Intracellular Tyrosine Kinase Domain Research Articles

Abstract IA012: Targeting AXL in platinum-resistant ovarian cancer: from the lab bench to Phase 3 trial

Abstract IA012: Targeting AXL in platinum-resistant ovarian cancer: from the lab bench to Phase 3 trial

Extracellular domain mutations of the EGF receptor differentially modulate high-affinity and low-affinity responses to EGF receptor ligands

The EGF receptor is mutated in a number of cancers. In most cases, the mutations occur in the intracellular tyrosine kinase domain. However, in glioblastomas, many of the mutations are in the extracellular ligand binding domain. To determine what changes in receptor function are induced by such extracellular domain mutations, we analyzed the binding and biological response to the seven different EGF receptor ligands in three common glioblastoma mutants--R84K, A265V, and G574V. Our data indicate that all three mutations significantly increase the binding affinity of all seven ligands. In addition, the mutations increase the potency of all ligands for stimulating receptor autophosphorylation, phospholipase Cγ, Akt, and MAP kinase activity. In all mutants, the rank order of ligand potency seen at the wild-type receptor was retained, suggesting that the receptors still discriminate among the different ligands. However, the low affinity ligands, EPR and EPG, did show larger than average enhancements of potency for stimulating Akt and MAPK, but not receptor autophosphorylation and phospholipase Cγ activation. Relative to the wild-type receptor, these changes lead to an increase in the responsiveness of these mutants to physiological concentrations of ligands and an alteration in the ratio of activation of the different pathways. This may contribute to their oncogenic potential. In the context of recent findings, our data also suggest that so-called ‘high’ affinity biological responses arise from activation by isolated receptor dimers, whereas ‘low’ affinity biological responses require clustering of receptors which occurs at higher concentrations of ligand.

Distinct interactions stabilize EGFR dimers and higher-order oligomers in cell membranes

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase with important roles in many cellular processes as well as in cancer and other diseases. EGF binding promotes EGFR dimerization and autophosphorylation through interactions that are well understood structurally. How these dimers relate to higher-order EGFR oligomers seen in cell membranes, however, remains unclear. Here, we used single-particle tracking (SPT) and Förster resonance energy transfer imaging to examine how each domain of EGFR contributes to receptor oligomerization and the rate of receptor diffusion in the cell membrane. Although the extracellular region of EGFR is sufficient to drive receptor dimerization, we find that the EGF-induced EGFR slowdown seen by SPT requires higher-order oligomerization-mediated in part by the intracellular tyrosine kinase domain when it adopts an active conformation. Our data thus provide important insight into the interactions required for higher-order EGFR assemblies involved in EGF signaling.

A Small Molecule Targeting the Intracellular Tyrosine Kinase Domain of ROR1 (KAN0441571C) Induced Significant Apoptosis of Non-Small Cell Lung Cancer (NSCLC) Cells.

The ROR1 receptor tyrosine kinase is expressed in embryonic tissues but is absent in normal adult tissues. ROR1 is of importance in oncogenesis and is overexpressed in several cancers, such as NSCLC. In this study, we evaluated ROR1 expression in NSCLC patients (N = 287) and the cytotoxic effects of a small molecule ROR1 inhibitor (KAN0441571C) in NSCLC cell lines. ROR1 expression in tumor cells was more frequent in non-squamous (87%) than in squamous (57%) carcinomas patients, while 21% of neuroendocrine tumors expressed ROR1 (p = 0.0001). A significantly higher proportion of p53 negative patients in the ROR1+ group than in the p53 positive non-squamous NSCLC patients (p = 0.03) was noted. KAN0441571C dephosphorylated ROR1 and induced apoptosis (Annexin V/PI) in a time- and dose-dependent manner in five ROR1+ NSCLC cell lines and was superior compared to erlotinib (EGFR inhibitor). Apoptosis was confirmed by the downregulation of MCL-1 and BCL-2, as well as PARP and caspase 3 cleavage. The non-canonical Wnt pathway was involved. The combination of KAN0441571C and erlotinib showed a synergistic apoptotic effect. KAN0441571C also inhibited proliferative (cell cycle analyses, colony formation assay) and migratory (scratch wound healing assay) functions. Targeting NSCLC cells by a combination of ROR1 and EGFR inhibitors may represent a novel promising approach for the treatment of NSCLC patients.

Precise assembly of inside-out cell membrane camouflaged nanoparticles via bioorthogonal reactions for improving drug leads capturing.

