankyrin Repeat Proteins (DARPins) have been designed to probe and target EGFR family originating complexes. New antibodies which target HER2 heterodimerization have already shown to have promising therapeutics efficacy for patients with metastatic breast cancer. EGFR family members provide critical regulatory

Correspondence: Antony W. Burgess, The Walter & Eliza Hall Institute of Medical Research, Burgess Lab Structural Biology, 1G Royal Parade, Parkville 3052, Australia. E-mail: [email protected]

History Received 17 February 2014 Accepted 17 February 2014 Published online 18 March 2014

signals during tissue development, morphogenesis and stem cell proliferation. The cross-talk between different family members has a significant role in cell migration. The cell surface regulation of activated EGFR and the interaction of the EGFR family signaling cascades with the changes which occur when tumor suppressor proteins are perturbed are critical elements of cancer progression. Jerusalem is a spectacular venue for any conference and, accordingly, provided a lively forum for the gathering of scientists to discuss the future directions of studies focused on the biochemistry, biology and medicine of the EGF receptor (EGFR, more generally erbB), family of receptor tyrosine kinases. These molecules are key hubs of cell signaling and as a consequence are involved in a major fraction of human tumors. In 2003, many EGFR structural biologists and biophysicists participated in a similar discussion at ‘‘ErbBs on the Beach’’ in Lorne, Australia and a decade later all were keen to join new colleagues, animal and cell biologists, and clinical researchers to define the current state of knowledge and challenges of EGFR biology and medicine. All 60 speakers and discussants addressed the opportunities for concerted investigations of the EGFR family (Table 1). The participation and the excellent facilities provided by the Israel Institute for Advanced Studies created an excitement for the topic, which is often difficult to achieve in larger meetings. Over a quarter of the scheduled program was allotted to discussing the talks and/or specific issues. These discussions provided fascinating glimpses of new biology, new anti-cancer drugs and encouraging clinical opportunities based on the erbB family.

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DOI: 10.3109/08977194.2014.896355

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Table 1. EGFR family: Future challenges and opportunities identified during discussions at the IIAS Meeting in Jerusalem, November 2013. Aggregation dynamics of the unligated receptor Role of receptor degradation in control of EGFR family signaling Role of phosphatases in control of EGFR family signaling Conformation of the cell surface receptor Hetero-oligomerization and antibody therapies Juxtamembrane events and kinase activation Conformation of the active erbB2:erbB3 kinase Role of membrane lipid composition and specific lipid–protein interactions Animal models for monitoring the action of EGFR family members in vivo Mouse with a fluorescent reporter for EGFR activity Cellular responses to EGFR induced cell cycling, filopodia formation and cell survival EGFR family signaling early and late metabolism responses Mechanisms by which factors inhibit EGFR family signaling: receptor degradation, cell surface inhibitors of the EGFR family (Lrig1), Mig6, phosphatase activation ligand traps Targeting the EGFR family for clinical benefit – amplification and mutation (focus on Brain tumors, and/or K-ras tumors) Identifying cancer patients likely to benefit from EGFR, erbB2, erb3 or erbB4 therapies Improving cancer therapies by targeting multiple EGFR family members Improving cancer therapies targeting EGFR family signaling pathways (pancreatic tumors) Combining cell surface and signaling therapeutics Harness the power of the immune system to target EGFR/HER2/HER3 tumors locally New approaches to EGFR family signaling inhibitors including receptor kinase independent signaling processes

The EGFR has been at the heart of cancer biology and medicine since the genetic era started in the early 1980s. In some ways it can be argued that the genetic era of cancer biology was ushered in by the genetic sequences of the EGFR family. Not surprisingly the EGFR family has provided the basis for several of the most successful targeted therapies such as the Herceptin and Cetuximab antibodies. Similarly, the three-dimensional structures of both the extracellular and intracellular domains of EGFR family members sparked a new era of receptor biology: with extraordinary conformational changes driving symmetry-breaking kinase activation processes which regulate cellular processes as diverse as migration, adhesion and proliferation. There has been significant progress in our understanding of EGF receptor biology and the talks and discussion at the meeting highlighted opportunities for using new structural, kinetic and therapeutic knowledge for improving cancer treatment. John Mendelsohn (University of Texas M D Anderson Cancer Center, Houston) opened the conference by highlighting the interplay between the advances in human genomics and the use of new agents to help cancer patients (Mendelsohn, 2013). The targeting of antibodies against erbB2, in women with breast cancers which overexpress the protein, has helped millions of women. More recently the oral EGFR tyrosine kinase inhibitors have benefited lung cancer patients with mutated receptors. We are approaching the time when most of the mutations that drive cancers will be identified at diagnosis. In 2011, a panel of 11 cancer genes was started to be sequenced for many cancer patients at M D Anderson, and in 2012, 46 cancer gene panels became available. In 2013, ion-proton sequencing of biopsy material is revealing mutations associated with 400 genes, and in the

