epidermal-growth-factor and Neoplasms

During the initial stages of gastrulation during embryonic differentiation and wound healing, Cripto-1 is a critical protein for human growth. The epithelial adhesion molecules' downregulation, the mesenchymal overexpression, and mobile proteins are important mechanisms by which Cripto-1 initiates epithelial to mesenchymal transition (EMT). As a result, the function of Cripto-1 for inducing EMT to increase cell migration is advantageous during embryogenesis; however, it is deleterious during the formation, growth, and malignant tumor metastasis. The majority of malignancies are reported to have elevated levels of Cripto-1. Cripto-1 can modify cancerous cells through its function in EMT, which enables these cells to migrate via the extracellular matrix, bloodstream, and lymphatic vessels, on their way for metastasizing to other organs. The goal of this review is to explain what role Cripto-1 plays in common cancers and to summarize how therapeutic strategies are used to interfere with this molecule to target cancers.

Topics: Cell Differentiation; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Humans; Neoplasm Proteins; Neoplasms

Behind the scenes of signaling cascades initiated by activated receptors, endocytosis determines the fate of internalized proteins through degradation in lysosomes or recycling. Over the years, significant progress has been made in understanding the mechanisms of endocytosis and deregulation in disease states. Here we review the role of the EGF-SNX3-EGFR axis in breast cancers with an extended discussion on deregulated EGFR endocytosis in cancer.

Topics: Endocytosis; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Signal Transduction

Abnormal regulation of cell signaling pathways on cell survival, proliferation and migration contributes to the development of malignant tumors. Among them, epidermal growth factor receptor (EGFR) is one of the most important biomarkers in many types of malignant solid tumors. Its over-expression and mutation status can be served as a biomarker to identify patients who can be benifit from EGFR tyrosine kinase inhibitors and anti-EGFR monocloncal antibody (mAb) therapy. For decades, researches on EGFR targeted ligands were actively carried out to identify potent candidates for cancer therapy. An ideal EGFR ligand can competitively inhibit the binding of endogenous growth factor, such as epidermal growth factor (EGF) and transforming growth factor-α(TGF-α) to EGFR, thus block EGFR signaling pathway and downregulate EGFR expression. Alternatively, conjugation of EGFR ligands on drug delivery systems (DDS) can facilitate targeting delivery of therapeutics or diagnostic agents to EGFR over-expression tumors via EGFR-mediated endocytosis. GE11 peptide is one of the potent EGFR ligand screened from a phage display peptide library. It is a dodecapeptide that can specifically binds to EGFR with high affinity and selectivity. GE11 has been widely used in the diagnosis and targeted delivery of drugs for radiotherapy, genetherapy and chemotherpy against EGFR positive tumors. In this review, the critical factors affecting the in vivo and in vitro targeting performance of GE11 peptide, including ligand-receptor intermolecular force, linker bond properties and physiochemical properties of carrier materials, are detailedly interpreted. This review provides a valuable vision for the rational design and optimization of GE11-based active targeting strategies for cancer treatment, and it will promote the translation studies of GE11 from lab research to clinical application.

Topics: Epidermal Growth Factor; ErbB Receptors; Humans; Ligands; Neoplasms; Peptide Library; Peptides; Protein Kinase Inhibitors; Transforming Growth Factor alpha

Previously, we showed that knockout mice homozygous for deficiency of the mercapturic acid pathway (MAP) transporter protein, RLIP (RLIP

Topics: Acetylcysteine; Adipokines; Animals; Carcinogenesis; Carcinogens; Carrier Proteins; Clathrin; Cytokines; Epidermal Growth Factor; GTPase-Activating Proteins; Humans; Infant; Insulins; Male; Mice; Neoplasms; Obesity; Oxidative Stress; Peptide Hormones; Transforming Growth Factor beta; Tumor Suppressor Protein p53

The pursual of novel anticancer molecules from natural sources has gained worthwhile appreciation, and a significant fraction of conceptual knowledge has revolutionized our understanding about heterogeneous nature of cancer. Betulinic acid has fascinated interdisciplinary researchers due to its tremendous pharmacological properties. Ground-breaking discoveries have unraveled previously unprecedented empirical proof-of-concept about momentous chemopreventive role of betulinic acid against carcinogenesis and metastasis. Deregulation of cell signaling pathways has been reported to play a linchpin role in cancer progression and colonization of metastatically competent cancer cells to the distant organs for the development of secondary tumors. Importantly, betulinic acid has demonstrated unique properties to mechanistically modulate oncogenic transduction cascades. In this mini-review, we have attempted to provide a sophisticated compendium of regulatory role of betulinic acid in cancer chemoprevention. We have partitioned this multi-component review into different sections in which we summarized landmark research-works which highlighted betulinic acid mediated regulation of JAK/STAT, VEGF, EGF/EGFR, TRAIL/TRAIL-R, AKT/mTOR and ubiquitination pathways in the inhibition of cancer. In parallel, betulinic acid mediated regulation of signaling cascades and non-coding RNAs will be critically analyzed in cell culture and animal model studies. Better comprehension of the pharmaceutical features of betulinic acid and mapping of the existing knowledge gaps will be valuable in the translatability of preclinical studies into rationally designed clinical trials.

Topics: Animals; Antineoplastic Agents; Betulinic Acid; Carcinogenesis; Epidermal Growth Factor; ErbB Receptors; Neoplasms; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A

Cancer is a complex group of diseases initiated by abnormal cell division with the potential of spreading to other parts of the body. The advancement in the discoveries of omics and bio- and cheminformatics has led to the identification of drugs inhibiting putative targets including vascular endothelial growth factor (VEGF) family receptors, fibroblast growth factors (FGF), platelet derived growth factors (PDGF), epidermal growth factor (EGF), thymidine phosphorylase (TP), and neuropeptide Y4 (NY4), amongst others. Drug resistance, systemic toxicity, and drug ineffectiveness for various cancer chemo-treatments are widespread. Due to this, efficient therapeutic agents targeting two or more of the putative targets in different cancer cells are proposed as cutting edge treatments. Heterocyclic compounds, both synthetic and natural products, have, however, contributed immensely to chemotherapeutics for treatments of various diseases, but little is known about such compounds and their multimodal anticancer properties. A compendium of heterocyclic synthetic and natural product multitarget anticancer compounds, their IC

Topics: Antineoplastic Agents; Biological Products; Epidermal Growth Factor; Fibroblast Growth Factors; Heterocyclic Compounds; Humans; Neoplasms; Platelet-Derived Growth Factor; Receptors, Neuropeptide Y; Thymidine Phosphorylase; Vascular Endothelial Growth Factor A

Epidermal growth factor receptor (EGFR) inhibitors were among the first type of targeted agents discovered in cancer and currently constitute the standard of care for a wide range of lung and colon malignancies. However, the therapeutic progress achieved with these drugs has been accompanied by the identification of an ever-increasing number of acquired resistance mechanisms that inevitably appear in nearly all patients. Increased knowledge on EGFR biochemistry, cellular crosstalk, and resistance pathways provides an opportunity to establish effective combination therapies and discover novel-acting inhibitors that prevent or overcome therapeutic resistance. One such strategy is the selective blockade of circulating growth factors such as EGF. In this review, we address the uses and limitations of approved EGFR inhibitors and explore the potential of drug combinations and new third avenues to block the activation of the EGFR.

Topics: Animals; Clinical Trials as Topic; Epidermal Growth Factor; ErbB Receptors; Humans; Molecular Targeted Therapy; Neoplasms; Protein Kinase Inhibitors

Cytoskeletal networks are dramatically reorganized upon EGF stimulation to enable complex cell behaviors such as cell-cell communication, migration and invasion, and cell division. In this issue of

Topics: Animals; Cell Proliferation; Cytoskeleton; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Proteome

Research on the epidermal growth factor (EGF) family and the family of receptors (EGFR) has progressed rapidly in recent times. New crystal structures of the ectodomains with different ligands, the activation of the kinase domain through oligomerisation and the use of fluorescence techniques have revealed profound conformational changes on ligand binding. The control of cell signaling from the EGFR-family is complex, with heterodimerisation, ligand affinity and signaling cross-talk influencing cellular outcomes. Analysis of tissue homeostasis indicates that the control of pro-ligand processing is likely to be as important as receptor activation events. Several members of the EGFR-family are overexpressed and/or mutated in cancer cells. The perturbation of EGFR-family signaling drives the malignant phenotype of many cancers and both inhibitors and antagonists of signaling from these receptors have already produced therapeutic benefits for patients. The design of affibodies, antibodies, small molecule inhibitors and even immunotherapeutic drugs targeting the EGFR-family has yielded promising new approaches to improving outcomes for cancer patients. In this review, we describe recent discoveries which have increased our understanding of the structure and dynamics of signaling from the EGFR-family, the roles of ligand processing and receptor cross-talk. We discuss the relevance of these studies to the development of strategies for designing more effective targeted treatments for cancer patients.

Topics: Animals; Antineoplastic Agents; Binding Sites; Drug Design; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Ligands; Mice; Models, Molecular; Neoplasms; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Signal Transduction; Structure-Activity Relationship

Members of the epidermal growth factor receptor (EGFR/ErbB) family play a critical role in normal cell growth and development. However, many ErbB family members, especially EGFR, are aberrantly expressed or deregulated in tumors and are thought to play crucial roles in cancer development and metastatic progression. In this chapter, we provide an overview of key mechanisms contributing to aberrant EGFR/ErbB signaling in transformed cells, which results in many phenotypic changes associated with the earliest stages of tumor formation, including several hallmarks of epithelial-mesenchymal transition (EMT). These changes often occur through interaction with other major signaling pathways important to tumor progression, causing a multitude of transcriptional changes that ultimately impact cell morphology, proliferation, and adhesion, all of which are crucial for tumor progression. The resulting mesh of signaling networks will need to be taken into account as new regimens are designed for targeting EGFR for therapeutic intervention. As new insights are gained into the molecular mechanisms of cross talk between EGFR signaling and other signaling pathways, including their roles in therapeutic resistance to anti-EGFR therapies, a continual reassessment of clinical therapeutic regimes and strategies will be required. Understanding the consequences and complexity of EGF signaling and how it relates to tumor progression is critical for the development of clinical compounds and establishing clinical protocols for the treatment of cancer.

Topics: Animals; Cell Proliferation; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Signal Transduction

P-glycoprotein (P-gp; multidrug resistance pump 1, MDR1; ABCB1) is a plasma membrane efflux pump that when activated in cancer cells exports chemotherapeutic agents. Transcription of the P-gp gene (MDR1) and activity of the P-gp protein are known to be affected by thyroid hormone. A cell surface receptor for thyroid hormone on integrin αvβ3 also binds tetraiodothyroacetic acid (tetrac), a derivative of L-thyroxine (T4) that blocks nongenomic actions of T4 and of 3,5,3'-triiodo-L-thyronine (T3) at αvβ3. Covalently bound to a nanoparticle, tetrac as nanotetrac acts at the integrin to increase intracellular residence time of chemotherapeutic agents such as doxorubicin and etoposide that are substrates of P-gp. This action chemosensitizes cancer cells. In this review, we examine possible molecular mechanisms for the inhibitory effect of nanotetrac on P-gp activity. Mechanisms for consideration include cancer cell acidification via action of tetrac/nanotetrac on the Na(+)/H(+) exchanger (NHE1) and hormone analogue effects on calmodulin-dependent processes and on interactions of P-gp with epidermal growth factor (EGF) and osteopontin (OPN), apparently via αvβ3. Intracellular acidification and decreased H(+) efflux induced by tetrac/nanotetrac via NHE1 is the most attractive explanation for the actions on P-gp and consequent increase in cancer cell retention of chemotherapeutic agent-ligands of MDR1 protein.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B; Cation Transport Proteins; Epidermal Growth Factor; Humans; Integrin alphaVbeta3; Neoplasm Proteins; Neoplasms; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; Thyroid Hormones

Epigen is the latest addition to the mammalian family of EGFR ligands. Epigen was initially identified as a novel expressed sequence tag with homology to the EGF family by high throughput sequencing of a mouse keratinocyte complementary DNA library, and received its name for its ability to act as an epithelial mitogen. In vitro studies attributed to epigen several unique features, such as persistent and potent biological actions involving low affinity receptor binding, as well as sub-maximal receptor activation and inactivation. Similarly to the other EGFR ligands, the expression of epigen is up-regulated by hormones and in certain cancer types. While the biological functions of epigen remain to be uncovered, it appears to play a role in epidermal structures, such as the mammary gland and the sebaceous gland. The latter organ, in particular, was greatly enlarged in transgenic mice overexpressing epigen. Interestingly, mice lacking epigen develop and grow normally, probably due to functional compensation by other EGFR ligands. Future studies are likely to reveal the biological roles of the unique receptor binding properties of epigen, as well as its potential harnessing during disease.

Topics: Animals; Epidermal Growth Factor; Epigen; ErbB Receptors; Gene Knockout Techniques; Humans; Neoplasms; Signal Transduction

Betacellulin was initially detected as a growth-promoting factor in the conditioned medium of a mouse pancreatic β-cell tumor cell line. Sequencing of the purified protein and of the cloned cDNA supported the assumption that betacellulin is a new ligand of the epidermal growth factor receptor (EGFR), which was later confirmed experimentally. As a typical EGFR ligand, betacellulin is expressed by a variety of cell types and tissues, and the soluble growth factor is proteolytically cleaved from a larger membrane-anchored precursor. Importantly, BTC can - in addition to the EGFR - bind and activate all possible heterodimeric combinations of the related ERBB receptors including the highly oncogenic ERBB2/3 dimer, as well as homodimers of ERBB4. While a large number of studies attest a role for betacellulin in the differentiation of pancreatic β-cells, the last decade witnessed the association of betacellulin with a large number of additional biological processes, ranging from reproduction to the control of neural stem cells.