Cell membrane camouflaged nanoparticles have been widely used in the field of drug leads discovery attribute to their unique biointerface targeting function. However, random orientation of cell membrane coating does not guarantee effective and appropriate binding of drugs to specific sites, especially when applied to intracellular regions of transmembrane proteins. Bioorthogonal reactions have been rapidly developed as a specific and reliable method for cell membrane functionalization without disturbing living biosystem. Herein, inside-out cell membrane camouflaged magnetic nanoparticles (IOCMMNPs) were accurately constructed via bioorthogonal reactions to screen small molecule inhibitors targeting intracellular tyrosine kinase domain of vascular endothelial growth factor recptor-2. Azide functionalized cell membrane acted as a platform for specific covalently coupling with alkynyl functionalized magnetic Fe3O4 nanoparticles to prepare IOCMMNPs. The inside-out orientation of cell membrane was successfully verified by immunogold staining and sialic acid quantification assay. Ultimately, two compounds, senkyunolide A and ligustilidel, were successfully captured, and their potential antiproliferative activities were further testified by pharmacological experiments. It is anticipated that the proposed inside-out cell membrane coating strategy endows tremendous versatility for engineering cell membrane camouflaged nanoparticles and promotes the development of drug leads discovery platforms.

Cancer regulator EGFR-ErbB4 heterodimer is stabilized through glycans at the dimeric interface.

EGFR and ErbB4 are the only two members of cancer-regulating ErbB RTKs that maintain the activation of their extracellular ligand-binding domain and intracellular tyrosine kinase domain. EGFR and ErbB4 could form homo and heterodimers upon their activation. Heterodimerization triggers more diverse intracellular pathways compared to homodimerization. Moreover, it is known that N-glycosylation is crucial for the stabilization and activation of EGFR and ErbB4 receptors. Herein, atomistic molecular dynamics were simulated to study the EGFR-ErbB4 heterodimer in the glycosylated and unglycosylated states. It was shown that the EGFR-ErbB4 heterodimer is highly stabilized by glycosylation. The increased stability is most significant at the dimeric interfaces, regulated by packing of three glycans attached to EGFR (Asn337) and ErbB4 (Asn333, Asn523) at the dimeric interface. Finally, it is proposed that heterodimerization is the persistent key player in the EGFR and ErbB4 activation. Thus, targeting the heterodimers in future therapeutic designs could be a promising approach against drug resistance to ErbB-positive cancers.

Activation of the human insulin receptor by non-insulin-related peptides

The human insulin receptor signalling system plays a critical role in glucose homeostasis. Insulin binding brings about extensive conformational change in the receptor extracellular region that in turn effects trans-activation of the intracellular tyrosine kinase domains and downstream signalling. Of particular therapeutic interest is whether insulin receptor signalling can be replicated by molecules other than insulin. Here, we present single-particle cryoEM structures that show how a 33-mer polypeptide unrelated to insulin can cross-link two sites on the receptor surface and direct the receptor into a signalling-active conformation. The 33-mer polypeptide engages the receptor by two helical binding motifs that are each potentially mimicable by small molecules. The resultant conformation of the receptor is distinct from—but related to—those in extant three-dimensional structures of the insulin-complexed receptor. Our findings thus illuminate unexplored pathways for controlling the signalling of the insulin receptor as well as opportunities for development of insulin mimetics.

The characterization and knockdown of a male gonad-specific insulin-like receptor gene in the white shrimp Penaeus vannamei

The insulin-like androgenic gland-specific peptide is a known primary regulator of sexual differentiation in crustaceans. However, the knowledge of the crucial elements involved in the insulin-like signaling cascade remains fragmented. Here, we characterized a novel insulin-like receptor gene in the white shrimp Penaeus vannamei and named as Pv-IR. The deduced amino acid sequence of Pv-IR contained conserved domains of IRs, including two ligand-binding domains, a furin-like domain, three fibronectin-3 domains, and an intracellular tyrosine kinase domain in order. Multiple sequence alignment of the first ligand-binding domain and the tyrosine kinase domain in Pv-IR revealed a high degree of similarity in all representative IRs. The phylogenetic analysis presented that Pv-IR was clustered into a separate branch with IRs from decapods. The tissue distribution analysis showed that Pv-IR was predominantly expressed in the sperm duct and terminal ampullae of the male reproductive system in P. vannamei. Meanwhile, Pv-IR knockdown was carried out in postlarvae. Although the sex reversal phenotype was not observed, the effects of Pv-IR silencing on the expressions of related-genes were analyzed by the comparative transcriptomic sequencing. Significantly, two down-regulated transcripts (clottable protein and insulin-like peptide) and two up-regulated transcripts (flotillin and thyroid stimulating hormone receptor) were focused as gonad-related differentially expressed unigenes in Pv-IR knockdown. The data provided a more theoretical basis for elucidating the mechanism of sexual differentiation in crustaceans.

