Ligand-independent oncogenic signaling by the epidermal growth factor receptor: v-ErbB as a paradigm

Relay of information from the extracellular environment into the cell often results from a peptide growth factor binding to its cognate cell surface receptor; this event is an integral mechanism by which many cellular functions occur, including cell growth, motility, and survival. In recent years, however, this requirement for ligand binding has been shown to be surpassed by several distinct mechanisms, including cell surface receptor cross-talk (e.g., between epidermal growth factor receptor [EGFR] and G-coupled receptors), receptor-extracellular matrix interactions (e.g., EGFR: integrin complexes), and finally by structural mutations within the receptor itself. While all of these pathways result in so-called ligand-independent signaling by the EGF receptor, to date, only structural mutations in the receptor have been shown to result in qualitative changes in downstream targets of the receptor, which specifically result in oncogenic signaling, transformation, and tumorigenicity. In this review, we describe aspects of the known signaling properties of the retroviral oncogene v-ErbB as a model of ligand-independent oncogenic signaling, and compare these properties to results emerging from ongoing studies on structurally related EGF receptor mutants originally identified in human tumors. A better understanding of the signaling pathways used by these uniquely oncogenic receptor tyrosine kinase mutants may ultimately reveal new targets for the development of novel therapeutics selective for the inhibition of tumor cell growth.     

Forced dimerization increases the activity of ΔEGFR/EGFRvIII and enhances its oncogenicity

Delta epidermal growth factor receptor (ΔEGFR), an in-frame deletion mutant of the extracellular ligand-binding domain, which occurs in about 30% of glioblastoma, is a potent oncogene that promotes tumor growth and progression. The signaling of ΔEGFR is ligand-independent and low intensity, allowing it to evade the normal mechanisms of internalization and degradation by the endocytic machinery and hence is persistent. The basis of the oncogenic potential of ΔEGFR remains incompletely understood, including whether dimerization plays an important role in its signal and whether its oncogenic potential is dependent on its relatively low intensity, when compared with the acutely activated wild-type receptor. To examine these two important questions, we have generated a chimeric ΔEGFR that allows forced dimerization via domains derived from variants of the FKBP12 protein that are brought together by FK506 derivatives. Forced dimerization of chimeric ΔEGFR significantly increased the intensity of its signal, as measured by receptor phosphorylation levels, suggesting that the naturally occurring ΔEGFR does not form strong or stable dimers as part of its low level signal. Interestingly, the increased activity of dimerized, chimeric ΔEGFR did not promote receptor internalization, implying that reduced rate of endocytic downregulation of ΔEGFR is an inherent characteristic. Significantly, forced dimerization enhanced the oncogenic signal of the receptor, implying that the ΔEGFR is a potent oncogene despite, not because of its low intensity.     

Epidermal Growth Factor Receptor (EGFR) Signaling Regulates Global Metabolic Pathways in EGFR-mutated Lung Adenocarcinoma

Genetic mutations in tumor cells cause several unique metabolic phenotypes that are critical for cancer cell proliferation. Mutations in the tyrosine kinase epidermal growth factor receptor (EGFR) induce oncogenic addiction in lung adenocarcinoma (LAD). However, the linkage between oncogenic mutated EGFR and cancer cell metabolism has not yet been clearly elucidated. Here we show that EGFR signaling plays an important role in aerobic glycolysis in EGFR-mutated LAD cells. EGFR-tyrosine kinase inhibitors (TKIs) decreased lactate production, glucose consumption, and the glucose-induced extracellular acidification rate (ECAR), indicating that EGFR signaling maintained aerobic glycolysis in LAD cells. Metabolomic analysis revealed that metabolites in the glycolysis, pentose phosphate pathway (PPP), pyrimidine biosynthesis, and redox metabolism were significantly decreased after treatment of LAD cells with EGFR-TKI. On a molecular basis, the glucose transport carried out by glucose transporter 3 (GLUT3) was downregulated in TKI-sensitive LAD cells. Moreover, EGFR signaling activated carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), which catalyzes the first step in de novo pyrimidine synthesis. We conclude that EGFR signaling regulates the global metabolic pathway in EGFR-mutated LAD cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, which is an attractive target for the development of more effective targeted therapies to treat patients with EGFR-mutated LAD.     