Topics: Animals; Betacellulin; Cell Differentiation; Epidermal Growth Factor; ErbB Receptors; Humans; Insulin-Secreting Cells; Neoplasms

Epiregulin is a 46-amino acid protein that belongs to the epidermal growth factor (EGF) family of peptide hormones. Epiregulin binds to the EGF receptor (EGFR/ErbB1) and ErbB4 (HER4) and can stimulate signaling of ErbB2 (HER2/Neu) and ErbB3 (HER3) through ligand-induced heterodimerization with a cognate receptor. Epiregulin possesses a range of functions in both normal physiologic states as well as in pathologic conditions. Epiregulin contributes to inflammation, wound healing, tissue repair, and oocyte maturation by regulating angiogenesis and vascular remodeling and by stimulating cell proliferation. Deregulated epiregulin activity appears to contribute to the progression of a number of different malignancies, including cancers of the bladder, stomach, colon, breast, lung, head and neck, and liver. Therefore, epiregulin and the elements of the EGF/ErbB signaling network that lie downstream of epiregulin appear to be good targets for therapeutic intervention.

Topics: Animals; Cell Proliferation; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Humans; Neoplasms; Signal Transduction

Tumor progression can be understood as a collaborative effort of mutations and growth factors, which propels cell proliferation and matrix invasion, and also enables evasion of drug-induced apoptosis. Concentrating on EGFR, we discuss downstream signaling and the initiation of transcriptional events in response to growth factors. Specifically, we portray a wave-like program, which initiates by rapid disappearance of two-dozen microRNAs, followed by an abrupt rise of immediate early genes (IEGs), relatively short transcripts encoding transcriptional regulators. Concurrent with the fall of IEGs, some 30-60 min after stimulation, a larger group, the delayed early genes, is up-regulated and its own fall overlaps the rise of the final wave of late response genes. This late wave persists and determines long-term phenotype acquisition, such as invasiveness. Key regulatory steps in the orderly response to growth factors provide a trove of potential oncogenes and tumor suppressors.

Topics: Animals; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genes, erbB-1; Genes, Immediate-Early; Humans; MicroRNAs; Neoplasms; Transcription, Genetic

This review focuses on the mechanisms by which the expression of specific genes is regulated by two proteins that are important in inflammation and cancer, namely the pro-inflammatory cytokine interleukin (IL)-1β and epidermal growth factor (EGF). In the review the receptors that recognize factors that cause inflammation are described with main focus on the receptors associated with activation of IL-1β. The function of IL-1β and pathways leading to activation of transcription factors, particularly NFκB and Elk-1 are analyzed. Then the mechanisms of EGF action, with particular emphasis of the activation of Elk-1 are illustrated. The link between aberrant signaling of EGF receptor family members and cancer development is explained. The relationship between inflammation and tumorigenesis is discussed.

Topics: Animals; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Humans; Inflammation; Interleukin-1; Neoplasms; Receptors, Interleukin

Within tumor microenvironment, a lot of growth factors such as hepatocyte growth factor and epidermal growth factor may induce similar signal cascade downstream of receptor tyrosine kinase (RTK) and trigger tumor metastasis synergistically. In the past decades, the intimate relationship of RTK-mediated receptor endocytosis with signal transduction was well established. In general, most RTK undergoes clathrin-dependent endocytosis and/or clathrin-independent endocytosis. The internalized receptors may sustain the signaling within early endosome, recycling to plasma membrane for subsequent ligand engagement or sorting to late endosomes/lysosome for receptor degradation. Moreover, receptor endocytosis influences signal transduction in a temporal and spatial manner for periodical and polarized cellular processes such as cell migration. The endosomal signalings triggered by various metastatic factors are quite similar in some critical points, which are essential for triggering cell migration and tumor progression. There are common regulators for receptor endocytosis including dynamin, Rab4, Rab5, Rab11 and Cbl. Moreover, many critical regulators within the RTK signal pathway such as Grb2, p38, PKC and Src were also modulators of endocytosis. In the future, these may constitute a new category of targets for prevention of tumor metastasis.

Topics: Disease Progression; Endocytosis; Endosomes; Epidermal Growth Factor; Fibroblast Growth Factors; Hepatocyte Growth Factor; Humans; Neoplasm Metastasis; Neoplasms; Platelet-Derived Growth Factor; Signal Transduction; Transforming Growth Factor beta

Since centuries, natural compounds from plants, animals and microorganisms were used in medicinal traditions to treat various diseases without a solid scientific basis. Recent studies have shown that plants that were used or are still used in the medieval European medicine are able to provide relieve for many diseases including cancer. Here we summarize impact and effect of selected purified active natural compounds from plants used in European medieval medicinal traditions on cancer hallmarks and enabling characteristics identified by Hanahan and Weinberg. The aim of this commentary is to discuss the pharmacological effect of pure compounds originally discovered in plants with therapeutic medieval use. Whereas many reviews deal with Ayurvedic traditions and traditional Chinese medicine, to our knowledge, the molecular basis of European medieval medicinal approaches are much less documented.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Cycle; Epidermal Growth Factor; Europe; Humans; Immune System; Molecular Targeted Therapy; Neoplasms; Neovascularization, Pathologic; Phytotherapy; Plants, Medicinal

Despite significant improvements in diagnosis, surgical techniques, and advancements in general patient care, the majority of deaths from cancer are caused by the metastases. There is an urgent need for an improved understanding of the cellular and molecular factors that promote cancer metastasis. The process of cancer metastasis depends on multiple interactions between cancer cells and host cells. Studies investigating the TGF α-EGFR signaling pathways that promote the growth and spread of cancer cells. Moreover, the signaling activates not only tumor cells, but also tumor-associated endothelial cells. TGF α-EGFR signaling in colon cancer cells creates a microenvironment that is conducive for metastasis, providing a rationale for efforts to inhibit EGFR signaling in TGF α-positive cancers. In this review, we describe the recent advances in our understanding of the molecular basis of cancer metastasis.

Topics: Animals; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasm Metastasis; Neoplasms; Signal Transduction; Tumor Microenvironment

The epidermal growth factor (EGF) pathway stimulates proliferation and differentiation of epidermal and epithelial tissues, and plays an important role in tumorigenesis. The association between EGF polymorphisms and cancer risk is controversial; thus, we performed this meta-analysis. Overall, 41 case-control studies with 9,779 cases and 15,932 controls were retrieved. We found that EGF +61A/G polymorphism increased overall cancer risk (G allele vs. A allele: OR=1.181, 95% CI=1.077-1.295, P(heterogeneity) < 0.001; GG vs. AA: OR=1.370, 95% CI=1.143-1.641, P(heterogeneity) < 0.001; GG+GA vs. AA: OR=1.175, 95% CI=1.047-1.318, P(heterogeneity) < 0.001). In the stratified analysis by cancer type, the +61 G allele was a risk factor for colorectal cancer, esophageal carcinoma, gastric cancer, and hepatocellular carcinoma. Individuals who carried +61G allele had higher cancer susceptibility in mixed and European racial subgroups. An increased association was detected in the hospital-based subgroup. No significant association was found among EGF -1380A/G, -1744G/A, rs6983267T/G polymorphisms and cancer risk.

Topics: Case-Control Studies; Epidermal Growth Factor; Genetic Association Studies; Genetic Predisposition to Disease; Genotype; Humans; Neoplasms; Odds Ratio; Polymerase Chain Reaction; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length; Risk Factors

Although the EGFR (epidermal growth factor receptor) was discovered over 30 years ago, its mechanism of activation is still the subject of intense study. There are many published studies on the mechanism of EGFR activation and regulation, including biochemical and biophysical analyses and crystallographic structures of EGFR in different activation states and conformations, mutated at various amino acids or bound to different pharmacological inhibitors. The cumulative biochemical, biophysical and structural data have led to a nearly complete account of the mechanism of activation of EGFR. The role of the JXM (juxtamembrane) domain in EGFR structure and activity has only recently begun to be elucidated through biochemical, biophysical and structural studies. In the present article, I review the studies that have highlighted the role of the JXM domain in EGFR activation.

Topics: Amino Acid Sequence; Calcium Signaling; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; Molecular Sequence Data; Neoplasms; Protein Binding; Protein Interaction Domains and Motifs; Protein Transport

Interfering with angiogenesis is an effective, widely used approach to cancer therapy, but antiangiogenic therapies have been associated with important systemic cardiovascular toxicities such as hypertension, left ventricular dysfunction, heart failure, and myocardial ischemia and infarction. As the use of vascular endothelial growth factor signaling pathway (VSP) inhibitors broadens to include older patients and those with existing cardiovascular disease, the adverse effects are likely to be more frequent, and cardiologists will increasingly be enlisted to help oncologists manage patients who develop adverse cardiovascular effects.. The Cardiovascular Toxicities Panel of the National Cancer Institute reviewed the published literature and abstracts from major meetings, shared experience gained during clinical development of VSP inhibitors, and contributed extensive clinical experience in evaluating and treating patients with cancer with cardiovascular disease. This report was edited and approved by the National Cancer Institute Investigational Drug Steering Committee. It presents the panel's expert opinion on the current clinical use and future investigation for safer, more expansive use of these drugs.. The panel recommends that physicians (1) conduct and document a formal risk assessment for existing cardiovascular disease and potential cardiovascular complications before VSP inhibitor treatment recognizing that preexisting hypertension and cardiovascular disease are common in patients with cancer, (2) actively monitor for blood pressure elevations and cardiac toxicity with more frequent assessments during the first treatment cycle, and (3) aggressively manage blood pressure elevations and early symptoms and signs of cardiac toxicity to prevent clinically limiting complications of VSP inhibitor therapy.

Topics: Antineoplastic Agents; Cardiovascular Diseases; Cardiovascular System; Disease Management; Epidermal Growth Factor; Global Health; Humans; Incidence; Neoplasms; Risk Factors; Signal Transduction

Metastatic, rather than primary tumours are responsible for ninety percent cancer deaths. Despite significant advances in the understanding of molecular and cellular mechanisms in tumour metastases, there are limitations in preventive treatment of metastatic tumours. Much evidence arising from laboratory and clinical studies suggests that growth factors and their receptors are implicated in cancer metastases development. We review the origin and production of growth factors and their receptors in all stages of cancer metastases including epithelial-mesenchymal transition, cancer cell invasion and migration, survival within the circulation, seeding at distant organs and metastatic tumour angiogenesis. The functions of growth factors and their receptors are also discussed. This review presents the efforts made in understanding this challenge to aid in the development of new treatment strategies for cancer metastases.

Topics: Angiopoietins; Animals; Apoptosis; Cell Movement; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Glucose-6-Phosphate Isomerase; Hepatocyte Growth Factor; Humans; Insulin-Like Growth Factor I; Intercellular Signaling Peptides and Proteins; Interleukin-8; Multienzyme Complexes; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Seeding; Neoplasms; Neoplastic Cells, Circulating; Neovascularization, Pathologic; Phosphodiesterase I; Phosphoric Diester Hydrolases; Pyrophosphatases; Receptor, IGF Type 1; Receptors, Growth Factor; Ribonuclease, Pancreatic; Smad Proteins; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The most ominous stage of cancer progression is metastasis, or the dissemination of carcinoma cells from the primary site into distant organs. Metastases are often resistant to current extirpative therapies and even the newest biological agents cure only a small subset of patients. Therefore a greater understanding of tumor biology that integrates properties intrinsic to carcinomas with tissue environmental modulators of behavior is needed. In no aspect of tumor progression is this more evident than the acquisition of cell motility that is critical for both escape from the primary tumor and colonization. In this overview, we discuss how this behavior is modified by carcinoma cell phenotypic plasticity that is evidenced by reversible switching between epithelial and mesenchymal phenotypes. The presence or absence of intercellular adhesions mediate these switches and dictate the receptivity towards signals from the extracellular milieu. These signals, which include soluble growth factors, cytokines, and extracellular matrix embedded with matrikines and matricryptines will be discussed in depth. Finally, we will describe a new mode of discerning the balance between epithelioid and mesenchymal movement.

Topics: Cadherins; Cell Adhesion; Cell Movement; Cell Transformation, Neoplastic; Cytokines; Desmosomes; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Extracellular Matrix Proteins; Gap Junctions; Hepatocyte Growth Factor; Humans; Insulin-Like Growth Factor I; Integrins; Neoplasm Metastasis; Neoplasms; Phenotype; Signal Transduction; Tight Junctions; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Overexpressed receptors, characteristic of many cancers, have been targeted by various researchers to achieve a more specific treatment for cancer. A common approach is to use the natural ligand for the overexpressed receptor as a cancer-targeting agent which can deliver a chemically or genetically conjugated toxic molecule. However, it has been found that the therapeutic efficacy of such ligand-drug molecular conjugates can be limited, since they naturally follow the intracellular trafficking pathways of the endogenous ligands. Therefore, a thorough understanding of the intracellular trafficking properties of these ligands can lead to novel design criteria for engineering ligands to be more effective drug carriers. This review presents a few commonly used ligand/receptor systems where intracellular trafficking considerations can potentially improve the therapeutic efficacy of the ligand-drug molecular conjugates.