EP16.03-019 Landscape of EGFR Extracellular Domain Mutations in Advanced Non Small Cell Lung Carcinoma

Plasma next generation sequencing (NGS) is a non-invasive method for tumor genomic profiling that offers personalized treatment options via cell free DNA analysis for patients with advanced cancers. Targeted therapies direct activity toward specific functional protein domain(s) to inhibit tumor cell survival. The EGFR protein is comprised of an extracellular domain (ECD) that spans amino acids 1-620 and an intracellular tyrosine kinase domain (TKD) that can be targeted in multiple cancer types. In advanced non-small cell carcinoma (NSCLC), EGFR directed-therapies mainly focus on mutations within the TKD.

Guidelines for HTRF technology in EGFR kinase assay

AbstractEpidermal growth factor receptor (EGFR) is among the first discovered transmembrane receptor tyrosine kinases. Ligand binding to the extracellular domain of EGFR results in the formation of homo‐dimeric or hetero‐dimeric complexes, activation, and auto‐phosphorylation in the intracellular tyrosine kinase domain, then regulates the cell's proliferation, differentiation, migration, and invasion. It has been reported that EGFR is overexpressed, amplified, or mutated in several epithelial tumors. EGFR tyrosine kinase inhibitors (EGFR‐TKIs) specifically bind to the tyrosine kinase domain of EGFR. Homogeneous time‐resolved fluorescence (HTRF) assay is successfully used in kinase assays and is a crucial tool in the drug discovery of various kinases. Moreover, the HTRF assay has been determined to exhibit many advantages, such as being easy to miniaturize, simple to read, and excellent to repeat. In these guidelines, the theory, materials, and reagent preparation of HTRF kinase assay are described and emphasized. Furthermore, the protocol and optimization of the assay are determined to establish an HTRF screening platform, then an optimized HTRF kinase assay is performed to test the compounds for screening potential EGFR‐TKIs. In addition, these guidelines can contribute to verifying the high‐throughput screening results of HTRF and help to reveal the underlying mechanism of the EGFR kinase and tested compounds.

Vandetanib drives growth arrest and promotes sensitivity to imatinib in chronic myeloid leukemia by targeting ephrin type‐B receptor 4

The oncogenic role of ephrin type‐B receptor 4 (EPHB4) has been reported in many types of tumors, including chronic myeloid leukemia (CML). Here, we found that CML patients have a higher EPHB4 expression level than healthy subjects. EPHB4 knockdown inhibited growth of K562 cells (a human immortalized myelogenous leukemia cell line). In addition, transient transfection of EPHB4 siRNA led to sensitization to imatinib. These growth defects could be fully rescued by EPHB4 transfection. To identify an EPHB4‐specific inhibitor with the potential of rapid translation into the clinic, a pool of clinical compounds was screened and vandetanib was found to be most sensitive to K562 cells, which express a high level of EPHB4. Vandetanib mainly acts on the intracellular tyrosine kinase domain and interacts stably with a hydrophobic pocket. Furthermore, vandetanib downregulated EPHB4 protein via the ubiquitin‐proteasome pathway and inhibited PI3K/AKT and MAPK/ERK signaling pathways in K562 cells. Vandetanib alone significantly inhibited tumor growth in a K562 xenograft model. Furthermore, the combination of vandetanib and imatinib exhibited enhanced and synergistic growth inhibition against imatinib‐resistant K562 cells in vitro and in vivo. These findings suggest that vandetanib drives growth arrest and overcomes the resistance to imatinib in CML via targeting EPHB4.

Correlation Epidermal Growth Factor Receptor Mutation with Non-small Cell Lung Cancer in Passive Smokers: A Review

Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein with an extracellular epidermal growth factor binding domain and an intracellular tyrosine kinase domain through signaling pathways to regulate cellular proliferation. Epidermal growth factor receptor binding to its ligand will result in autophosphorylation by intrinsic tyrosine kinase activity, thereby triggering multiple signal transduction cascades. Constant or sustained activation of these downstream target sequences is thought to result in a more aggressive tumor phenotype. Mutations in EGFR are associated with non-small cell lung cancer in passive smokers.