A structural perspective on targeting the RTK/Ras/MAP kinase pathway in cancer

Precision oncology is premised on identifying and drugging proteins and pathways that drive tumorigenesis or are required for survival of tumor cells. Across diverse cancer types, the signaling pathway emanating from receptor tyrosine kinases on the cell surface to RAS and the MAP kinase pathway is the most frequent target of oncogenic mutations, and key proteins in this signaling axis including EGFR, SHP2, RAS, BRAF, and MEK have long been a focus in cancer drug discovery. In this review, we provide an overview of historical and recent efforts to develop inhibitors targeting these nodes with an emphasis on the role that an understanding of protein structure and regulation has played in inhibitor discovery and characterization. Beyond its well‐established role in structure‐based drug design, structural biology has revealed mechanisms of allosteric regulation, distinct effects of activating oncogenic mutations, and other vulnerabilities that have opened new avenues in precision cancer drug discovery.     

The anti-EGFR monoclonal antibody blocks cisplatin-induced activation of EGFR signaling mediated by HB-EGF

Cisplatin is a key agent in combination chemotherapy for various types of solid tumor. We now show that cisplatin activates signaling by the epidermal growth factor receptor (EGFR) by inducing cleavage of heparin-binding epidermal growth factor-like growth factor (HB-EGF). Matuzumab, a monoclonal antibody to EGFR, inhibited cisplatin-induced EGFR signaling, likely through competition with the soluble form of HB-EGF for binding to EGFR. Matuzumab enhanced the antitumor effect of cisplatin in nude mice harboring human non-small cell lung cancer xenografts. Our findings shed light on the mechanism by which monoclonal antibodies to EGFR might augment the efficacy of cisplatin.     

Triepitopic Antibody Fusions Inhibit Cetuximab-Resistant BRAF and KRAS Mutant Tumors via EGFR Signal Repression

Dysregulation of epidermal growth factor receptor (EGFR) is a hallmark of many epithelial cancers, rendering this receptor an attractive target for cancer therapy. Much effort has been focused on the development of EGFR-directed antibody-based therapeutics, culminating in the clinical approval of the drugs cetuximab and panitumumab. Unfortunately, the clinical efficacy of these drugs has been disappointingly low, and a particular challenge to targeting EGFR with antibody therapeutics has been resistance, resulting from mutations in the downstream raf and ras effector proteins. Recent work demonstrating antibody cocktail-induced synergistic downregulation of EGFR motivated our design of cetuximab-based antibody-fibronectin domain fusion proteins that exploit downregulation-based EGFR inhibition by simultaneously targeting multiple receptor epitopes. We establish that, among our engineered multiepitopic formats, trans-triepitopic antibody fusions demonstrate optimal efficacy, inducing rapid EGFR clustering and internalization and consequently ablating downstream signaling. The combined effects of EGFR downregulation, ligand competition, and immune effector function conspire to inhibit tumor growth in xenograft models of cetuximab-resistant BRAF and KRAS mutant cancers. Our designed triepitopic constructs have the potential to enhance the efficacy and expand the scope of EGFR-directed therapies, and our multiepitopic may be readily applied to other receptor targets to formulate a new class of antibody-based therapeutics.     

Can EGFR be a therapeutic target in breast cancer?

Epidermal growth factor receptor (EGFR) is highly expressed in certain cancer types and is involved in regulating the biological characteristics of cancer progression, including proliferation, metastasis, and drug resistance. Various medicines targeting EGFR have been developed and approved for several cancer types, such as lung and colon cancer. To date, however, EGFR inhibitors have not achieved satisfactory clinical results in breast cancer, which continues to be the most serious malignant tumor type in females. Therefore, clarifying the underlying mechanisms related to the ineffectiveness of EGFR inhibitors in breast cancer and developing new EGFR-targeted strategies (e.g., combination therapy) remain critical challenges. Various studies have demonstrated aberrant expression and maintenance of EGFR levels in breast cancer. In this review, we summarize the regulatory mechanisms underlying EGFR protein expression in breast cancer cells, including EGFR mutations, amplification, endocytic dysfunction, recycling acceleration, and degradation disorders. We also discuss potential therapeutic strategies that act directly or indirectly on EGFR, including reducing EGFR protein expression, treating the target protein to mediate precise clearance, and inhibiting non-EGFR signaling pathways. This review should provide new therapeutic perspectives for breast cancer patients with high EGFR expression.     