Topics: Antineoplastic Agents; Biological Transport, Active; Biomedical Engineering; Diphtheria Toxin; Doxorubicin; Drug Carriers; Epidermal Growth Factor; ErbB Receptors; Exotoxins; Folic Acid; Folic Acid Transporters; Humans; Interleukin-13; Ligands; Models, Biological; Neoplasms; Receptors, Interleukin-13; Receptors, Transferrin; Ricin; Signal Transduction; Transferrin; Transforming Growth Factor alpha

Cancer therapies have led to remarkable results due to improved toxicity profiles and effects on survival. While these medical, surgical, and radiation protocols are chiefly responsible for these noteworthy contributions, an unexpected constellation of toxicities has emerged. Most notably, dermatologic adverse events have gained considerable attention, due to their high frequency, visibility, and impact on physical and psychosocial health, all of which affect dose intensity and possibly clinical outcome. Consequently, increased attention to cutaneous health in oncology has resulted in supportive oncodermatology clinical programs and toxicity-driven investigations, aiming to mitigate these untoward events and permit the continued optimization of cancer treatments.

Topics: Antineoplastic Agents; Epidermal Growth Factor; Humans; Neoplasms; Neoplasms, Radiation-Induced; Postoperative Complications; Radiodermatitis; Radiotherapy; Skin Diseases

Serine protease inhibitor Kazal type 1 (SPINK1) was originally identified as a trypsin inhibitor by Kazal et al. in 1948. SPINK1 is strongly elevated in pancreatitis and the elevation correlates with the severity of disease. In 2000, mutations in the SPINK1 gene were shown to be associated with chronic pancreatitis. Since then, there have been many reports on association between mutations in the SPINK1 genes and patients with pancreatitis. In 1982, SPINK1 was shown to be identical to tumor associated trypsin inhibitor (TATI). In addition, sequence similarities were detected between human epidermal growth factor (EGF) and human SPINK1 in 1983. Actually, SPINK1 was shown to stimulate growth of several cell lines including cancer cells in 1985. Recent clinical studies showed that high levels of SPINK1 protein in serum or urine were associated with adverse outcome in various cancer types. However, there was little evidence that showed in vivo function of SPINK1. Surprisingly, mice deficient in Spink3 (a mouse homologue gene of human SPINK1) showed excessive autophagy, but not pancreatitis in the exocrine pancreas, leading to autophagic cell death. We also demonstrated that SPINK1 acts as a growth factor through EGFR signaling. These data indicate that the role of the SPINK1 is not just as a trypsin inhibitor, but also as a growth factor as well as a negative regulator of autophagy. In this review, we summarize the roles of SPINK1/Spink3 in pancreatic diseases based on the data obtained from analyses using mouse models.

Topics: Animals; Autophagy; Carrier Proteins; Epidermal Growth Factor; ErbB Receptors; Glycoproteins; Humans; Mice; Mutation; Neoplasms; Pancreatitis, Chronic; Prostatic Secretory Proteins; Signal Transduction; Stimulation, Chemical; Trypsin Inhibitor, Kazal Pancreatic

EGF promoter polymorphisms are observed to modulate EGF levels and thought to have effect on susceptibility to various carcinomas but the results are inconsistent. In this meta-analysis, we assessed published studies of the association between three EGF polymorphisms and cancer risk from 21 studies with 14,609 subjects for EGF G61A, from two studies with 2,535 subjects for G-1380A and A-1744G, respectively. For EGF G61A, the contrast of homozygote (OR=0.80, 95% CI=0.65-0.98), allele (OR=0.90, 95% CI=0.81-0.99) and dominant model (OR=0.86, 95% CI=0.74-0.99) produced significant association among 21 studies with relatively large heterogeneity (Pheterogeneity<0.001). Through the stratified analysis, heterogeneity decreased significantly. In the stratified analysis by racial descent, the significant risks were found among Asians for homozygote contrast (OR=0.83, 95% CI=0.69-0.99, Pheterogeneity=0.506) and Americans for the contrast of homozygote (OR=0.50, 95% CI=0.30-0.84, Pheterogeneity=0.051), allele (OR=0.70, 95% CI=0.51-0.96, Pheterogeneity=0.008) and dominant model (OR=0.57, 95% CI=0.42-0.77, Pheterogeneity=0.28). No significant associations were found in all Caucasians genetic models. In the subgroup analyses by cancer types, for gastric cancer and esophageal cancer significant associations were found in all genetic models without heterogeneity. Significant risk was also found in the contrast of homozygote (OR=0.41, 95% CI=0.20-0.81, Pheterogeneity=0.184) and recessive model (OR=0.53, 95% CI=0.33-0.85, Pheterogeneity=0.384) for hepatoma and recessive model (OR=0.72, 95% CI=0.53-0.99, Pheterogeneity=0.474) for glioma. For EGF G-1380A and A-1744G, no significant associations were found in all genetic models. This meta-analysis suggests that the EGF G61A polymorphism most likely contributes to decreased susceptibility to cancers among Asians and Americans, and A allele may be a protective factor for gastric cancer, esophageal cancer, hepatoma and glioma. Both EGF G-1380A and A-1744G is marginally associated with cancer susceptibility.

Topics: Clinical Trials as Topic; Epidermal Growth Factor; Ethnicity; Genetic Predisposition to Disease; Humans; Neoplasms; Polymorphism, Genetic; Prognosis; Promoter Regions, Genetic; Risk Factors; Survival Rate

Tumor microenvironment plays a critical role in tumor initiation and progression. Components in the microenvironment can modulate the growth of tumor cells, their ability to progress and metastasize. A major venue of communication between tumor cells and their microenvironment is through polypeptide growth factors and receptors for these growth factors. This article discusses three major classes of growth-stimulatory polypeptide growth factors and receptors for these growth factors. It also discusses how deregulation of these growth factors or their receptors can drive malignant transformation and progression.

Topics: Animals; Epidermal Growth Factor; ErbB Receptors; Fibroblast Growth Factors; Humans; Models, Biological; Neoplasms; Platelet-Derived Growth Factor; Receptors, Fibroblast Growth Factor; Receptors, Platelet-Derived Growth Factor

Growth factors are low molecular peptides active in the stimulation of cell proliferation and in the regulation of embryonic development and cellular differentiation. Significant progress has been made in developing effective strategies to treat human malignancies with new chemical compounds based on a rationale directed against various components of signaling pathways. Many of these drugs target a growth factor receptor--for instance, in the form of monoclonal antibodies or inhibitors of tyrosine kinases, such as monoclonal antibodies against epidermal growth factor receptors used in treating certain types of breast cancer. Imatinib mesylate [Gleevec]) is an excellent example of mediators of signal transduction, such as tyrosine kinases. Growth factors proper are used to ameliorate various and sometimes fatal side effects of cytotoxic and/or myelosuppressive chemotherapy. Basic characteristics of several growth families are discussed with therapeutic modalities based on growth factor activity or, more often, inhibition of such activity.

Topics: Animals; Antineoplastic Agents; Epidermal Growth Factor; Fibroblast Growth Factors; Humans; Intercellular Signaling Peptides and Proteins; Neoplasms; Platelet-Derived Growth Factor; Progranulins; Receptors, Growth Factor; Signal Transduction; Somatomedins; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Numerous studies have investigated the risk of cancer associated with the polymorphism of epidermal growth factor (EGF) 61A>G, but the results have been inconsistent. We performed this meta-analysis to drive a more precise estimation of association between this polymorphism and risk of cancer.. Electronic searches of PubMed and EMBASE were conducted to select studies. Case-control studies containing available genotype frequencies of EGF 61A>G were chose, and Odds ratio (OR) with 95% confidence interval (CI) was used to assess the strength of this association.. 23 case-control studies including 5578 cases and 7306 controls were identified. This meta-analysis showed significant effect of EGF 61A>G on cancer risk (GG vs. AA: OR=1.34, 95%CI=1.05-1.72; GG vs. GA+AA: OR=1.23, 95%CI=1.03-1.47; GG+GA vs. AA: OR=1.18, 95%CI=1.02-1.38). In subgroup analysis, significant increased risk was found in gastric cancer and glioma in additive model (OR=1.54, 95%CI=1.13-2.12; OR=1.69, 95%CI=1.21-2.37) and in recessive model (OR=1.29, 95%CI=1.10-1.52; OR=1.54, 95%CI=1.16-2.04).. This meta-analysis suggested that the EGF 61G allele is a risk factor of cancer, especially for gastric cancer and glioma.

Topics: Case-Control Studies; Epidermal Growth Factor; Genetic Predisposition to Disease; Humans; Neoplasms; Polymorphism, Genetic

Cripto-1 is critical for early embryonic development and, together with its ligand Nodal, has been found to be associated with the undifferentiated status of mouse and human embryonic stem cells. Like other embryonic genes, Cripto-1 performs important roles in the formation and progression of several types of human tumors, stimulating cell proliferation, migration, epithelial to mesenchymal transition, and tumor angiogenesis. Several studies have demonstrated that cell fate regulation during embryonic development and cell transformation during oncogenesis share common signaling pathways, suggesting that uncontrolled activation of embryonic signaling pathways might drive cell transformation and tumor progression in adult tissues. Here we review our current understanding of how Cripto-1 controls stem cell biology and how it integrates with other major embryonic signaling pathways. Because many cancers are thought to derive from a subpopulation of cancer stem-like cells, which may re-express embryonic genes, Cripto-1 signaling may drive tumor growth through the generation or expansion of tumor initiating cells bearing stem-like characteristics. Therefore, the Cripto-1/Nodal signaling may represent an attractive target for treatment in cancer, leading to the elimination of undifferentiated stem-like tumor initiating cells.

Topics: Animals; Disease Progression; Embryonic Development; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; GPI-Linked Proteins; Humans; Hypoxia; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Neoplasm Proteins; Neoplasms; Neoplastic Stem Cells; Nodal Protein; Receptors, Notch; Signal Transduction; Stem Cells; Wnt Proteins

Several studies have shown that cell fate regulation during embryonic development and oncogenic transformation share common regulatory mechanisms and signaling pathways. Indeed, an embryonic gene member of the EGF-Cripto-1/FRL1/Cryptic family, Cripto-1, has been implicated in embryogenesis and in carcinogenesis. Cripto-1 together with the TGF-beta ligand Nodal is a key regulator of embryonic development and is a marker of undifferentiated human and mouse embryonic stem cells. While Cripto-1 expression is very low in normal adult tissues, Cripto-1 is re-expressed at high levels in several different human tumors, modulating cancer cell proliferation, migration, epithelial-to-mesenchymal transition and stimulating tumor angiogenesis. Therefore, inhibition of Cripto-1 expression using blocking antibodies or antisense expression vectors might be a useful modality not only to target fully differentiated cancer cells but also to target a subpopulation of tumor cells with stem-like characteristics.

Topics: Amino Acid Sequence; Animals; Embryo, Mammalian; Embryonic Development; Epidermal Growth Factor; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Humans; Intercellular Signaling Peptides and Proteins; Mammary Glands, Human; Membrane Glycoproteins; Mice; Molecular Sequence Data; Neoplasm Proteins; Neoplasms; Neovascularization, Pathologic

Emerging evidence has clearly implicated an inappropriate activation of embryonic regulatory genes during cell transformation in adult tissues. An example of such a case is the embryonic gene Cripto-1. Cripto-1 is critical for embryonic development and is considered a marker of undifferentiated embryonic stem cells. Critpo-1 is expressed at low levels in adult tissues, but is re-expressed at a high frequency in a number of different types of human carcinomas, therefore, representing an attractive therapeutic target in cancer.. This review surveys different approaches that have been used to target Cripto-1 in cancer as reflected by the relevant patent literature as well as peer-reviewed publications. Potential involvement and targeting of Cripto-1 in neurodegenerative and degenerative muscle diseases are also discussed.. The reader will gain an overview of different mAbs, vaccines or oligonucleotides antisense targeting Cripto-1. A humanized anti-Cripto-1 antibody is currently being tested in a Phase I clinical trial in cancer patients.. Targeting Cripto-1 in human tumors has the potential to eliminate not only differentiated cancer cells but also destroy an undifferentiated subpopulation of cancer cells with stem-like characteristics that support tumor initiation and self-renewal.

Topics: Animals; Antibodies, Monoclonal; Cancer Vaccines; Drug Delivery Systems; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Neoplasm Proteins; Neoplasms; Oligonucleotides, Antisense; Patents as Topic

Breast, prostate, pancreatic, colorectal, lung, and head and neck cancers exploit deregulated signaling by ErbB family receptors and their ligands, EGF family peptide growth factors. EGF family members that bind the same receptor are able to stimulate divergent biological responses both in cell culture and in vivo. This is analogous to the functional selectivity exhibited by ligands for G-protein coupled receptors. Here we review this literature and propose that this functional selectivity of EGF family members is due to distinctions in the conformation of the liganded receptor and subsequent differences in the sites of receptor tyrosine phosphorylation and receptor coupling to signaling effectors. We also discuss the roles of divergent ligand activity in establishing and maintaining malignant phenotypes. Finally, we discuss the potential of mutant EGF family ligands as cancer chemotherapeutics targeted to ErbB receptors.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Drug Delivery Systems; Epidermal Growth Factor; ErbB Receptors; Humans; Ligands; Neoplasms; Signal Transduction

Human Cripto-1 is a cell membrane protein that has been shown to be overexpressed in different types of human tumors. Because Cripto-1 is expressed at low levels in normal tissues, it represents a promising candidate for therapeutic intervention in cancer. The present patent describes a novel approach to target Cripto-1 in cancer using a vaccine. Immunization with Cripto-1 modified by addition of a foreign peptide to overcome T-cell tolerance for self-proteins has the potential to generate an antibody-based immune response that ultimately will block Cripto-1 activity in cancer cells. Although targeting Cripto-1 with a vaccine in cancer patients is promising, several experimental and clinical studies need to be done to validate this approach.