Glioblastoma mutations alter EGFR dimer structure to prevent ligand bias.

The epidermal growth factor receptor (EGFR) is frequently mutated in human cancer1,2, and is an important therapeutic target. EGFR inhibitors have been successful in lung cancer, where mutations in theintracellular tyrosine kinase domain activate the receptor1, but not in glioblastoma multiforme (GBM)3, where mutations occur exclusively in the extracellular region. Here we show that common extracellular GBM mutations prevent EGFR from discriminating between its activating ligands4. Different growth factor ligands stabilize distinct EGFR dimer structures5 that signal with different kinetics to specify or bias outcome5,6. EGF itself induces strong symmetric dimers that signal transiently to promote proliferation. Epiregulin (EREG) induces much weaker asymmetric dimers that drive sustained signalling and differentiation5. GBM mutations reduce the ability of EGFR to distinguish EREG from EGF in cellular assays, and allow EGFR to form strong (EGF-like) dimers in response to EREG and other low-affinity ligands. Using X-ray crystallography, we further show that the R84K GBM mutation symmetrizes EREG-driven extracellular dimers so that they resemble dimers normally seen with EGF. By contrast, a second GBM mutation, A265V, remodels key dimerization contacts to strengthen asymmetric EREG-driven dimers. Our results argue for an important role of altered ligand discrimination by EGFR in GBM, with potential implications for therapeutic targeting.

FLT4 Mutations Are Associated with Segmental Lymphatic Dysfunction and Initial Lymphatic Aplasia in Patients with Milroy Disease.

This study explored mutations in the Fms-related tyrosine kinase 4/vascular endothelial growth factor receptor 3 gene (FLT4) and lymphatic defects in patients with Milroy disease (MD). Twenty-nine patients with lower limb lymphedema were enrolled. Sixteen patients had a familial history of MD, while 13 patients exhibited sporadic MD. Clinical signs, FLT4 mutations, indocyanine green (ICG) lymphography findings, and skin tissue immunohistochemical staining results were evaluated. Twenty-eight variants in FLT4 were identified. Twelve of these have previously been reported, while 16 are novel. Of the 28 variants, 26 are missense mutations, and the remaining two comprise a splicing mutation and a non-frame shift mutation. Twenty-five variants are located in the intracellular protein tyrosine kinase domain; three are located in the extracellular immunoglobulin domain. Substantially delayed contrast-enhanced tortuous lymphatic vessels were visualized to the ankle or knee level in 15 of 23 patients who underwent ICG lymphography. No initial lymphatic vessels were visualized in skin specimens from four patients who did not exhibit lymphatic vessels during imaging analyses. No specific variant was identified in relation to the unique clinical phenotype. Segmental dysfunction of lymphatic vessels and initial lymphatic aplasia are present in MD patients with FLT4 mutations.

CAR T Cells Targeting FLT3 on AML Confer Potent Anti-Leukemic Activity and Act Synergistically with the FLT3 Inhibitor Midostaurin