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near future, Nextgen sequencing may identify all of the mutations associated with tumors both at initial diagnosis and at relapse. However, the technology is moving so fast that its utility for clinical applications needs careful consideration. At present, a smaller number of genes with reliably identified mutations (e.g. 50) may provide more clinical impact than whole exosome sequencing. John Mendelsohn pointed out that consideration is being given to redesigning clinical trial schemes to take advantage of the genetic information: soon it will not be sufficient to base treatment simply on the tissue of origin or the stage of the cancer. Genetic or proteomic information is useful and soon will be required to support medical decision-making and for directing the choice of treatment combinations capable of killing specific tumors. Further knowledge of signaling mechanisms associated with the EGFR family is required for managing treatment of breast, lung, head and neck, colon, pancreatic and brain cancers. The links between EGFR family inhibition and the mechanisms causing cancer cell death are as yet insufficiently defined, yet once this is achieved, the outcomes for patients are likely to improve significantly. The asymmetry of activation of the EGFR family has been an area of interest for many years. Mark Lemmon (University of Pennsylvania) described processes that explained how the binding of one ligand induces allosteric structural changes that reduce the affinity of the second ligand, i.e. in a process of negative cooperativity (Alvarado et al., 2010). Next, John Kuriyan (University of California, Berkeley) presented a thorough description of the asymmetric changes that activate the kinase domain in response to ligand or mutation (Arkhipov et al., 2013; Lorenz et al., 2013). The intricate interactions between the transmembrane, juxtamembrane, kinase and C-terminal domains are reaching a stage of engineering capable of predicting the effects of specific mutations. John Kuriyan’s analysis of the EGFR C-terminus identified that a tyrosine residue at 1086 is essential for EGFR signaling – the role of this residue in the signaling processes had not previously been recognized. The structures for all of the ligand-binding regions of the EGFR family members have now been determined. Dan Leahy (Johns Hopkins University) described conformational variability associated with these regions (Leahy, 2013; Liu et al., 2012). These conformational variations reflected significant differences in dimer interactions in the published crystal structures of the EGFR and ErbB4 extracellular domains. These differences are likely to distinguish singly- and doubly-ligated EGFR/ErbB homo- and heterodimers. He pointed out that although it is a holy grail to obtain 3D-structures for full-length, unligated and ligated EGFR family members, the crystallography may not be possible owing to intrinsic flexibility of full-length EGFR. However, advanced (e.g. cryo-electron) microscopy techniques and the use of carefully designed lipid vesicles or bilayers with the appropriate phospholipid composition ¨ nal Coskun, Paul Langerhans Institute, (as described by U Dresden) are reaching a promising stage, and may fill this gap (Sezgin et al., 2012). It is critical to the EGFR field that someone determines the structures for both the inactive and active forms for all family members and the appropriate complexes.