Topics: Animals; Cancer Vaccines; Drug Delivery Systems; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Neoplasm Proteins; Neoplasms; Patents as Topic; Self Tolerance; T-Lymphocytes

PAI-1 (plasminogen activator inhibitor-1) is a member of plasminogen cascade with an inhibitory role in plasmin activation. Plasmin is a protease capable of acting on wide range of substrates and, together with metaloproteinases, is a main proteolytic enzyme. Except its role in plasminogen cascade, PAI-1 has an affinity to vitronectin and uPA/uPAR what involves PAI-1 in cell's motility. PAI-1 gene is regulated in response to cytokines, hormones and many growth factors among which TGFbeta is the most important one. The PAI-1 promoter contains SBE, CAGA box, HRE, ERE, NFkB - binding sites, Sp-1, AP-1 and other. Cooperation between transcription factors bound to promoter and cross-talks between kinases and other upstream proteins decide about gene expression. This work describes the present knowledge in this field.

Topics: Animals; Cell Adhesion; Cell Cycle; Cell Movement; Cytokines; Enzyme Activation; Epidermal Growth Factor; Fibrinolysin; Gene Expression Regulation; Humans; Neoplasms; Plasminogen Activator Inhibitor 1

Topics: Epidermal Growth Factor; ErbB Receptors; Genes, Neoplasm; Humans; Ligands; Neoplasms

Members of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and their ligands (EGFR ligands) are known to play crucial roles in the regulation of cell proliferation and differentiation, and in the survival of many types of cancer. HER family members are activated in cancer cells and are now considered to be useful molecular targets for cancer therapy. Recently, several new drugs, including monoclonal antibodies and small-molecule inhibitors that target HER members, have been developed and clinically used to treat solid tumors. Members of a disintegrin and metalloproteinase (ADAM) family are thought to mediate the shedding of EGFR ligands and this event is critical for the production of soluble functional EGFR ligands. In melanoma cells, UV irradiation activates some ADAM members and induces melanoma cell growth through EGFR ligand shedding by activated ADAMs. These findings suggest that ADAM inhibitors are also candidate anticancer drugs acting via the blockade of HER family signaling pathways. After shedding of EGFR ligands by ADAMs, the carboxy-terminal fragments (CTFs) of EGFR ligands in the cytoplasm are translocated to the nucleus and induce cell proliferation by binding and exporting repressors and activating cyclin A and c-Myc. Based on these findings, the present molecular targeting therapy against HER members, EGFR and HER2, may not be sufficient, while ADAMs and nuclear translocation of the CTF of EGFR ligands are potential targets for the treatment of cancer, particularly malignancies that are dependent on the EGF family.

Topics: ADAM Proteins; Animals; Antibodies, Monoclonal; Antineoplastic Agents; Epidermal Growth Factor; ErbB Receptors; Humans; Ligands; Neoplasms; Protease Inhibitors; Signal Transduction

Although drugs inhibiting ErbB receptors such as epidermal growth factor receptor (EGFR) and HER2 have been developed as anticancer agents targeting the EGF family, they are not effective for all types of cancer and instead target only certain types. We propose the following four main reasons for these observations: (i) although seven EGFR ligands exist, effective inhibition of specific EGFR ligands may occur because their expression levels differ in different malignancies; (ii) suppressing EGFR ligands inhibits aggregation of EGFR and other ErbB receptors and activation of ERK and Akt signals; (iii) EGFR ligands may have various combinations for signal transduction through the EGFR pathway and other receptor signals; and (iv) the intracellular C-terminals of EGFR ligands move into the nucleus and strongly regulate cell proliferation. In this review, we describe important implications for targeted cancer therapy against EGFR ligands and describe the current situation in the development of ligand-based therapies for cancer.

Topics: Animals; Antineoplastic Agents; Drug Delivery Systems; Epidermal Growth Factor; ErbB Receptors; Humans; Ligands; Neoplasms

The past 5 years have seen an explosion of phosphoproteomics methods development. In this review, using epidermal growth-factor signaling as a model, we will discuss how phosphoproteomics, along with bioinformatics and computational modeling, have impacted key aspects of oncogenic signaling such as in the temporal fine mapping of phosphorylation events, and the identification of novel tyrosine kinase substrates and phosphorylation sites. We submit that the next decade will see considerable exploitation of phosphoproteomics in cancer research. Such a phenomenon is already happening as exemplified by its use in promoting the understanding of the molecular etiology of cancer and target-directed therapeutics.

Topics: Animals; Cell Transformation, Neoplastic; Computational Biology; Computer Simulation; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Oncogenes; Phosphoproteins; Phosphorylation; Proteomics; Signal Transduction

Activin, myostatin and other members of the TGF-beta superfamily signal through a combination of type II and type I receptors, both of which are transmembrane serine/threonine kinases. Activin type II receptors, ActRIIA and ActRIIB, are primary ligand binding receptors for activins, nodal, myostatin and GDF11. ActRIIs also bind a subset of bone morphogenetic proteins (BMPs). Type I receptors that form complexes with ActRIIs are dependent on ligands. In the case of activins and nodal, activin receptor-like kinases 4 and 7 (ALK4 and ALK7) are the authentic type I receptors. Myostatin and GDF11 utilize ALK5, although ALK4 could also be activated by these growth factors. ALK4, 5 and 7 are structurally and functionally similar and activate receptor-regulated Smads for TGF-beta, Smad2 and 3. BMPs signal through a combination of three type II receptors, BMPRII, ActRIIA, and ActRIIB and four type I receptors, ALK1, 2, 3, and 6. BMPs activate BMP-specific Smads, Smad1, 5 and 8. Smad proteins undergo multimerization with co-mediator Smad, Smad4, and translocated into the nucleus to regulate the transcription of target genes in cooperation with nuclear cofactors. The signal transduction pathway through activin type II receptors, ActRIIA and ActRIIB, with type I receptors is involved in various human diseases. In this review, we discuss the role of signaling through activin receptors as therapeutic targets of intractable neuromuscular diseases, endocrine disorders and cancers.

Topics: Activin Receptors; Activins; Animals; Antineoplastic Agents; Bone Morphogenetic Proteins; Drug Delivery Systems; Epidermal Growth Factor; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Models, Biological; Musculoskeletal Diseases; Myostatin; Neoplasm Proteins; Neoplasms; Protein Binding; Protein Kinase Inhibitors; Signal Transduction; Transforming Growth Factor beta

Tumor angiogenesis plays an important role in tumor growth, aggression and metastasis. Many molecules have been demonstrated as positive regulators of angiogenesis, including vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and others. In recent years, significant progress has been made in the research on anti-angiogenic strategies for tumor therapies. In this review, anti-angiogenic active immunotherapies for tumors based on vaccination with xenogeneic homologous molecules and non-xenogeneic homologous molecules are discussed.

Topics: Animals; Cancer Vaccines; Epidermal Growth Factor; Fibroblast Growth Factors; Humans; Immunotherapy; Neoplasms; Neovascularization, Pathologic; Vascular Endothelial Growth Factor A

The epidermal growth factor (EGF) family and the EGF receptor (EGFR, ErbB) tyrosine kinase family have been spearheading the studies of signal transduction events that determine cell fate and behavior in vitro and in vivo. The EGFR family and their signaling pathways are giving us tremendous advantages in developing fascinating molecular target strategies for cancer therapy. Currently, two important types of EGFR inhibitors are in clinical use: neutralizing antibodies of EGFR or ErbB2, and synthetic small compounds of tyrosine kinase inhibitors designed for receptors. On the other hand, basic research of the EGF family ligands presents new challenges as membrane-anchored growth factors. All members of the EGF family have important roles in development and diseases and are shed from the plasma membrane by metalloproteases. The ectodomain shedding of the ligands has emerged as a critical component in the functional transactivation of EGFRs in interreceptor cross-talk in response to various shedding stimulants such as G-protein coupled receptor agonists, growth factors, cytokines, and various physicochemical stresses. Among the EGFR-ligands, heparin-binding EGF-like growth factor (HB-EGF) is a prominent ligand in our understanding of the pathophysiological roles of ectodomain shedding in cancer, wound healing, cardiac diseases, etc. Here we focus on ectodomain shedding of the EGF family ligands, especially HB-EGF by disintegrin and metalloproteases, which are not only key events of receptor cross talk, but also novel intercellular signaling by their carboxy-terminal fragments to regulate gene expression directly.

Topics: Animals; Cytoplasm; Disintegrins; Epidermal Growth Factor; ErbB Receptors; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Ligands; Membranes; Metalloproteases; Models, Biological; Neoplasms; Signal Transduction; Transcriptional Activation

The FRS2 family of adaptor/scaffold proteins has two members, FRS2alpha and FRS2beta. Both proteins contain N-terminal myristylation sites for localization on the plasma membrane and a PTB domain for binding to limited species of receptor tyrosine kinases (RTKs), including the FGF receptor, the neurotophin receptor, RET, and ALK. Activation of these RTKs allows FRS2 proteins to become phosphorylated of tyrosine residues and then bind to Grb2 and Shp2, a SH2 domain-containing adaptor and a tyrosine phosphatase, respectively. Subsequently, Shp2 activates a Ras/ERK pathway and Grb2 activates a Ras/ERK, phosphatidyl inositol (PI)-3 kinase and ubiquitination/degradation pathways by binding to SOS, Gab1, and Cbl via the SH3 domains of Grb2. FRS2alpha acts as 'a conning center' in FGF signaling mainly because it induces sustained levels of activation of ERK via Shp2-binding sites and Grb2-binding sites, though the contribution of the former is greater. Indeed, FRS2alpha knockout mice and mice with mutated Shp2-binding sites exhibit a variety of phenotypes due to defects in FGF signaling in vivo. Although FRS2beta binds to the EGF receptor, it does not induce tyrosine phosphorylation on the receptor. Instead, it inhibits EGF signaling, resulting in inhibition of EGF-induced cell proliferation and cell transformation. Based on these findings, the involvement of FRS2 proteins in tumorigenesis should be studied extensively to be validated as candidate biomarkers for the effectiveness of treatments targeting RTKs such as the FGF receptor and EGF receptor.

Topics: Adaptor Proteins, Signal Transducing; Animals; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Humans; Membrane Proteins; Neoplasms; Phosphorylation; Protein Structure, Tertiary; Receptors, Fibroblast Growth Factor; Signal Transduction

An intracellular signal transduction network constitutes an assembled machinery to control the dynamics of kinase-phosphatase cascade and gene expression. Spatio-temporal analyses of the cellular process can explain the biochemical role of the receptor tyrosine kinases in cancer development from a system point of view.

Topics: Animals; Dimerization; Disease Progression; Epidermal Growth Factor; ErbB Receptors; Feedback, Physiological; Humans; Models, Biological; Neoplasms; Signal Transduction

Over the last two decades, several lines of experimental evidence have suggested that the gastrin-releasing peptide (GRP) may act as a growth factor in many types of cancer. For that reason, gastrin-releasing peptide receptor (GRPR) antagonists have been developed as anticancer candidate compounds, exhibiting impressive antitumoral activity both in vitro and in vivo in various murine and human tumors. In this article, the GRPR cell surface expression profile in human malignancies is reviewed aiming at the identification of potential tumor types for future clinical trials with GRP analogues and antagonists. In this review, we summarize the current literature regarding the GRPR status in human malignancies. Source data were obtained by searching all published material available through Medline, PubMed and relevant articles from 1971 to 2006. The data available demonstrated a high expression of GRPRs in a large spectrum of human cancers, demonstrating the potential relevance of this intracellular signaling pathway in various human tumor models. The GRPR may be an interesting target for therapeutic intervention in human malignancies, as carriers for cytotoxins, immunotoxins or radioactive compounds, being also a potential tool for tumor detection.

Topics: Antineoplastic Agents; Bombesin; Clinical Trials as Topic; Drug Delivery Systems; Epidermal Growth Factor; Humans; Neoplasms; Receptors, Bombesin; Signal Transduction; Vascular Endothelial Growth Factor A

In this review, we describe the critical functions assumed by the interplay of epidermal growth factor, hedgehog, Wnt/beta-catenin, tumor growth factor-beta and integrin signaling cascades in tumorigenic and migrating cancer progenitor cells and activated stromal cells during carcinogenesis. These growth factors provide an important role for the sustained growth and survival of tumorigenic cancer progenitor cells and their progeny by up-regulating numerous mitotic and antiapoptotic signaling cascades. Furthermore, these potent morphogens may cooperate for inducing the molecular events associated with the epithelial-mesenchymal program in cancer cells including the alterations in epithelial cell shape and motility through the dissociation of intercellular adherens junctions. Of therapeutic interest, new strategies for the development of more effective clinical treatments against the locally aggressive and invasive cancers based on the molecular targeting of deregulated signaling elements in tumorigenic and migrating cancer cells and their local microenvironment are also described.

Topics: Antineoplastic Combined Chemotherapy Protocols; beta Catenin; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cytokines; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Growth Substances; Hedgehog Proteins; Humans; Integrins; Mesoderm; Neoplasm Metastasis; Neoplasms; Neoplastic Stem Cells; Signal Transduction

Growth factor receptors and their ligands not only regulate normal cell processes but have been also identified as key regulators of human cancer formation. The epidermal growth factor receptor (EGFR/ErbB1/HER1) belongs to the ErbB/HER-family of tyrosine kinase receptors (RTKs). These trans-membrane proteins are activated following binding with peptide growth factors of the EGF-family of proteins. Several evidences suggest that cooperation of multiple ErbB receptors and ligands is required for the induction of cell transformation. In this respect, EGFR, upon activation, sustains a complex and redundant network of signal transduction pathways with the contribution of other trans-membrane receptors. EGFR has been found to be expressed and altered in a variety of malignancies and clearly it plays a significant role in tumor development and progression, including cell proliferation, regulation of apoptotic cell death, angiogenesis and metastatic spread. Moreover, amplification of the EGFR gene and mutations in the EGFR tyrosine kinase domain have been recently reported in human carcinomas. As a result, investigators have developed approaches to inhibit the effects of EGFR activation, with the aim of blocking tumor growth and invasion. A number of agents targeting EGFR, including specific antibodies directed against its ligand-binding domain and small molecules inhibiting its tyrosine kinase activity are either in clinical trials or are already approved for clinical treatment. This article reviews the EGFR role in carcinogenesis and tumor progression as rational bases for the development of specific therapeutic inhibitors.