Background: FMS-like tyrosine kinase 3 (FLT3) is a homodimeric transmembrane protein that is expressed on normal hematopoietic stem (HSC) and progenitor cells. FLT3 is also uniformly present on malignant blasts in acute myeloid leukemia (AML) providing a target for immunotherapy with chimeric antigen receptor (CAR)-modified T cells. FLT3 is a driver of leukemia-genesis in AML cases with internal tandem duplication (FLT3-ITD) and other gain-of-function mutations in the intracellular tyrosine kinase domain. Accordingly, the FLT3 inhibitor midostaurin has recently been approved in combination with chemotherapy to treat FLT3-mutated AML. Here, we explored the antileukemia efficacy of FLT3 CAR-T cells against FLT3 wild-type (wt) and FLT3-ITD+ AML, alone and in combination with midostaurin to determine whether the two therapeutic modalities could be used synergistically, particularly in high-risk FLT3-ITD+ AML.Methods: A FLT3-CAR (BV10 scFv_IgG4 hinge_CD28_CD3ζ_EGFRt) was encoded in the lentiviral vector epHIV7 for gene-transfer into T cells of healthy donors and AML patients (n>6). CAR-T cell cytolytic activity was evaluated in FACS-/luminescence-based assays, cytokine production analyzed by ELISA and proliferation assessed by CFSE dye dilution. Immunodeficient NSG mice were engrafted with MOLM-13/ffLuc AML cells and treated with 5x106 CAR-modified or control T cells (CD4:CD8 ratio = 1:1). MOLM-13 cells were cultured in the presence of 10nM midostaurin and doses titrated up to 50nM to induce resistance (MOLM-13R).Results: We confirmed specific recognition and high-level cytolytic activity of CD8+ FLT3 CAR-T cells against a panel of AML cell lines that included THP-1 (FLT3 wt), MOLM-13 (FLT3-ITD+/-) and MV4;11 (FLT3-ITD+/+), as well as FLT3-ITD+ primary AML blasts obtained from multiple patients. Both CD8+ and CD4+ FLT3 CAR-T cells produced IFN-γ and IL-2, and underwent proliferation after stimulation with native AML cell lines and primary AML blasts.We then treated AML cell lines with midostaurin and detected a significant increase in FLT3 expression in MOLM-13 and MV4;11 by flow cytometry (delta MFI = 8986 and 1442, respectively, p<0.05), but not THP-1 cells, indicating that upregulation specifically occurred in FLT3-ITD+ AML. We sought to determine whether higher FLT3 expression on midostaurin-resistant AML target cells translated into superior antileukemia efficacy of FLT3 CAR-T cells. Indeed, we observed superior cytolytic activity, cytokine production and proliferation of FLT3 CAR-T cells after stimulation with MOLM-13R compared to native MOLM-13 cells (n=3 experiments; p<0.05). In contrast to FLT3, expression of CD33 and CD123 as alternative CAR target antigens was decreased on MOLM-13R.We confirmed upregulation of FLT3 occurred on native MOLM-13 AML cells in vivo during midostaurin treatment in NSG mice, and observed synergistic antileukemia efficacy of FLT3 CAR-T cells and midostaurin in combination therapy. Intriguingly, we detected higher frequencies of CAR-T cells in all mice that received the combination treatment, compared to mice that received FLT3 CAR-T cells alone. At the end of the observation period, all mice in the combination treatment group had achieved complete remission of AML from peripheral blood, bone marrow and spleen.Importantly, we also show that FLT3 CAR-T cells recognize normal HSCs and interfere with hematopoiesis in colony formation assays in vitro . However, midostaurin treatment did not lead to an increase of FLT3 on normal HSC consistent with their expression of wt FLT3.Summary: Collectively, our data demonstrate that FLT3 CAR-T cells mediate potent reactivity against FLT3 wt and FLT3-ITD+ AML cells in vitro and in vivo . We further demonstrate that FLT3 CAR-T cells and midostaurin act synergistically, i.e. midostaurin-induced upregulation of FLT3 in FLT3-ITD+ AML cells enhances their recognition and elimination by FLT3 CAR-T cells. Due to recognition of normal HSC, the clinical use of FLT3 CAR-T cells is likely restricted to a defined therapeutic window prior to allogeneic HSC transplantation, followed by CAR-T cell depletion and hematopoietic reconstitution. Our data provide an operational model to augment the antileukemia efficacy of FLT3 CAR-T cells in high-risk FLT3-ITD+ AML patients, and to mitigate the risk for relapse with FLT3-negative AML variants that may otherwise develop under therapeutic pressure. DisclosuresNo relevant conflicts of interest to declare.

Role of Glycans on Key Cell Surface Receptors That Regulate Cell Proliferation and Cell Death.

Cells undergo proliferation and apoptosis, migration and differentiation via a number of cell surface receptors, most of which are heavily glycosylated. This review discusses receptor glycosylation and the known roles of glycans on the functions of receptors expressed in diverse cell types. We included growth factor receptors that have an intracellular tyrosine kinase domain, growth factor receptors that have a serine/threonine kinase domain, and cell-death-inducing receptors. N- and O-glycans have a wide range of functions including roles in receptor conformation, ligand binding, oligomerization, and activation of signaling cascades. A better understanding of these functions will enable control of cell survival and cell death in diseases such as cancer and in immune responses.

Pyrimidine-based EGFR TK inhibitors in targeted cancer therapy.

Despite significant improvements of new treatment options, cancer continues to represent as one of the most common and fatal disease. The EGFR signaling pathway is considered as a significant approach in targeted therapy of cancers. Blocking the EGFR-driven pathway by inhibiting the intracellular tyrosine kinase domain of EGFR have shown considerable improvement in cancer therapy. In an effort to identify EGFR tyrosine kinase inhibitors (TKI), several small molecules especially pyrimidine containing derivatives have been designed by applying molecular simulation and evaluated the emergence of epigenetic mutation and resistance problems restricted the long-term effectiveness of such medication and explained the need for further investigations in this field. In recent years, the studies have been focused on genetic alterations on EGFR tyrosine kinase domain, which led to the design and synthesis of more selective and effective inhibitors. Herein, we give an overview of the importance and status of EGFR inhibitors in cancer therapy. In addition, we provide an update of the recent advances in design, discovery and development of novel pyrimidine containing compounds as promising selective EGFR TK inhibitors.