Paracrine regulation of growth factor signaling by shed leucine-rich repeats and immunoglobulin-like domains 1

Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a recently discovered negative regulator of growth factor signaling. The LRIG1 integral membrane protein has been demonstrated to regulate various oncogenic receptor tyrosine kinases, including epidermal growth factor (EGF) receptor (EGFR), by cell-autonomous mechanisms. Here, we investigated whether LRIG1 ectodomains were shed, and if LRIG1 could regulate cell proliferation and EGF signaling in a paracrine manner. Cells constitutively shed LRIG1 ectodomains in vitro, and shedding was modulated by known regulators of metalloproteases, including the ADAM17 specific inhibitor TAPI-2. Furthermore, shedding was enhanced by ectopic expression of Adam17. LRIG1 ectodomains appeared to be shed in vivo, as well, as demonstrated by immunoblotting of mouse and human tissue lysates. Ectopic expression of LRIG1 in lymphocytes suppressed EGF signaling in co-cultured fibroblastoid cells, demonstrating that shed LRIG1 ectodomains can function in a paracrine fashion. Purified LRIG1 ectodomains suppressed EGF signaling without any apparent downregulation of EGFR levels. Taken together, the results show that the LRIG1 ectodomain can be proteolytically shed and can function as a non-cell-autonomous regulator of growth factor signaling. Thus, LRIG1 or its ectodomain could have therapeutic potential in the treatment of growth factor receptor-dependent cancers.     

Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1

Receptor desensitization is accomplished by accelerated endocytosis and degradation of ligand-receptor complexes. An in vitro reconstituted system indicates that Cbl adaptor proteins directly control downregulation of the receptor for the epidermal growth factor (EGFR) by recruiting ubiquitin-activating and -conjugating enzymes. We infer a sequential process initiated by autophosphorylation of EGFR at a previously identified lysosome-targeting motif that subsequently recruits Cbl. This is followed by tyrosine phosphorylation of c-Cbl at a site flanking its RING finger, which enables receptor ubiquitination and degradation. Whereas all three members of the Cbl family can enhance ubiquitination, two oncogenic Cbl variants, whose RING fingers are defective and phosphorylation sites are missing, are unable to desensitize EGFR. Our study identifies Cbl proteins as components of the ubiquitin ligation machinery and implies that they similarly suppress many other signaling pathways.     

Inhibition of SHP2 leads to mesenchymal to epithelial transition in breast cancer cells

The Src homology phosphotyrosyl phosphatase 2 (SHP2) is an essential transducer of mitogenic and cell survival signaling in the epidermal growth factor receptor (EGFR) signaling pathway. However, the role of SHP2 in aberrant EGFR and human EGFR2 (HER2) signaling and cancer, particularly in breast cancer, has not been investigated. Here, we report that SHP2 is required for mitogenic and cell survival signaling and for sustaining the transformation phenotypes of breast cancer cell lines that overexpress EGFR and HER2. Inhibition of SHP2 suppressed EGF-induced activation of the Ras-ERK and the phosphatidylinositol 3 kinase-Akt signaling pathways, abolished anchorage-independent growth, induced epithelial cell morphology and led to reversion to a normal breast epithelial phenotype. Furthermore, inhibition of SHP2 led to upregulation of E-cadherin (epithelial marker) and downregulation of fibronectin and vimentin (mesenchymal markers). These results indicate that SHP2 promotes breast cancer cell phenotypes by positively modulating mitogenic and cell survival signaling, by suppressing E-cadherin expression which is known to play a tumor suppressor role and by sustaining the mesenchymal state as evidenced by the positive impact on fibronectin and vimentin expression. Therefore, SHP2 promotes epithelial to mesenchymal transition, whereas its inhibition leads to mesenchymal to epithelial transition. On the basis of these premises, we propose that interference with SHP2 function might help treat breast cancer.     