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Humans; Mutation; Neoplasms; Protein-Tyrosine Kinases; Signal Transduction

Topics: Angiogenesis Inhibitors; Animals; Clinical Trials as Topic; Epidermal Growth Factor; Humans; Neoplasms; Neovascularization, Pathologic; Protein Kinase Inhibitors; Receptors, Vascular Endothelial Growth Factor; Vascular Endothelial Growth Factor A

To review the biology of the EGFR, its structure, and the associated signal transduction pathways. To provide an overview of the role of EGFR in normal physiology and the pathophysiology of malignancy. Current anti-EGFR treatments are also discussed.. Research articles.. EGFR is a valid target in the treatment of solid tumors. EGFR abnormalities and dysfunction are involved in various aspects of carcinogenesis and tumor progression, and EGFR is overexpressed in several tumor types. The development of anti-EGFR therapies represents an important advance in cancer therapy.. Anti-EGFR therapy is currently available in the clinical setting. Nurses involved in the care of patients with cancer can benefit from an increased understanding of the normal and abnormal function of EGFR in the body and the mechanisms by which anti-EGFR therapies act.

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Oncology Nursing; Protein Kinase Inhibitors; Signal Transduction

HB-EGF, a member of the EGF family of growth factors, exerts its biological activity through activation of the EGFR and other ErbB receptors. HB-EGF participates in diverse biological processes, including heart development and maintenance, skin wound healing, eyelid formation, blastocyst implantation, progression of atherosclerosis and tumor formation, through the activation of signaling molecules downstream of ErbB receptors and interactions with molecules associated with HB-EGF. Recent studies have indicated that HB-EGF gene expression is significantly elevated in many human cancers and its expression level in a number of cancer-derived cell lines is much higher than those of other EGFR ligands. Several lines of evidence have indicated that HB-EGF plays a key role in the acquisition of malignant phenotypes, such as tumorigenicity, invasion, metastasis and resistance to chemotherapy. Studies in vitro and in vivo have indicated that HB-EGF expression is essential for tumor formation of cancer-derived cell lines. CRM197, a specific inhibitor of HB-EGF, and an antibody against HB-EGF are both able to inhibit tumor growth in nude mice. These results indicate that HB-EGF is a promising target for cancer therapy, and that the development of targeting tools against HB-EGF could represent a novel type of therapeutic strategy, as an alternative to targeting ErbB receptors.

Topics: Animals; Cell Movement; Epidermal Growth Factor; Female; Genes, erbB; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Mice; Models, Biological; Neoplasms; Ovarian Neoplasms; Phenotype; Transcriptional Activation

Epidermal growth factor (EGF)-like proteins comprise a group of structurally similar growth factors, which contain a conserved six-cysteine residue motif called the EGF-domain. EGF-like factors are synthesized as transmembrane precursors, which can undergo proteolytic cleavage at the cell surface to release a mature soluble ectodomain; a process often referred to as "ectodomain shedding". Ectodomain shedding of EGF-like factors has been linked to multiple zinc-binding metalloproteases of the matrix metalloprotease (MMP) and a disintegrin and metalloprotease (ADAM) families. Shedding can be activated by a variety of pharmacological and physiological stimuli and these activation events have been linked to the enhancement of metalloprotease activity, possibly via the action of intracellular signaling modules. Once shed from the cell surface, EGF-like factors bind to a family of four cell surface receptors named ErbB-1, -2, -3 and -4. Heterodimerization or homodimerization of these receptors following ligand binding drives intracellular signal transduction cascades, which eventuate in diverse cell fates including proliferation, differentiation, migration and inhibition of apoptosis. In addition to its role in driving normal developmental processes, a wealth of evidence now exists showing that de-regulated ErbB signaling is associated with the formation of tumors in a variety of tissues and that ectodomain shedding of EGF-like factors plays a critical event in this process. Thus, knowledge of the molecular mechanisms by which EGF-like factors are shed from the cell surface and the nature of the proteases and cellular signals that govern this process is crucial to understanding ErbB receptor signaling and potentially also in the development of novel cancer therapeutics targeting the ErbB pathway. This review focuses on the structure and function of EGF-like factors, and the mechanisms that govern the shedding of these transmembrane molecules from the cell surface.

Topics: Amino Acid Sequence; Animals; Epidermal Growth Factor; ErbB Receptors; Growth and Development; Humans; Metalloproteases; Molecular Sequence Data; Neoplasms; Protein Structure, Tertiary; Receptor, ErbB-2; Receptor, ErbB-3; Signal Transduction

There is a balance between cell death and survival in living organisms. The ability of cells to sense their environment and decide to survive or die is dependent largely upon growth factors. Epidermal growth factor (EGF) is a key growth factor regulating cell survival. Through its binding to cell surface receptors, EGF activates an extensive network of signal transduction pathways that include activation of the PI3K/AKT, RAS/ERK and JAK/STAT pathways. These pathways predominantly lead to activation or inhibition of transcription factors that regulate expression of both pro- and anti-apoptotic proteins effectively blocking the apoptotic pathway. In cancer, EGF signaling pathways are often dysfunctional and targeted therapies that block EGF signaling have been successful in treating cancers. In this review, we will discuss the EGF survival signaling network, how it cross-talks with the apoptotic signaling pathways and the therapeutic drugs targeting the EGF survival pathway used to treat cancers.

Topics: Animals; Apoptosis; Cell Survival; Epidermal Growth Factor; Humans; Models, Biological; Neoplasms; Phosphatidylinositol 3-Kinases; ras Proteins; Signal Transduction

Topics: Animals; Antineoplastic Agents; Aorta; Atherosclerosis; Biomechanical Phenomena; Chondroitin Sulfate Proteoglycans; Chondroitin Sulfates; Cytokines; Disease Progression; Epidermal Growth Factor; Extracellular Matrix; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Lipoproteins, LDL; Neoplasms; Signal Transduction; Versicans

Cellular receptors for the Epidermal Growth Factor (EGF-R) are members of the ErbB receptor family and are considered important targets for the experimental treatment of human cancer. Monoclonal antibodies as well as small tyrosine kinase inhibitors (TKIs) have been developed and have undergone extensive evaluation in preclinical and clinical studies based on the general idea that EGF-R plays a critical role on the growth and survival of human tumors. This assumption has been derived by the successful development of BCR/ABL tyrosine kinase inhibitors in human chronic myeloid leukemia as well as on the activity of therapy with monoclonal antibodies (mAb) in breast cancer and lymphoproliferative diseases. It is now becoming clear that factors regulating sensitivity to kinase inhibitors may differ from monoclonal antibodies and that the molecules targeted by interfering drugs must be prioritaire for growth and survival of those specific tumors in order to achieve valuable results. In this article, we will describe the signal transduction pathways regulated by EGF-R and the principal pharmacological and biotechnological agents directed against EGF-R. We will discuss the significance of targeting the EGF-R driven survival pathways and the compensatory intracellular survival mechanisms that counteract the specific EGF-R inhibition and are the cause of the poor clinical results derived from study based on the use of these agents. We will describe new multipotent TKIs that target also other members of ErbB family (i.e. ErbB2) blocking one of the compensatory mechanism that can be triggered in cancer cells. Moreover, we will report new patent on bispecific mAbs that bind EGF-R and immune effectors in order to increase the immunological function of this agent that could be the basis of the different clinical results achieved with the use of TKI and mAbs. Finally, we will propose a pharmacological model able to make cancer cells dependent on EGF-R for their survival and proliferation and we will discuss the relevance of patenting also new therapeutical strategies and not only the simple drug.

Topics: Animals; Antibodies, Blocking; Antineoplastic Agents; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Patents as Topic

One of the recent, significant advances in cancer immunotherapy is the identification of molecules as targets which regulate cell growth by induction of proliferation and survival signalling pathways. Among them, epidermal growth factor receptor and Her2 have been effectively targeted by monoclonal antibodies. Currently, the treatment of cancer has limitations and most cancer deaths result from the local invasion and distant metastasis of tumour cells. An important insight for the understanding of tumour invasion and metastasis came from the recent discovery that the phenotypic changes of increased motility and invasiveness of cancer cells are reminiscent of the epithelial-mesenchymal transition (EMT) that occurs during embryonic development. The human Cripto, a member of the epidermal growth factor-Cripto, Frl1, and Cryptic (EGF-CFC) protein family and a signalling protein during early embryonic development, plays an important role in cancers. Cripto is attached to the cell membrane through a glycosyl-phosphatidylinositol motif, and is upregulated in a wide range of epithelial cancers. In this paper the authors review the role of Cripto expression in tumourigenesis and in EMT to promote tumour invasion, with emphasis that the unique EGF-like region of Cripto plays a critical role in Cripto signalling-mediated tumour growth and EMT. Therefore, the region should be regarded as a therapeutic point for interruption of the oncogenic and metastatic potential of Cripto for cancer immunotherapy.

Topics: Amino Acid Sequence; Animals; Biomarkers, Tumor; Epidermal Growth Factor; GPI-Linked Proteins; Humans; Immunotherapy; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Molecular Sequence Data; Neoplasm Proteins; Neoplasms

Human Cripto-1 (CR-1), a member of the epidermal growth factor (EGF)-CFC family, has been implicated in embryogenesis and in carcinogenesis. During early vertebrate development, CR-1 functions as a co-receptor for Nodal, a transforming growth factor beta (TGFbeta) family member and is essential for mesoderm and endoderm formation and anterior-posterior and left-right axis establishment. In adult tissues, CR-1 is expressed at a low level in all stages of mammary gland development and expression increases during pregnancy and lactation. Overexpression of CR-1 in mouse mammary epithelial cells leads to their transformation in vitro and, when injected into mammary glands, produces ductal hyperplasias. CR-1 can also enhance migration, invasion, branching morphogenesis and epithelial to mesenchymal transition (EMT) of several mouse mammary epithelial cell lines. Furthermore, transgenic mouse studies have shown that overexpression of a human CR-1 transgene in the mammary gland under the transcriptional control of the mouse mammary tumor virus (MMTV) promoter results in mammary hyperplasias and papillary adenocarcinomas. Finally, CR-1 is expressed at high levels in approximately 50 to 80% of different types of human carcinomas, including breast, cervix, colon, stomach, pancreas, lung, ovary, and testis. In conclusion, EGF-CFC proteins play dual roles as embryonic pattern formation genes and as oncogenes. While during embryogenesis EGF-CFC proteins perform specific and regulatory functions related to cell and tissue patterning, inappropriate expression of these molecules in adult tissues can lead to cellular proliferation and transformation and therefore may be important in the etiology and/or progression of cancer.

Topics: Epidermal Growth Factor; GPI-Linked Proteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Neoplasm Proteins; Neoplasms

Metastasis development requires the migratory activity of tumor cells. It is therefore important to understand the molecular mechanisms of this migration in order to prevent metastasis development, which is the pernicious step in most solid tumor diseases. A lot of methods have been invented to investigate tumor cell migration, but not all are equally suited and no method alone is able to deliver a complete picture of tumor cell migration. We herein suggest a combination of three-dimensional in vitro and in vivo methods for the investigation of tumor cell migration and summarize the knowledge, which has been reached so far.

Topics: Animals; Cell Adhesion; Cell Movement; Epidermal Growth Factor; Humans; In Vitro Techniques; Luciferases; Mice; Mice, Inbred BALB C; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms; Rats; Signal Transduction

It is increasingly evident that genes known to perform critical roles during early embryogenesis, particularly during stem cell renewal, pluripotentiality and survival, are also expressed during the development of cancer. In this regard, oncogenesis may be considered as the recapitulation of embryogenesis in an inappropriate temporal and spatial manner. The epidermal growth factor-Cripto-1/FRL1/cryptic family of proteins consists of extracellular and cell-associated proteins that have been identified in several vertebrate species. During early embryogenesis, epidermal growth factor-Cripto-1/FRL1/cryptic proteins perform an obligatory role as coreceptors for the transforming growth factor-beta subfamily of proteins, which includes Nodal. Cripto-1 has also been shown to function as a ligand through a Nodal/Alk4-independent signaling pathway that involves binding to glypican-1 and the subsequent activation through src of phosphoinositol-3 kinase/Akt and ras/mitogen-activated protein kinase intracellular pathways. Expression of Cripto-1 is increased in several human cancers and its overexpression is associated with the development of mammary tumors in mice. Here, we review the role of Cripto-1 during embryogenesis, cell migration, invasion and angiogenesis and how these activities may relate to cellular transformation and tumorigenesis. We also briefly discuss evidence suggesting that Cripto-1 may be involved in stem cell maintenance.