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Antibodies are excellent probes for exploring the conformation of the EGFR family. Kathryn Ferguson (University Pennsylvania) described the properties of several antibodies which distinguish different states of the EGFR (Bagchi et al., 2014). One antibody (mAb108) was raised against overexpressed, C-terminally truncated EGFR (i.e. lacking a cytoplasmic domain), which bound to domain III and blocked high-affinity binding of the ligand. Another antibody (mAb2E9) raised against overexpressed EGFR on A431 membranes, binds to EGFR domain I and only blocks lowaffinity ligand binding. Both antibodies bind to all of the EGFR on the cell surface, but when bound to the receptor, the antibodies constrain the extracellular domain to either highor low-affinity binding. Kathryn Ferguson described research in conjunction with Paul van Bergen en Henegouwen (Utrecht University), where nanobodies 7D12, EgA1 and 9G8 bind to the EGFR (Schmitz et al., 2013). Whereas 7D12 bound to domain III, the other two nanobodies inhibited the activation of the EGFR by binding to the domain II/III junction. Kate Ferguson also described the characteristics of some new EGFR mutants identified in gliomas that either enhanced dimer formation or increased the affinity of EGFR binding. Nanobodies are being produced to improve the imaging of EGFR or HER2 expressing tumors (Paul van Bergen en Henegouwen, Utrecht University) (Schmitz et al., 2013). An alternative binding scaffold, DARPins, are also being used to probe and alter the orientations and conformations of the EGFR family members on the cell surface and thus to induce cytoxicity in the target cell (Christian Jost, University of Zurich) (Jost et al., 2013). The new HER2-nanobodies have a high affinity and are cleared from the circulation within 4 h; thus, staining with the fluorescent tagged HER2 nanobody can be used to guide the surgical removal of the tumors. Paul van Bergen en Henegouwen also described the potential of EGFR and HER2 nanobody fusion proteins with albumin, trail or toxins to create anticancer drugs with improved therapeutic properties. The compositional diversity of the biological membrane and specific protein–lipid interactions are important and need to be accommodated in our current thinking and methodologies when studying receptor dynamics and activation mechanisms. The allosteric regulation of the human EGFR by direct protein–lipid interactions was demonstrated by ¨ nal Coskun (Paul Langerhans Institute, Dresden) in plasma U membrane derived vesicles of A431 cells and EGFR proteoliposomes with defined lipid compositions (Coskun et al., 2011). The ganglioside GM3 directly exerts a strong allosteric inhibition of EGFR autophosphorylation activity by perturbing the dimerization dynamics of the EGFR transmembrane domains, without affecting ligand-binding properties of EGFR ectodomain dimerization. In contrast to the inhibitory action of the outer leaflet lipid GM3, polyphosphorylated phosphoinositides in the inner leaflet modulate the high-affinity ligand binding state of the EGFR through direct interactions with the intracellular juxtamembrane region of the receptor. Philippe Bastiaens (Max Planck Institute for Molecular Physiology, Dortmund) explained that since cell surface receptor kinases are constrained to a membrane, the reduction in the dimensionality of their movement increases their

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effective concentration and potentiates activation. Thus, both the orientation of the EGFR extracellular domain and the excess of intracellular tyrosine phosphatases combine to avoid spontaneous signaling and allow sensitive, robust ligand signaling with low noise levels. Philippe Bastiaens also described the effects of rab11 and cbl on the activation and trafficking of the EGFR (Blu¨mer et al., 2013; Zimmermann et al., 2013). Distinct EGFR aggregation states can be detected, and homoFRET studies indicate that higher order oligomers traffic differently than activated monomeric receptors. Interestingly, the receptor kinase activation stops recycling to the cell surface and initiates transfer to the late endosome, where cbl-mediated ubiquitination during endicytosis mediates receptor degradation (see also Alex Sorkin, University of Pittsburgh) (Huang et al., 2013). The dynamics of the clustering and movement of the EGFR on the surface of cells is being studied by fluorescence lifetime microscopy (Donna Arndt-Jovin and Thomas Jovin, Max Planck Institute for Biophysical Chemistry) (Ziomkiewicz et al., 2013) and multi-color microscopy using quantum dots (Diane Lidke, University of New Mexico) (Cutler et al., 2013; Valley et al., 2013). The unligated EGFR extra-cellular domain appears to be oriented closer to the plane of the membrane than the ligated dimer. Donna Arndt-Jovin explained that the ligated EGFR dimer is in the extended conformation and thus does not appear to be oriented parallel to the membrane surface (Ziomkiewicz et al., 2013). Diane Lidke used single molecule tracking with ligand labeled with multi-colored quantum dots to follow the interactions between EGFRs. Although some receptors formed stable complexes, there appear to be many interactions in which the receptors interact transiently (Cutler et al., 2013). The types of signaling associated with these distinct types of events need further investigation, especially with respect to the kinetics of the assembly and activation of the intra-cellular complexes. The distribution of erbB3 was characterized by large patches on the cell surface. EGFR kinase mutations, which cause oncogenic activation (e.g. L858R) also, alter the receptor dynamics and phosphorylation. Even in the absence of ligand, L858R-EGFR forms stable dimers on the cell surface. Hyperspectral imaging can track simultaneously the movement of up to eight different quantum dots, such that it will be possible to monitor the interaction dynamics between EGFR family members and their ligands on both normal and cancerous cells. The structures of the monomeric, dimeric and higher order EGFR oligomers on the cell surface were discussed (Kozer et al., 2013; Walker et al., 2012). The issues considered included the relative proportions of tethered and untethered monomers, the orientation(s) of the monomer and dimers to the cell surface, and, in particular, whether a preferential inclination of a single-liganded and thereby asymmetric EGFR dimer account for negative cooperativity, i.e. interference with the binding of a second ligand (Alvarado et al., 2010). [Mentioned but not discussed was evidence for positive cooperativity of binding to the EGFR, as would be predicted by the initial structure-based models of extracellular events.] Answers to many of these questions await higher resolution data from crystallographic studies and/or electron microscopy of full-length receptors reconstituted in lipid