Endocytic downregulation of ErbB receptors: mechanisms and relevance in cancer

ErbB receptors (EGFR (ErbB1), ErbB2, ErbB3, and ErbB4) are important regulators of normal growth and differentiation, and they are involved in the pathogenesis of cancer. Following ligand binding and receptor activation, EGFR is endocytosed and transported to lysosomes where the receptor is degraded. This downregulation of EGFR is a complex and tightly regulated process. The functions of ErbB2, ErbB3, and ErbB4 are also regulated by endocytosis to some extent, although the current knowledge of these processes is sparse. Impaired endocytic downregulation of signaling receptors is frequently associated with cancer, since it can lead to increased and uncontrolled receptor signaling. In this review we describe the current knowledge of ErbB receptor endocytic downregulation. In addition, we outline how ErbB receptors can escape endocytic downregulation in cancer, and we discuss how targeted anti-cancer therapy may induce endocytic downregulation of ErbB receptors.     

小分子酪氨酸激酶抑制剂耐药机制

表皮生长因子受体（epidermal growth factor receptor,EGFR）通路异常在肿瘤发生、发展过程中起到非常重要的作用,特异性抑制该通路的小分子受体酪氨酸激酶抑制剂在肿瘤治疗上取得了显著的效果,但是该药在临床上已经出现耐药现象,现将有关EGFR基因突变、EGFR旁路信号通路的激活、下游信号分子的结构性活化3个方面对EGFR抑制剂耐药机制的研究进展进行综述。     

Membrane-tethered Notch1 exhibits oncogenic property via activation of EGFR–PI3K–AKT pathway in oral squamous cell carcinoma

Notch proteins are highly conserved cell surface receptors which play essential roles in cellular differentiation, proliferation, and apoptotic events at all stages of development. Recently, NOTCH1 mutations have been extensively observed in oral squamous cell carcinoma (OSCC) and are hinted to be Notch1-inactivating mutations. However, little is known about the biological effect of these reported mutations in OSCC. To mimic the inactivation of Notch1 due to inappropriate mutations and to determine the potential mechanisms, we utilized wild-type Notch1 vectors (Notch1^WT) or mutant Notch1 vectors (Notch1^V1754L) to transfect into OSCC cell lines. Membrane-tethered Notch1 induced by mutation was analyzed by immunofluorescence staining. γ-Secretase inhibitor PF-03084014 was utilized to determine the phenotype in the absence of endogenous Notch1 activation. Here we demonstrated that membrane-tethered Notch1 inactivated the canonical Notch1 signaling and oncogenic phenotypes were identified by promoting cell proliferation and invasion and by inducing epithelial-to-mesenchymal transition in cells. The γ-secretase inhibitor PF-03084014 also showed distinct oncogenic property after treatment. Importantly, both membrane-tethered Notch1 and PF-03084014 inhibitor activated the epidermal growth factor receptor (EGFR)–phosphoinositide 3-kinase (PI3K)–protein kinase B (AKT) signaling pathway, which has been confirmed as an overwhelming modulator in OSCC. This was the first time that we clearly simulated the mutated Notch1 activities and determined the oncogenic phenotypes of membrane-tethered Notch1. Compared with wild-type Notch1, membrane-tethered Notch1 was strongly associated with activated EGFR–PI3K–AKT signaling pathway.     

Tumor cell killing mechanisms of epidermal growth factor receptor (EGFR) antibodies are not affected by lung cancer-associated EGFR kinase mutations

The epidermal growth factor receptor (EGFR) serves as a molecular target for novel cancer therapeutics such as tyrosine kinase inhibitors (TKI) and EGFR Abs. Recently, specific mutations in the EGFR kinase domain of lung cancers were identified, which altered the signaling capacity of the receptor and which correlated with clinical response or resistance to TKI therapy. In the present study, we investigated the impact of such EGFR mutations on antitumor cell activity of EGFR Abs. Thus, an EGFR-responsive cell line model was established, in which cells with tumor-derived EGFR mutations (L858R, G719S, delE746-A750) were significantly more sensitive to TKI than wild-type EGFR-expressing cells. A clinically relevant secondary mutation (T790M) abolished TKI sensitivity. Significantly, antitumor effects of EGFR Abs, including signaling and growth inhibition and Ab-dependent cellular cytotoxicity, were not affected by any of these mutations. Somatic tumor-associated EGFR kinase mutations, which modulate growth inhibition by TKI, therefore do not impact the activity of therapeutic Abs in vitro.