Topics: Activin Receptors, Type I; Cell Movement; Cell Transformation, Neoplastic; DNA-Binding Proteins; Embryonic Development; Epidermal Growth Factor; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Membrane Glycoproteins; Milk Proteins; Neoplasm Proteins; Neoplasms; Neovascularization, Physiologic; Nodal Protein; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Smad2 Protein; Trans-Activators; Transforming Growth Factor beta; Wnt Proteins

Over the past 30 years, a relatively simple growth factor and its cognate receptor have provided seminal insights into the understanding of the genetic basis of cancer, as well as growth factor signalling. The epidermal growth factor (EGF), its cognate receptor (EGFR) and related family members have been shown to be important in normal, as well as the malignant growth of many cell types including: glioblastomata, astrocytomas, medulloblastomata, non-small cell lung carcinoma (NSCLC) and breast cancer. This review summarises the history of the EGFR gene and the v-ErbB oncogene, as well as diverse approaches developed to inhibit EGFR activity. The two most advanced therapies use either small-molecule cell membrane permeable kinase inhibitors or antibodies which prevent receptor activation. Recent clinical trials indicate that certain NSCLC patients have mutations in the EGFR gene which makes them more responsive to kinase inhibitors. These mutations appear to enhance the ability of the ligand to activate EGFR activity and also prolong the binding of the EGFR inhibitor to the kinase domain. Evidence to date suggests that these EGFR mutations in NSCLC occur more frequently in Japan than in the western hemisphere. Although these mutations are correlated with enhanced efficacy to the inhibitors in NSCLC, they can not explain or predict the sensitivity of many other cancer patients to the beneficial effects of the EGFR kinase inhibitors or antibody mediated therapy. As with as other small-molecule kinase inhibitors and susceptible diseases (e.g., imatinib and chronic myeloid leukaemia), resistance to EGFR inhibitors has been reported recently, documenting the requirement for development of multi-pronged therapeutic approaches. EGFR kinase inhibitors are also being evaluated as adjuvants in hormonal therapy of breast cancer - especially those which overexpress EGFR. Genetically engineered antibodies specific for the EGFR family member ErbB2 have been developed which show efficacy in the treatment of primary, and prevent the relapse of, breast cancer. Clearly, the EGF/EGFR signalling cascade has, and continues to play, an important role in the development of novel anticancer targeted therapies.

Topics: Antineoplastic Agents; Epidermal Growth Factor; Humans; Multigene Family; Neoplasms; Pharmacogenetics

The success of molecular target-based cancer therapy exampled by Herceptin targeting Her2 indicates that cancer immunotherapy involves identifying and targeting key molecular drivers of cancer. Recently, the human Cripto, a founding member of the epidermal growth factor-Cripto-FRL1-Cryptic (EGF-CFC) protein family has been demonstrated to be a unique molecule and could be targeted by anti-Cripto monoclonal antibodies for the treatment of cancer. Cripto plays an important role in embryonic development, tumorigenesis, cancer cell proliferation and survival. Cripto is upregulated in most epithelial cancers but is absent or weakly expressed in normal cells. Cripto expression is associated with tumorigenesis and invasion. Cripto is also involved in tumor metastasis, which is strongly supported by the recent discovery that the phenotypic changes of increased motility and invasiveness of cancer cells are reminiscent of the epithelial mesenchymal transition (EMT) that occurs during embryonic development. In this review, we emphasize that the EGF-like region of Cripto plays a critical role in Cripto signaling-mediated tumor growth and EMT. Therefore the EGF-like region should be regarded as a therapeutic point for monoclonal antibody (MAb) intervention. The mechanisms of action of these MAbs to the EGF-like region of Cripto are most likely through intervention of c-Src signaling. The CFC region of Cripto is another region to be targeted for treatment of cancer, as the Cripto can bind to activin B through the CFC region to stimulate cell proliferation. The MAbs to the CFC region block the binding of Cripto and activin B and result in an inhibition of cell growth. Therefore the MAbs to Cripto may be of value in the treatment of various cancers.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Cell Proliferation; Cell Survival; Epidermal Growth Factor; Gene Expression Regulation; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasms; Recombinant Proteins; Signal Transduction

The role of dysregulated epidermal growth factor receptor-tyrosine kinase (EGFR-TK) activity in promoting tumor resistance to radiation therapy is discussed, and evidence supporting the rationale for the use of gefitinib (IRESSA, ZD1839) to enhance tumor radiosensitivity is reviewed.. A review of the literature regarding the role of EGFR-TK signaling in tumor response to radiation therapy was conducted, and results were summarized from preclinical and clinical studies of gefitinib in the treatment of solid tumors alone and in combination with radiation therapy.. Preclinical results indicate that EGFR-TK activity in tumors can block the cytotoxic effects of radiation therapy and enhance tumor repopulation, resulting in failure of local tumor control. In xenograft tumor models, gefitinib in combination with ionizing radiation resulted in additive to synergistic growth inhibition. In randomized clinical trials, gefitinib has demonstrated efficacy with favorable tolerability as monotherapy for patients with advanced non-small-cell lung cancer or head-and-neck carcinomas who had previously received standard therapies.. These results indicate that there is potential for improved responses by combining gefitinib with radiation therapy in non-small-cell lung cancer, head-and-neck cancers, and other solid tumors.

Topics: Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Combined Modality Therapy; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Glioblastoma; Head and Neck Neoplasms; Humans; Lung Neoplasms; Neoplasm Proteins; Neoplasms; Quinazolines; Radiation Tolerance; Radiation-Sensitizing Agents

Topics: Animals; Antineoplastic Agents; Epidermal Growth Factor; Humans; Neoplasms; Receptor Protein-Tyrosine Kinases; Vascular Endothelial Growth Factor A

In healthy tissues, there is a balance between cell survival and death. This balance ensures epithelial cells survive in the right milieu, but undergo programmed cell death (apoptosis) when the environment is no longer supportive. Cells sense these changes primarily through receptors on the cell surface that bind to specific ligands present in the extracellular environment. These receptors, through signal transduction pathways, lead to promotion of cell survival or induction of cell death. One of the most important types of receptors regulating cell survival is the epidermal growth factor (EGF) receptors, while one of the most important types of receptors regulating apoptosis is the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptors. EGF receptors activate survival signaling pathways including PI3K/AKT, Ras/MAPK, and JAK/STAT signaling pathways leading to cell survival. TRAIL activates apoptotic signaling pathways leading to caspase activation and mitochondrial dysfunction. The balance between these two signaling pathways determine whether a cell survives or dies. In disease states, this balance is altered. For example, epithelial-derived cancer cells often have increased expression of EGF receptors and are resistant to apoptosis. Understanding the interactions between survival and apoptotic signaling pathways mediated by EGF receptors and TRAIL death receptors will be essential to explain the role these pathways play in healthy and diseased cells.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Cell Survival; Drug Interactions; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Humans; Membrane Glycoproteins; Neoplasms; Receptors, Tumor Necrosis Factor; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Transcription Factors; Tumor Necrosis Factor-alpha

Angiogenesis is required for multistage carcinogenesis. The inducible enzyme cyclooxygenase-2 (COX-2) is an important mediator of angiogenesis and tumor growth. COX-2 expression occurs in a wide range of preneoplastic and malignant conditions; and the enzyme has been localized to the neoplastic cells, endothelial cells, immune cells, and stromal fibroblasts within tumors. The proangiogenic effects of COX-2 are mediated primarily by three products of arachidonic metabolism: thromboxane A(2) (TXA(2)), prostaglandin E(2) (PGE(2)), and prostaglandin I(2) (PGI(2)). Downstream proangiogenic actions of these eicosanoid products include: (1) production of vascular endothelial growth factor; (2) promotion of vascular sprouting, migration, and tube formation; (3) enhanced endothelial cell survival via Bcl-2 expression and Akt signaling; (4) induction of matrix metalloproteinases; (5) activation of epidermal growth factor receptor-mediated angiogenesis; and (6) suppression of interleukin-12 production. Selective inhibition of COX-2 activity has been shown to suppress angiogenesis in vitro and in vivo. Because these agents are safe and well tolerated, selective COX-2 inhibitors could have clinical utility as antiangiogenic agents for cancer prevention, as well as for intervention in established disease alone or in combination with chemotherapy, radiation, and biological therapies.

Topics: Angiogenesis Inhibitors; Animals; Anticarcinogenic Agents; Celecoxib; Cell Movement; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dinoprostone; Epidermal Growth Factor; Epoprostenol; Humans; Interleukin-12; Isoenzymes; Lactones; Matrix Metalloproteinases; Membrane Proteins; Neoplasms; Neovascularization, Pathologic; Prostaglandin-Endoperoxide Synthases; Pyrazoles; Signal Transduction; Sulfonamides; Sulfones; Thromboxane A2; Vascular Endothelial Growth Factor A

Accurate and rapid detection of tumors is of great importance for interrogating the molecular basis of cancer pathogenesis, preventing the onset of complications, and implementing a tailored therapeutic regimen. In this era of molecular medicine, molecular probes that respond to, or target molecular processes are indispensable. Although numerous imaging modalities have been developed for visualizing pathologic conditions, the high sensitivity and relatively innocuous low energy radiation of optical imaging method makes it attractive for molecular imaging. While many human diseases have been studied successfully by using intrinsic optical properties of normal and pathologic tissues, molecular imaging of the expression of aberrant genes, proteins, and other pathophysiologic processes would be enhanced by the use of highly specific exogenous molecular beacons. This review focuses on the development of receptor-specific molecular probes for optical imaging of tumors. Particularly, bioconjugates of probes that absorb and fluoresce in the near infrared wavelengths between 750 and 900 nm will be reviewed.

Topics: Animals; Bombesin; Carbocyanines; Carrier Proteins; Cell Membrane; Coloring Agents; Contrast Media; Diagnostic Imaging; Disease Models, Animal; Epidermal Growth Factor; Fluorescent Dyes; Folic Acid; Humans; Light; Models, Chemical; Models, Molecular; Molecular Probes; Neoplasms; Neurotensin; Peptides; Photosensitizing Agents; Somatostatin; Xanthenes

Topics: Animals; Cell Differentiation; Cell Movement; Cell Survival; Embryonic Development; Epidermal Growth Factor; ErbB Receptors; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Mesoderm; Mice; Mice, Transgenic; Models, Genetic; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasms; Phosphorylation; Receptor, ErbB-4; Signal Transduction

Topics: Active Transport, Cell Nucleus; Animals; Cell Membrane; Cell Nucleus; Cytoplasm; Epidermal Growth Factor; Fibroblasts; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Mice; Mice, Knockout; Models, Biological; Neoplasms; Peptide Hydrolases; Phenotype; Protein Binding; Protein Structure, Tertiary; Signal Transduction; Wound Healing

The epidermal growth factor receptor (EGFR) inhibitors are among the most intensely studied new molecular therapeutic agents. Although response rates have been somewhat disappointing when EGFR inhibitors are used as single-agent therapy for advanced disease, these inhibitors may be more effective as chemo- and radiosensitizers. The first phase III randomized trial evaluating EGFR inhibitors as radiosensitizers in patients with locally advanced head and neck cancer was strongly positive, indicating significant potential of this class of agents to improve outcome with radiotherapy. However, optimal implementation of EGFR inhibitors as radiosensitizers depends, in part, on a better understanding of the mechanisms of radiosensitization. Preclinical studies provide important observations with regard to potential mechanisms. The phenotypic cellular changes associated with EGFR inhibition are impressively consistent between different model systems, with almost all studies showing that EGFR inhibitors affect proliferation, angiogenesis, and cell survival. Whether EGFR inhibitors influence response to radiation directly, or whether the improved response is a result of additive effects of the two modalities, remains unclear. However, cell-cycle arrest, endothelial cell sensitivity, and apoptotic potential are all important factors in radiation response of epithelial tumors. Furthermore, less-studied effects of EGFR inhibitors on DNA repair suggest that modulation of DNA damage response to cytotoxic injury might result in radio- or chemosensitization. This review will explore potential mechanisms of radiosensitization by EGFR inhibitors.

Topics: Antineoplastic Agents; Combined Modality Therapy; DNA Repair; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Radiation-Sensitizing Agents; Signal Transduction

The cell-surface associated molecule Cripto is overexpressed in a wide range of epithelial cancers, yet little is known about potential mechanisms by which Cripto expression might enhance tumorigenesis. A new study reveals that binding of Cripto to the TGF-beta ligand Activin B can block Activin B-mediated suppression of cell proliferation. Furthermore, this study also demonstrates that antibody blockade of Cripto function may prove useful in the inhibition of tumorigenesis.

Topics: Activins; Amino Acid Motifs; Animals; Cell Division; Epidermal Growth Factor; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Models, Biological; Neoplasm Proteins; Neoplasms; Nodal Protein; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Epidermal growth factor receptor (EGFR), a member of a family of membrane receptors with tyrosine kinase activity, is emerging as a target candidate for anti-cancer therapy, due to its overexpression in many carcinomas and its relationship with several hallmark properties of malignant behavior such as continuous cell proliferation, escape from apoptosis, cell migration and angiogenesis. Specially appealing is the overexpression of EGFR in tumors such as lung, colon, kidney and head and neck carcinomas which are mostly resistant to current chemotherapy. Several anti-EGFR agents are already in clinical testing: small molecule tyrosine kinases inhibitors, monoclonal antibodies and cancer vaccines. Early results provide evidence of antitumor activity in humans, to be confirmed in larger trials. Toxicity profiles do not overlap with chemotherapy or radiotherapy, but skin rash and diarrhea can be severe. Future investigations should clarify optimal schedules and explore combinations with standard onco-specific treatments. The ultimate challenge will be to combine diverse therapeutic interventions dealing with a regulatory system which is complex, highly redundant and robust. Combinations between vaccines and antibodies, or between vaccines to several molecular components of the system should be evaluated, as well as combinations between inhibitors of the EGFR signaling pathway and inhibitors of other regulatory pathways related to cell proliferation, apoptosis and angiogenesis.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Breast Neoplasms; Cancer Vaccines; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Ligands; Neoplasms; Protein-Tyrosine Kinases; Signal Transduction

Topics: Epidermal Growth Factor; Humans; Neoplasms

The activation of the epidermal growth factor receptor (REGF) participates in oncogenesis by inducing cell proliferation, cell mobility and angiogenesis, and inhibiting apoptosis. This activation might be due to numerous abnormalities, including increased expression of its ligand. Although based on retrospective analyses with no standard technique of evaluation, the level of REGF expression is a prognosis factor for several tumors. It appears to be an indicator of poor prognosis which might influence treatments in head and neck tumors, cancers of the cervix, oesophagus, bladder and ovary. Its prognostic value is not observed in non-small cell lung cancer and remains to be demonstrated in adenocarcinomas such as colorectal tumors and beast cancer. Because of its role in oncogenesis and its prognostic value, REGF might in the future become a therapeutic target.