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bilayers (disks, etc.) of defined composition. In the meantime, molecular dynamics simulations are being used to explore the flexibility and aggregation of the EGFR embedded in a phospholipid bilayer (Yibing Shan, D E Shaw Research, New York). The molecular dynamics calculations predict two conformations for the intracellular domain – an inactive form in which the kinase substrate site is occluded by the membrane and an active state in which the kinase domain is located at a distance from the membrane. On ligand binding, an interesting oligomerization of the EGFR occurred during the simulation with ligated EGFR monomers ‘‘sandwiched’’ between unligated EGFR dimers, interacting with each other via donor–acceptor domains resembling those observed in ligand–receptor interactions (Arkhipov et al., 2013; Endres et al., 2013). These EGFR ‘‘rafts’’ were predicted to induce significant curvature of the membrane. During the last decade, alternative protein recognition domains with non-immunoglobulin fold have been created to expand the range of formats and applications beyond what is possible with immunoglobulin-based proteins. In particular, proteins based on ankyrin repeats (DARPins) have been used to select high-affinity binders to a multitude of targets (Christian Jost, University of Zurich). DARPins can be selected, e.g., to bind to specific extracellular domains of HER2 (Jost et al., 2013). Christian Jost described a fusion protein in which DARPins specific for the extracellular domains I and IV of HER2 were linked by a short peptide. The binding of such DARPin fusion proteins, as elucidated by means of protein crystallography, bridges and distorts pairs of HER2 molecules and thereby brings the receptor into a signaling-incompetent state. By these means, the bispecific DARPin induce strong cell-specific cytotoxicity and demonstrate powerful anti-tumor activity in vivo, without any further toxic moiety, which holds promise for developing new therapeutic candidates. The kinase active form of erbB2 has a low diffusivity in membranes that is altered when erbB2 is inhibited by lapatinib (Inhee Chung, Genentech Inc., San Francisco, CA). When overexpressed, erbB2 moves into confined domains on the membrane, which becomes so distorted that regions of corrugated protrusions form on the cell surface (Chung et al., 2014). These corrugated regions reduce the adhesion of the cells and increase their mobility. An overexpressed, kinase dead mutant of erbB2 also induces membrane curvature and the cell surface corrugations. Mark Sliwkowski (Genentech Inc.) made the observation that it would be interesting to know if simply anchoring the erbB2 extracellular domain via a GPI-anchor would also induce membrane curvature or whether transmembrane interactions were required for the effect. By combining the MD simulations of Yibing Shan with the erbB2 membrane biophysical approaches described by Inhee Chung, Diane Lidke and Donna Arndt-Jovin, and the DARPin technology described by Christian Jost, it may be feasible to develop models of the structure and dynamics of EGFR and erbB2 complexes on the cell surface. These models could become sufficiently reliable to provide structural predictions at a higher resolution. EGFR complexes are involved in the directed migration of many cell types. Cell surface EGFR oligomers become associated with actin fibers (Marisa Martin-Fernandez,