The Ablation of VEGFR-1 Signaling Promotes Pressure Overload-Induced Cardiac Dysfunction and Sudden Death.

Molecular mechanisms involved in cardiac remodelling are not fully understood. To study the role of vascular endothelial growth factor receptor 1 (VEGFR-1) signaling in left ventricular hypertrophy (LVH) and heart failure, we used a mouse model lacking the intracellular VEGFR-1 tyrosine kinase domain (VEGFR-1 TK−/−) and induced pressure overload with angiotensin II infusion. Using echocardiography (ECG) and immunohistochemistry, we evaluated pathological changes in the heart during pressure overload and measured the corresponding alterations in expression level and phosphorylation of interesting targets by deep RNA sequencing and Western blot, respectively. By day 6 of pressure overload, control mice developed significant LVH whereas VEGFR-1 TK−/− mice displayed a complete absence of LVH, which correlated with significantly increased mortality. At a later time point, the cardiac dysfunction led to increased ANP and BNP levels, atrial dilatation and prolongation of the QRSp duration as well as increased cardiomyocyte area. Immunohistochemical analyses showed no alterations in fibrosis or angiogenesis in VEGFR-1 TK−/− mice. Mechanistically, the ablation of VEGFR-1 signaling led to significantly upregulated mTOR and downregulated PKCα phosphorylation in the myocardium. Our results show that VEGFR-1 signaling regulates the early cardiac remodelling during the compensatory phase of pressure overload and increases the risk of sudden death.

A novel uniquely ovary-expressed insulin-like receptor in the female prawn, Macrobrachium rosenbergii (Decapoda, Palaemonidae)

Abstract The insulin signalling pathway is one of the most studied pathways, including ovary maturation and female reproduction. However, downstream elements involved in this cascade remain unknown. Here, we identified and characterized an insulin-like receptor (IR) in female Macrobrachium rosenbergii, and named as Mro-IR. The deduced Mro-IR contained conserved domains of IR proteins, including two ligand-binding domains, a furin-like domain, two fibronectin-3 domains, a transmembrane domain and an intracellular tyrosine kinase domain in order. Multiple sequence alignment of the first ligand-binding domain and the tyrosine kinase domain in Mro-IR revealed a high degree of similarity to other representative IRs. Mro-IR was clustered into a separate branch with IRs from decapods by phylogenetic analysis. Additionally, tissue distribution analysis showed that Mro-IR was uniquely expressed in the ovary of M. rosenbergii. This study would possibly provide a new highlight to the molecular mechanism of insulin-like signalling cascade, regulating female sexual differentiation in crustaceans.

IL-8 secreted by tumor associated macrophages contribute to lapatinib resistance in HER2-positive locally advanced breast cancer via activation of Src/STAT3/ERK1/2-mediated EGFR signaling

Locally advanced breast cancer (LABC) is an aggressive disease characterized by late clinical presentation, large tumor size, treatment resistance and low survival rate. Expression of EGFR/HER2 and activation of intracellular tyrosine kinase domains in LABC are associated with poor prognosis. Thus, target therapies such as the anti-receptor tyrosine kinases lapatinib drug have been more developed in the past decade. The response to lapatinib involves the inhibition of RTKs and subsequently signaling molecules such as Src/STAT3/Erk1/2 known also to be activated by the cytokines in the tumor microenvironment (TME). The aim of the present study is to identify the major cytokine that might contribute to lapatinib resistance in EGFR+/HER2+ LABC patients. Indeed, tumor associated macrophages (TAMs) are the main source of cytokines in the TME. Herein, we isolated TAMs from LABC during modified radical mastectomy (MRM). Cytokine profile of TAMs revealed that IL-8 is the most prominent highly secreted cytokine by TAMs of LABC patients. Using in-vitro cell culture model we showed that recombinant IL-8 (50 and 100 ng/mL) at different time intervals interfere with lapatinib action via activation of Src/EGFR and signaling molecules known to be inhibited during treatment. We proposed that to improve LABC patients' response to lapatinib treatment it is preferred to use combined therapy that neutralize or block the action of IL-8.