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Male; Neoplasm Proteins; Neoplasms; Organ Specificity; Prognosis; Receptor, ErbB-2; Transforming Growth Factor alpha

ImClone, in collaboration with licensees, Merck KGaA and Bristol-Myers Squibb (BMS), are developing cetuximab, a chimeric monoclonal antibody that blocks the epidermal growth factor receptor, for the potential treatment of various cancers, including colorectal, and head and neck tumors [179103]. The companies are evaluating the product both as a single agent, and in combination with radiation and a variety of chemotherapeutic agents. By January 2002, phase III trials had been initiated in head and neck cancer, and phase II trials were ongoing for colorectal and other cancers [427710], [437833]. In November 2001, ImClone completed the filing of a rolling BLA with the FDA for cetuximab in combination with irinotecan to treat irinotecan-refractory colorectal cancer. In December 2001, however, the FDA advised ImClone that its BLA was not acceptable for filing. In January 2002, it was reported that resubmission of the BLA was expected within 3 months [434999], and by February 2002, ImClone was expecting to use data from an ongoing phase II study in colorectal cancer patients conducted by Merck KGaA. In March 2002, Merck had anticipated European launch in 2003 [444653]; however, in April 2002, Merck reported that it was delaying its application to the European Agency for the Evaluation of Medicinal Products from the last half of 2002 to the first half of 2003, so that it could primarily include its own colorectal cancer data rather than ImClone's data. European launch was thus expected in 2004 [449226]. In May 2002, Lehman Brothers predicted global peak sales of US $2 billion [454652], while in the same month, Bear Stearns estimated that sales for Merck KGaA would reach 285 million Euros in 2007 [453500].

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Cetuximab; Clinical Trials as Topic; Drug Industry; Epidermal Growth Factor; Humans; Neoplasms

There is a need for new, selective anticancer agents that differentiate between malignant and nonmalignant cells. The benefits of such agents would include a higher therapeutic index and lower toxicity than conventional therapies. Although expressed in nonmalignant cells, the epidermal growth factor receptor (EGFR) is highly expressed in a variety of tumors, and its expression correlates with poor response to treatment, disease progression, and poor survival. Evidence for a role for the EGFR in the inhibition and pathogenesis of various cancers has led to the rational design and development of agents that selectively target this receptor. Activation of the EGFR signaling pathway in cancer cells has been linked with increased cell proliferation, angiogenesis, and metastasis, and decreased apoptosis. Preclinical data show that anti-EGFR therapies can inhibit these effects in vitro and in vivo. In addition, preclinical data confirm that many such agents have the potential to increase the effectiveness of current cytotoxic agents. Following accelerated drug development programs, phase III trials are now under way for a number of EGFR-targeted therapies, including the monoclonal antibody IMC-C225 and the EGFR-tyrosine kinase inhibitors ZD1839 (Iressa) and OSI-774. Thus, the rationale for EGFR-targeted approaches to cancer treatment is apparent and now well established, and there is increasing evidence that they may represent a significant contribution to cancer therapy.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Cetuximab; Clinical Trials as Topic; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Neoplasms; Quinazolines; Signal Transduction

The integrity of oral and digestive mucosa depend on many salivary components like the Epidermal Growth Factor (EGF). Sometimes indicative, sometimes stimulated or modulated factor of oral and digestive health, EGF appears as a clinical marker in neoplastic and inflammatory diseases. As cellular growth factor, it protects the digestive mucosa with stimulation of mucus production and with inhibition of gastric secretion. Equally implicated in healing process, it enhances this one, and determines, in patients, more or less sensibility to inflammatory damages. Its strategic place in various pathologies, as stomach ulcer and tumoral process, open research prospects with a real potential of repair and pronostic.

Topics: Biomarkers; Digestive System Physiological Phenomena; Epidermal Growth Factor; Gastric Mucosa; Humans; Inflammation; Intestinal Mucosa; Mouth Mucosa; Neoplasms

Systemic gene delivery systems are needed for therapeutic applications; in some situations, target cells might be spread throughout the organism, as in the case of cancer metastases, which can be reached only via the systemic route. Within the class of nonviral vectors, polymer-based transfection particles named DNA polyplexes and lipid-based systems named DNA lipoplexes are being developed for this purpose. For systemic circulation, masking the surface charge of DNA complexes has to be accomplished to avoid interactions with plasma components, erythrocytes, and the reticuloendothelial system. Among other vector formulations, polyplexes based on polyethylenimine (PEI), shielded with polyethylene glycol (PEG), and linked to the receptor binding ligands transferrin (Tf) or epidermal growth factor (EGF) have been developed. Complexes were found to mediate efficient gene transfer into tumor cell lines in a receptor-dependent and cell-cycle-dependent manner. Systemic administration of surface-shielded Tf-PEI polyplexes into the tail vein of mice resulted in preferential gene delivery into distantly growing subcutaneous tumors. In contrast, application of positively charged PEI polyplexes directed gene transfer primarily to the lung.

Topics: Animals; Cell Nucleus; DNA; Epidermal Growth Factor; Gene Transfer Techniques; Genetic Therapy; Humans; Ligands; Macromolecular Substances; Mice; Neoplasms; Polyamines; Polyelectrolytes; Polyethylene Glycols; Polyethyleneimine; Receptors, Cytoplasmic and Nuclear; Transferrin

Epidermal growth factor (EGF) is being tried as a vaccine in cancer immunotherapy with the aim of inducing neutralizing antibodies that might affect EGF-dependent tumors. Here we summarize our experience using the EGF self-molecule as an autoimmunigen. We report here that IgG anti-EGF antibodies are prevalent in healthy people and that augmentation of the response to EGF requires conjugation to an effective carrier and an adjuvant. Paradoxically, the response to EGF immunization could be enhanced by an 'immunosuppressive' treatment with cyclophosphamide, most probably by suppressing active control mechanisms. EGF is expressed in the thymus. Thus, EGF may be added to the immunological homunculus, the class of self-antigens to which there is both natural autoimmunity and natural regulation of the autoimmunity. The results using EGF as a vaccine can teach us about the homunculus and how to activate it.

Topics: Adjuvants, Immunologic; Animals; Autoantibodies; Autoimmunity; Cancer Vaccines; Cyclophosphamide; Epidermal Growth Factor; Humans; Immunodominant Epitopes; Immunosuppressive Agents; Neoplasms

Cripto, a growth factor with an EGF-like domain, and the first member of the EGF-CFC family of genes to be sequenced and characterized, contributes to deregulated growth of cancer cells. A role for Cripto in tumor development has been described in the human and the mouse. Members of the EGF-CFC family are found only in vertebrates: CFC proteins in zebrafish, Xenopus, chick, mouse and human have been characterized and indicate some common general functions in development. Cripto expression was first found in human and mouse embryonal carcinoma cells and male teratocarcinomas, and was demonstrated to be over-expressed in breast, cervical, ovarian, gastric, lung, colon, and pancreatic carcinomas in contrast to normal tissues where Cripto expression was invariably low or absent. Cripto may play a role in mammary tumorigenesis, since in vitro, Cripto induces mammary cell proliferation, reduces apoptosis, increases cell migration, and inhibits milk protein expression. This prediction is strengthened by observations of Cripto expression in 80% of human and mouse mammary tumors. At least three important roles for Cripto in development have created considerable interest, and each activity may be distinct in its mechanism of receptor signaling. One role is in the patterning of the anterior-posterior axis of the early embryo, a second is a crucial role in the development of the heart, and a third is in potentiating branching morphogenesis and modulating differentiation in the developing mammary gland. Whether these properties are functions of different forms of Cripto, different Cripto receptors or the distinct domains within this 15-38 kDa glycoprotein are examined here, but much remains to be revealed about this evolutionarily conserved gene product. Since all Cripto receptors have not yet been determined with certainty, future possible uses as therapeutic targets remain to be developed. Cripto is released or shed from expressing cells and may serve as an accessible marker gene in the early to mid-progressive stages of breast and other cancers. Meanwhile some speculations on possible receptor complexes for Cripto signaling in mammary cells are offered here as a spur to further discoveries.

Topics: Animals; Autocrine Communication; Epidermal Growth Factor; Fibroblast Growth Factors; Gene Expression; GPI-Linked Proteins; Growth Substances; Homeodomain Proteins; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Membrane Proteins; Multigene Family; Neoplasm Proteins; Neoplasms; Receptors, Cell Surface; Signal Transduction; Transcription Factors; Xenopus Proteins; Zebrafish Proteins

This review article details the diagnostical significance of immunohistochemistry, which has developed during the last quarter of the century. Certainly, the advancement of monoclonal antibody technology has been of great significance in assuring the place of immunohistochemistry in the modern accurate microscopic diagnosis of human neoplasms, as a method of choice in histopathology. The fact still remains that in order to properly assess any immunohistochemical reactivity used for differential diagnostic purposes, the target cells have to be identified as neoplastically transformed cells by routine histopathological techniques. Selected groups of target molecules of great significance in cancer biology are discussed. The discovery of neoplasm-associated antigens has not only made the more accurate diagnosis of human cancer feasible but has also shed light on the extensive immunophenotypical heterogeneity of even the most closely linked human malignancies. The identification of disseminated neoplastically transformed cells by immunohistochemistry has allowed for a clearer picture of cancer invasion and metastasis, as well as the evolution of the tumour cell associated immunophenotype towards increased malignancy. Some possibilities of neoplasm-associated antigen targeted, receptor-directed immunotherapy are discussed and reviewed in this manuscript. Future antineoplastic therapeutical approaches should see the inclusion of a variety of immunotherapies, in the form of an individualised 'cocktail' specific for the particular immunophenotypical pattern associated with each individual patient's neoplastic disease.

Topics: Animals; Apoptosis; Epidermal Growth Factor; ErbB Receptors; Genes, p53; Humans; Immunohistochemistry; Immunotherapy; Neoplasms; Neovascularization, Pathologic

Topics: Adenocarcinoma; Amides; Aniline Compounds; Animals; Antineoplastic Agents; Breast Neoplasms; Drug Design; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Female; Genistein; Humans; Leukemia; Macaca fascicularis; Mice; Mice, Inbred BALB C; Mice, SCID; Models, Molecular; Molecular Structure; Neoplasm Proteins; Neoplasms; Nitriles; Protein Conformation; Protein Structure, Tertiary; Quinazolines; Recombinant Fusion Proteins; Sequence Alignment; Species Specificity; Structure-Activity Relationship; Tumor Cells, Cultured; Tumor Stem Cell Assay; Xenograft Model Antitumor Assays

Overexpression of epidermal growth factor receptor (EGFr) has been demonstrated on many human tumors, and the increase in receptor expression levels has been linked with a poor clinical prognosis. Blocking the interaction of EGFr and the growth factors could lead to the arrest of tumor growth and possibly result in tumor cell death. To this end, using XenoMouse technology, ABX-EGF, a human IgG2 monoclonal antibody (mAb) specific to human EGFr, has been generated. ABX-EGF binds EGFr with high affinity (5x10(-11) M), blocks the binding of both EGF and transforming growth factor-alpha (TGF-alpha) to various EGFr-expressing human carcinoma cell lines, and inhibits EGF-dependent tumor cell activation, including EGFr tyrosine phosphorylation, increased extracellular acidification rate, and cell proliferation. In vivo ABX-EGF prevents completely the formation of human epidermoid carcinoma A431 xenografts in athymic mice. More importantly, administration of ABX-EGF without concomitant chemotherapy results in complete eradication of established tumors. No tumor recurrence was observed for more than 8 months following the last antibody injection, further indicating complete tumor cell elimination by the antibody. Inhibition of human pancreatic, renal, breast and prostate tumor xenografts which express different levels of EGFr by ABX-EGF was also achieved. Tumor expressing more than 17000 EGFr molecules per cell showed significant growth inhibition when treated with ABX-EGF. ABX-EGF had no effect on EGFr-negative tumors. The potency of ABX-EGF in eradicating well-established tumors without concomitant chemotherapy indicates its potential as a monotherapeutic agent for treatment of multiple EGFr-expressing human solid tumors, including those where no effective chemotherapy is available. Utilization of mAbs directed to growth factor receptors as cancer therapeutics has been validated recently by the tumor responses obtained from clinical trials with Herceptin, the humanized anti-HER2 antibody, in patients with HER2 overexpressing metastatic breast cancer. Being a fully human antibody, ABX-EGF is anticipated to exhibit a long serum half-life and minimal immunogenicity with repeated administration, even in immunocompetent patients. These results demonstrate the potent anti-tumor activity of ABX-EGF and its therapeutic potential for the treatment of multiple human solid tumors that overexpress EGFr.