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STFC Rutherford Appleton Laboratory, Oxfordshire, UK) (Needham et al., 2013). The linear arrays of cell surface EGFR appear to undergo directed motion. Using similar techniques (Rolfe et al., 2011) it is possible to determine that erbB2 is considerably more mobile than the EGFR (Christopher Tynan, Research Complex at Harwell, Oxfordshire, UK). Receptor clustering processes can also be used to follow the interactions between different receptor subunits. The type II transforming growth factor-b receptor (TGF-bR) can form homodimers on the cell surface and the concentration of the homodimers is influenced by ligands and by intracellular interactions involving the cytoplasmic domain (Yoav Henis, Tel Aviv University). Although the intracellular domain of the type I TGF-bR does not affect homodimer formation, it does affect the formation of type I–II TGF-bR heterodimers (Ehrlich et al., 2011). Immunofluorescence co-patching and patch-FRAP studies show that preformed type I and type II TGF-bR homodimers are present at the cell surface (26 and 45% of the total receptors, respectively), and that ligand binding enhances their formation, except for the type I homodimers (which do not bind ligand). Of note, these experiments also determined the dynamics of complex formation, demonstrating that the TGF-bR oligomers are significantly more stable than the related bone morphogenetic protein receptors. This may reflect on signaling, inasmuch as homomeric complexes may play an inhibitory role. Mark Sliwkowski (Genentech Inc.) discussed the discovery and development of second generation approaches to target HER2. Pertuzumab (PerjetaÕ , Genentech Inc.) is a monoclonal antibody that blocks HER2’s role as a co-receptor (Sliwkowski, 2012). Clinical trials demonstrate that pertuzumab in combination with trastuzumab and taxotere is highly effective in the treatment of patients with first-line metastatic breast cancer. This regimen is now regarded as the standard of care, and has gained health authority approval in more than 30 countries throughout the world. Moreover in September, the label was extended in the US to include neoadjuvant treatment of early HER2-positive breast cancer. The notion of antibody drug or toxin conjugates has been described since the discovery of monoclonal antibodies. T-DM1 (adotrastuzumab emtansine; KadcylaÕ , Genentech Inc.) is an antibody drug conjugate composed of a maytansinoid derivative (a very potent microtubule destabilizing agent) that is conjugated to trastuzumab with a non-reducible thioether linker (DiGiulio, 2013). Randomized clinical trials in advanced metastatic breast cancer patients demonstrate that KadcylaÕ is a highly effective treatment with a very favorable safety profile. These data led to FDA approval of KadcylaÕ in February 2013. Ongoing studies are investigating the combination of PerjetaÕ with KadcylaÕ . If successful, it will be the first time a chemotherapy-free regimen is approved for the treatment of women with first-line metastatic breast cancer (Arteaga et al., 2012; Sliwkowski, 2012). EGFR family members appear to be associated with specific membrane structures. Indeed, the EGFR and erbB3 are present on filopodia of many cell types. It appears that the presence of EGFR or erbB2 on cellular protrusions might lead to the formation of structures that accelerate cell migration and metastasis. Antagonists capable of inhibiting both EGFR

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and erbB2 may prove valuable agents for reducing the escape of tumors from primary sites. The role of EGFR in the control of cell–cell and cell–matrix interactions was discussed by several speakers. The deletion of EGFR in hepatocytes reduces claudin-3 and occludin expression (Maria Sibilia, Medical University of Vienna). Conversely, when the EGFR is expressed and activated in hemopoietic cells it can induce b-catenin expression and consequentially direct E-cadherin to the membrane, including in filopodia and at lamellopodia (Tony Burgess, Walter & Eliza Institute, Melbourne). EGFR signaling is an essential stimulator of tissue formation. Intestinal crypts can be produced from stem cells in the presence of EGF, wnt3a and R-spondin. The roles of these cytokines are yet to be defined, yet EGFR signaling in cell–cell adhesion and cell–matrix interactions during cell production movement clearly plays a critical role in the formation of cell location and dynamics in the crypt (Hirokawa et al., 2014; Tan et al., 2013). Maria Sibilia explained that the role of EGFR family signaling in whole animals is influenced by the genetic background of organisms (e.g. EGFR / mice from 129/Sv mice die during gestation, EGFR / C57Bl mice die soon after birth, whereas EGFR / CH3/MF1 mice survive to weaning, but have bone defects) (Makki et al., 2013; Schreier et al., 2013). In the brain, EGFR / mice suffer from massive hemorrhages. Brain cell specific EGFR / conditional knockout mice (e.g. specific to astrocytes or neural cells) survive; although, it was noted that the EGFR produced before the induction of gene loss can persist for more than 2 months. The hypomorphic allele of the EGFR wa-2 (a V741G mutation) reduces the kinetics and severity of tumor formation in mice expressing elevated levels of SOS under the action of the K5 (keratin promoter). The tumorigenic action of SOS and the stimulation of tumor formation by the actions of the EGFR are related to the stimulation of Akt by the EGFR. A prediction from these animal models is that mutant K-ras-associated tumors should be inhibited by therapeutic strategies aimed at the EGFR. Colon cancer is driven by the deletion of the tumor suppressor Apc. Interestingly, when cre deletion of the Apc gene is driven by the promoter of an inhibitor of the EGFR – Lrig1 (Robert Coffey, Vanderbilt University), the transgenic mice develop adenomas in both the colon and small intestine (Powell et al., 2012; Wang et al., 2013). There is considerable cross-talk between the EGFR family members, required in part because of the lack of a ligand for erbB2 and the defective kinase erbB3. The heterooligomerization associated with ligand activation leads to an activation of signaling from more than one family member at a time. However, in several cancers, EGFR signaling appears to be unbalanced by over-expression of a particular family member EGFR, erbB2 or erbB3. Nancy Hynes (Friedrich Miescher Institute, Basel) asked the question: ‘‘Why do erbB2 overexpressing cells metastasize?’’ In part, her answer was associated with the expression and properties of Memo (mediator of motility) (Marone et al., 2004). Memo is a cofilin-interacting protein that influences PLCg1 and cofilin activities, and is essential for maintaining directionality during ErbB2-induced tumor-cell migration

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(Haenzi et al., 2014; Jiang et al., 2013; Schlatter et al., 2012). Memo acts downstream of erbB2 and of other RTKs including EGFR, PDGFR and FGFR and is essential for migration induced by these receptors. Thus, Memo appears to be a general cell motility regulator, but may potentially be interfering with RhoA activation. The cross-talk between the EGFR family and other tyrosine kinase receptors, e.g. FGFR, Ins/IGF1R and PDGFR is also important for driving proliferation and metastasis (Haenzi et al., 2014). Nancy Hynes showed that in breast cancer it may be necessary to block both the EGFR and FGFR systems to reduce the activation of Akt and the S6 kinase, thereby blocking tumor growth and metastatic potential. EGFRs are internalized from the cell surface by both clathrin-dependent and -independent mechanisms (Alexander Sorkin, University of Pittsburgh). Receptors entering early endosomes are either recycled back to the membrane or progress to late endosomes and lysosomes for degradation (Huang et al., 2013; Sorkin & Puthenveedu, 2013). Although EGFR signaling is usually quenched by receptor degradation, a fusion protein consisting of the EGFR and a de-ubiquitin enzyme recycles and signals continuously. It is important that receptor overexpression and high ligand concentrations are avoided in endocytosis studies, as some of the proteins participating in internalization and lysosomal degradation can be saturated. Ligands other than EGF are believed to dissociate from the EGFR in the early endosomes; however, Grb2 remains attached to EGFR activated by various ligands and continues to direct signaling from the endosomes (Fortian & Sorkin, 2014). Down-regulation of EGFR is a significant feature of growth factor-induced cell cycling. EGFR signaling is reduced at the cell surface both by receptor degradation and feedback inhibitors (Yosef Yarden, Weizmann Institute of Science, Rehovot) (Mellman & Yarden, 2013). The initial EGFR signaling is inhibited by delayed feedback from DUSP (also called MKP) phosphatases (Hu et al., 2013), which dephosphorylate active MAP-kinases, and Mig6, an endogenous inhibitor of the EGFR’s kinase domain. However, to pass through to S-phase and mitosis, EGFR signaling must still be significant 6 h after the initial stimulation. They found that EGFR signaling enables p53 to rapidly (within less than 60 min) associate with the chromatin, such that the chromatinassociated p53 permits the induction of anti-proliferative genes (Ben-Chetrit et al., 2013). As a consequence, cells cannot progress to DNA synthesis and mitosis, unless EGFR activation is prolonged. By using a 2-pulse scenario of stimulation with EGF, it was possible to show that the later pulse of EGF relieves the p53-mediated constraint, thereby allowing cell cycle progression. Thus, unlike normal cells, which require long (46 h) exposure to a growth factor, cancer cells, in which p53 is deleted, commit to cell cycle progression after a short exposure to EGF. The kinetics of signaling from EGFR family members can be quite different (Nguyen et al., 2013; Wagner et al., 2013). In particular, neuregulin-1 produces a sustained intracellular response (levels of c-fos remain high even 90 min after the initial stimulus (Boris Kholodenko, Systems Biology Ireland, University College Dublin, Belfield, Dublin). The timing of different signaling pulses from EGFR, erbB2 and erbB3 appear to control cell proliferation and cell morphology

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(Klinger et al., 2013). It is now possible to develop computer models of EGF and nrg-1 signaling that can predict the fate of cells (e.g. survival, proliferation or migration): where Raf, phosphorylated Raf and Rassf1A are regulated upstream from the EGFR and downstream by LATS1 feedback inhibition. The modeling of EGFR signaling by classical differential equations can be quite complex (Richard Posner, Translational Genomics Research Institute and Northern Arizona University), so it is essential that biologists participate in the processes of simulating specific components of EGFR actions (Whatcott et al., 2013). New tools for creating pathway simulations are available in high level language formats (e.g. BioNetGen), so with minimal mathematical knowledge, biologists can now develop realistic models of the EGFR signaling processes. Many squamous carcinomas (e.g. head and neck cancers) and gliomas are characterized by high levels of EGFR expression; similarly, almost 25% of breast cancers overexpress erbB2. It is reasonable to expect that anti-cancer agents targeting these overexpressed cell surface proteins will have a significant therapeutic advantage. Poly-IC can be targeted to tumors by conjugation to anti-EGFR, anti-Her2 affibodies or EGF (Alex Levitzki, The Hebrew University, Jerusalem) (Zigler et al., 2013). Severe combined immunodeficiency (SCID)-mice reconstituted with human peripheral blood stem cells can carry human tumor xenografts. Treatment of the reconstituted, tumor-bearing mice with the poly-IC-polyethyleneimide-anti-EGFR-affibody kills the tumor with minimal side-effects on the mice. It is important to kill all of the tumor cells, otherwise resistance builds up to anti-EGFR therapies through the development of K-ras, B-raf, PI3K or PTEN mutations (see also Alberto Bardelli, Department of Oncology, University of Torino – IRCC, Candiolo, Italy). When colorectal cancer patients are treated with Cetuximab, although there is an initial tumor response, 80% of the patients relapse with K-ras mutations within 12 months. The patient primary tumors are heterogeneous and it is probable that a subset of tumors, with lower levels of EGFR, escape the Cetuximab treatment via the K-ras oncogene (Alberto Bardelli) (Blair et al., 2014; Misale et al., 2013; Valtorta et al., 2013). It is now clear that free-DNA released by tumors circulates in the blood and this free-DNA can be used for identifying patients in relapse, several months earlier than current clinical techniques (MRI, CT or PET). Once the genetic drivers of a tumor are known (e.g. EGFR and/or K-rasonc), the data from animal models suggests that combination treatments (e.g. MEK inhibitor and Cetuximab) should be more effective than single agents. Although K-ras, N-Ras, B-Raf, PI3K, Met and erbB3 mutations have been detected, it is puzzling that no activating mutations of the EGFR appear to occur in colorectal cancer. Cancers that are driven by oncogenic K-ras or Braf are potential targets for anti-EGFR therapeutics. K-ras activation induces the autocrine ligand TGF-a, which appears to be necessary for many tumors. A new anti-cancer agent, Salirasib, blocks the activation of K-ras (Faigenbaum et al., 2013). Salirasib is now in clinical trial (Yoel Kloog, Tel Aviv University). An interesting interaction has been discovered between nucleolin, the EGFR family members and K-ras

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(Ronit Pinkas-Kramarski and Sari Schokoroy, Tel Aviv University) (Schokoroy et al., 2013). Previously thought to be solely a nucleolar protein, it is clear that nucleolin shuttles between the membrane, cytosol and nucleus. On the membrane, nucleolin binds to the EGFR and enhances signaling. Ligand-independent phosphorylation of the EGFR is associated with the formation of an EGFR-oncogenic K-ras-nucleolin complex. Overexpression of nucleolin in K-ras transformed cells increases anchorage-independent growth, which correlates with increased oncogenicity of cells in mouse xenografts. Although EGFR activates an intracellular phosphotyrosine cascade, other signaling systems are triggered from ligandactivated EGFR (e.g. glucose transport). Under hypoxic conditions, activation of the human EGFR perturbs the maturation of miRNAs (Mien-Chie Hung, University of Texas M D Anderson Cancer Center) (Shen et al., 2013). miRNA maturation plays an important role in fundamental biological processes and cancer development. Other activities of the EGFR have been proposed, in particular, direct transcriptional modification of promoter complexes by the EGFR kinase; however, more data are required before a definitive role for nuclear EGFR in transcriptional processes can be considered likely. Although anti-EGFR therapy has already helped many colon and lung cancer patients, there are significant sideeffects (e.g. severe skin rash), which limit the clinical studies from building on new information, e.g. the 3D-structures of the extra-cellular domain (Gan et al., 2012). The mechanism of binding of a ‘‘tumor-specific’’ anti-EGFR antibody (e.g. mAb806) has been determined (Andrew Scott Ludwig Institute for Cancer Research and Tony Burgess Walter & Eliza Hall Institute, Melbourne) (Walker et al., 2012). The mAb806 epitope also provides a probe of the EGFR conformation during ligand activation and heterodimerization. The 806 antibody binds to the EGFR on tumors which overexpress the receptor, where the receptor is truncated or where there is continuous autocrine activation (e.g. brain tumors) (Lee et al., 2010). Neither the chimeric nor the humanized form of antibody 806 are taken up by the liver or cause skin rashes. Animal models indicate that mAb806 improves the efficacy of radiotherapy (Johns et al., 2010). The tumor specificity of mAb806 has allowed the conjugation of a toxin to the antibody (ABT414); the conjugate accumulates in xenografted human gliomas growing in mice and kills the tumors. Several new anti-cancer antibodies and alternative binding scaffolds were presented at the meeting (e.g. to deliver a toxin via erbB2, a dual specificity EGFR/HER3 antibody, poly-IC-anti-EGFR and – erbB2 affibodies, nanobodies derived from heavy chain only antibodies (Vosjan et al., 2011) and DARPins inducing an inactive state of HER2). The dual action antibody or two-in-one antibody against EGFR/ erbB3 is more effective than the combined actions of Cetuximab and anti-erbB3 (Gabriele Schaefer, Genentech Inc., San Francisco, CA) (Jaiswal et al., 2013; Kamath et al., 2012; Phillips et al., 2014). Interestingly, the dual action antibody is effective on Cetuximab-resistant tumors and it was most effective on two patients whose tumors had the highest levels of neuregulin-1.

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These antibody-drug conjugates and dual-specificity antibodies represent a new era in targeted cancer diagnostics, imaging and targeted therapies. When we are able to combine the efficacy of these cell surface directed drugs with drugs which interfere with cancer-specific signaling processes (e.g. oncogenic K-ras, oncogenic PI3kinase or oncogenic B-raf), we will clearly be approaching more effective, rational treatments for cancer. There is a wealth of genetic, signaling and cell biology knowledge associated with the EGFR family; synthesis and use of this knowledge is starting to impact on clinical studies. The decade ahead will be an exciting era for the scientists and clinicians studying the EGFR family.

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Acknowledgements The conference organizers wish to acknowledge the enthusiasm and logistical support provided by the team at the Israel Institute of Advanced Studies. Their commitment to excellence, care for the conference participants and their facilities helped to create the wonderful atmosphere which stimulated our discussions.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. The Institute for Advanced Studies and the Israel Science Foundation, Eli Lilly and Genentech Inc. provided the generous financial support which made the conference possible.

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