Topics: Animals; Antibodies, Heterophile; Antibodies, Monoclonal; Antineoplastic Agents; Epidermal Growth Factor; Humans; Mice; Neoplasms; Panitumumab

The epidermal growth factor (EGF) family of peptides encodes several proteins that can function as growth factors. The EGF-like peptides, with the exception of proteins of the EGF-CFC subfamily, bind and activate tyrosine kinase receptors that belong to the erbB family. The EGF-like peptides are overexpressed in a majority of human carcinomas as compared with their nontransformed counterpart. By using different approaches, it has been shown that several different EGF-like peptides function as autocrine growth factors in carcinoma cell lines of different histological origin. Direct evidence that the EGF-like growth factors might function as transforming genes has been provided by in vitro and in vivo studies. In particular, the development of different transgenic mouse lines in which EGF-like growth factors have been overexpressed by means of tissue-specific or nonspecific promoters has provided invaluable information relating to their ability to function as dominantly transforming oncogenes. Cooperation of the EGF-like peptides with cellular protooncogenes in determining cell transformation has been demonstrated by using both in vitro and transgenic mice systems. Taken together, these data strongly suggest that the EGF-like peptides are involved in the pathogenesis of human carcinomas, and that they might represent suitable targets for novel therapeutic approaches.

Topics: Amphiregulin; Animals; Betacellulin; EGF Family of Proteins; Epidermal Growth Factor; Epiregulin; Glycoproteins; GPI-Linked Proteins; Growth Substances; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Membrane Proteins; Neoplasm Proteins; Neoplasms; Neuregulins; Transforming Growth Factor alpha

Topics: Bacterial Infections; Biotechnology; Epidermal Growth Factor; Humans; Neoplasms; Recombinant Proteins; Wound Healing

Topics: Amino Acid Sequence; Animals; Breast; Breast Neoplasms; DNA, Complementary; Embryonic and Fetal Development; Epidermal Growth Factor; Female; Gastrointestinal Neoplasms; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Lactation; Male; Membrane Glycoproteins; Molecular Sequence Data; Neoplasm Proteins; Neoplasms; Pseudogenes; Recombinant Proteins; Sequence Alignment; Spleen; Testis

The EGF-CFC gene family encodes a group of structurally related proteins that serve as important competence factors during early embryogenesis in Xenopus, zebrafish, mice and humans. This multigene family consists of Xenopus FRL-1, zebrafish one-eyed-pinhead (oep), mouse cripto (Cr-1) and cryptic, and human cripto (CR-1) and criptin. FRL-1, oep and mouse cripto are essential for the formation of mesoderm and endoderm and for correct establishment of the anterior/posterior axis. In addition, oep and cryptic are important for the establishment of left-right (L/R) asymmetry. In zebrafish, there is strong genetic evidence that oep functions as an obligatory co-factor for the correct signaling of a transforming growth factor-beta (TGFbeta)-related gene, nodal, during gastrulation and during L/R asymmetry development. Expression of Cr-1 and cryptic is extinguished in the embryo after day 8 of gestation except for the developing heart where Cr-1 expression is necessary for myocardial development. In the mouse, cryptic is not expressed in adult tissues whereas Cr-1 is expressed at a low level in several different tissues including the mammary gland. In the mammary gland, expression of Cr-1 in the ductal epithelial cells increases during pregnancy and lactation and immunoreactive and biologically active Cr-1 protein can be detected in human milk. Overexpression of Cr-1 in mouse mammary epithelial cells can facilitate their in vitro transformation and in vivo these Cr-1-transduced cells produce ductal hyperplasias in the mammary gland. Recombinant mouse or human cripto can enhance cell motility and branching morphogenesis in mammary epithelial cells and in some human tumor cells. These effects are accompanied by an epithelial-mesenchymal transition which is associated with a decrease in beta-catenin function and an increase in vimentin expression. Expression of cripto is increased several-fold in human colon, gastric, pancreatic and lung carcinomas and in a variety of different types of mouse and human breast carcinomas. More importantly, this increase can first be detected in premalignant lesions in some of these tissues. Although a specific receptor for the EGF-CFC proteins has not yet been identified, oep depends upon an activin-type RIIB and RIB receptor system that functions through Smad-2. Mouse and human cripto have been shown to activate a ras/raf/MAP kinase signaling pathway in mammary epithelial cells. Activation of phosphatidylinositol 3-kinase and Akt a

Topics: Amino Acid Sequence; Animals; Cell Differentiation; Embryo, Mammalian; Embryonic and Fetal Development; Epidermal Growth Factor; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Molecular Sequence Data; Neoplasms; Sequence Homology, Amino Acid

The receptor for the epidermal growth factor (EGF) and related ligands (EGFR), the prototypal member of the superfamily of receptors with intrinsic tyrosine kinase activity, is widely expressed on many cell types, including epithelial and mesenchymal lineages. Upon activation by at least five genetically distinct ligands (including EGF, transforming growth factor-alpha (TGF alpha) and heparin-binding EGF (HB-EGF)), the intrinsic kinase is activated and EGFR tyrosyl-phosphorylates itself and numerous intermediary effector molecules, including closely-related c-erbB receptor family members. This initiates myriad signaling pathways, some of which attenuate receptor signaling. The integrated biological responses to EGFR signaling are pleiotropic including mitogenesis or apoptosis, enhanced cell motility, protein secretion, and differentiation or dedifferentiation. In addition to being implicated in organ morphogenesis, maintenance and repair, upregulated EGFR signaling has been correlated in a wide variety of tumors with progression to invasion and metastasis. Thus, EGFR and its downstream signaling molecules' are targets for therapeutic interventions in wound repair and cancer.

Topics: Endocytosis; Epidermal Growth Factor; ErbB Receptors; Humans; Isoenzymes; Ligands; Neoplasms; Phospholipase C gamma; Phosphorylation; Receptor Protein-Tyrosine Kinases; Signal Transduction; Type C Phospholipases; Wound Healing

Recent technological advances, together with the discovery of the important role many growth factors play in modulating cell proliferation and differentiation, have led to the development of novel therapeutic agents for the treatment of cancer. In particular, advances in hybridoma technology and molecular engineering have permitted the development of humanized or chimeric monoclonal antibodies capable of interfering with growth factor signaling pathways. One promising target of interest is the epidermal growth factor receptor (EGFr), which is activated by the ligands EGF and TGF-alpha. This ligand receptor interaction plays a crucial role in the growth and survival of many human cancers. A chimeric (human/mouse) monoclonal antibody p6tuximab (IMC-C225) targets the EGFr and has potential clinical value as an anticancer agent.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Cell Division; Cell Survival; Cetuximab; Clinical Trials as Topic; Epidermal Growth Factor; ErbB Receptors; Humans; Mice; Neoplasms; Protein Engineering; Recombinant Fusion Proteins; Signal Transduction; Transforming Growth Factor alpha

The process of angiogenesis plays a critical role in tumor growth and metastasis. Recently, there has been much interest in the possible use of angiogenic growth factors as tumor markers. This paper will review the results thus far of attempts at measuring various angiogenic factors in bodily fluids. In the future, angiogenic factors will most likely be useful as a monitor of therapy and/or a predictor of outcome after cancer has been diagnosed.

Topics: Biomarkers, Tumor; Cytokines; E-Selectin; Endothelial Growth Factors; Epidermal Growth Factor; Fibroblast Growth Factors; Hepatocyte Growth Factor; Humans; Lymphokines; Neoplasms; Neovascularization, Pathologic; Transforming Growth Factors; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

c-Src, the prototype of the cytoplasmic, membrane-associated,non-receptor tyrosine kinases, is a co-transducer of mitogenic signals emanating from a number of tyrosine kinase polypeptide growth factor receptors. Examples of such receptors include those that bind the platelet-derived growth factor (PDGF), colony stimulating factor-1 (CSF-1), and epidermal growth factor (EGF). Investigations into the mechanisms by which c-Src contributes to receptor signaling suggest that interactions between the two proteins are bidirectional, i.e., that c-Src can bind, phosphorylate, and activate the receptor, and vice versa. The consequences of these interactions appear to be enhanced phosphorylation of specific substrates. Delineating which cellular proteins are substrates of which tyrosine kinase and determining the consequences of tyrosine phosphorylation on the function of specific substrates are the goals of current investigations. Utilizing the murine C3H10T fibroblast model, in which a panel of wild type and mutant c-Src/EGF receptor overexpressors has been studied for temporal and spatial second messenger responses to EGF, distinctions between substrates of c-Src and the EGF receptor and the effects of tyrosine phosphorylation on substrate function are beginning to emerge. In the 10T model, preferred substrates of c-Src are almost exclusively comprised of those molecules that associate with the actin cytoskeleton or with focal adhesions, such as cortactin, p190RhoGAP, and p130CAS, while preferred substrates of the EGF receptor include the receptor itself, SHC, phospholipase C-gamma and p62DOK. While the major mitogenic signaling pathway is thought to proceed directly from the receptor (through SHC/GRB2/SOS/Ras/Raf/MEK/MAPkinase/Elk1), more evidence is accumulating to suggest that proteins involved in regulating the actin cytoskeleton (such as c-Src substrates) also participate in mitogenesis, either as unique transducers of growth signals and/or as monitors of anti-apoptotic conditions (substratum attachment). How c-Src may contribute to the EGF mitogenic response through tyrosine phosphorylation of or association with its specific substrates is discussed. Cellular Src (c-Src), prototype for a family of intracellular membrane-associated tyrosine kinases, is required for mitogenesis initiated by multiple growth factor receptors, including the receptors for epidermal growth factor (EGF), platelet-derived growth factor (PDGF), colony stimulating factor-1 (CSF-1), an

Topics: Animals; Cell Division; Cytoskeletal Proteins; Epidermal Growth Factor; Humans; Mice; Neoplasms; Proto-Oncogene Proteins pp60(c-src); Receptor Protein-Tyrosine Kinases; Signal Transduction

It is known that long-term withdrawal of choline from the diet induces hepatocellular carcinomas in animal models in the absence of known carcinogens. We hypothesize that a choline deficient diet (CD) alters the balance of cell growth and cell death in hepatocytes and thus promotes the survival of clones of cells capable of malignant transformation. When grown in CD medium (5 microM or 0 microM choline) CWSV-1 rat hepatocytes immortalized with SV40 large T-antigen underwent p53-independent apoptosis (terminal dUTP end-labeling of fragmented DNA; laddering of DNA in agarose gel). CWSV-1 cells which were adapted to survive in 5 microM choline acquired resistance to CD-induced apoptosis and were able to form hepatocellular carcinomas in nude mice. These adapted CWSV-1 cells express higher amounts of both the 32 kDa membrane-bound and 6 kDa mature form of TGF alpha compared to cells made acutely CD. Control (70 microM choline) and adapted cells, but not acutely deficient hepatocytes, could be induced to undergo apoptosis by neutralization of secreted TGF alpha. Protein tyrosine phosphorylation is known to protect against apoptosis. We found decreased EGF receptor tyrosine phosphorylation in acutely choline deficient CWSV-1 cells. TGF beta 1 is an important growth-regulator in the liver. CWSV-1 cells express TGF beta 1 receptors and this peptide induced cell detachment and death in control and acutely deficient cells. Hepatocytes adapted to survive in low choline were also resistant to TGF beta 1, although TGF beta 1 receptors and protein could be detected in the cytoplasm of these cells. The non-essential nutrient choline is important in maintaining plasma membrane structure and function, and in intracellular signaling. Our results indicate that acute withdrawal of choline induces p53-independent programmed cell death in hepatocytes, whereas cells adapted to survive in low choline are resistant to this form of apoptosis, as well as to cell death induced by TGF beta 1. Our results also suggest that CD may induce alterations (mutations?) in growth factor signaling pathways which may enhance cell survival and malignant transformation.

Topics: Animals; Apoptosis; Choline; Choline Deficiency; Diet; Epidermal Growth Factor; Humans; Neoplasms; Signal Transduction; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Suppressor Protein p53

HB-EGF is a heparin-binding member of the EGF family that was initially identified in the conditioned medium of human macrophages. Soluble mature HB-EGF is proteolytically processed from a larger membrane-anchored precursor and is a potent mitogen and chemotactic factor for fibroblasts, smooth muscle cells but not endothelial cells. HB-EGF activates two EGF receptor subtypes, HER1 and HER4 and binds to cell surface HSPG. The transmembrane form of HB-EGF is a juxtacrine growth and adhesion factor and is uniquely the receptor for diphtheria toxin. HB-EGF gene expression is highly regulated, for example by cytokines, growth factors, and transcription factors such as MyoD. HB-EGF has been implicated as a participant in a variety of normal physiological processes such as blastocyst implantation and wound healing, and in pathological processes such as tumor growth, SMC hyperplasia and atherosclerosis.

Topics: Amino Acid Sequence; Animals; Arteriosclerosis; Binding Sites; Cell Adhesion; Cell Division; Cell Membrane; Chromosome Mapping; Epidermal Growth Factor; Gene Expression Regulation; Genes; Heparan Sulfate Proteoglycans; Heparin; Heparin-binding EGF-like Growth Factor; Humans; Hyperplasia; Intercellular Signaling Peptides and Proteins; Molecular Sequence Data; Muscular Diseases; Neoplasms; Promoter Regions, Genetic; Protein Binding; Receptors, Cell Surface; Reproduction; Signal Transduction; Wound Healing

Topics: Animals; Antibodies, Neoplasm; Antibody-Dependent Cell Cytotoxicity; Cytokines; Epidermal Growth Factor; Humans; Immunoconjugates; Immunotherapy, Adoptive; Interleukin-2; Killer Cells, Lymphokine-Activated; Lymphotoxin-alpha; Mice; Mice, SCID; Neoplasm Transplantation; Neoplasms; Neuroblastoma; Recombinant Fusion Proteins; Superantigens; T-Lymphocytes, Cytotoxic

Topics: Animals; Brain Neoplasms; Breast Neoplasms; Cell Division; Cell Transformation, Neoplastic; Digestive System Neoplasms; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Growth Substances; Head and Neck Neoplasms; Humans; Lung Neoplasms; Melanoma; Neoplasm Proteins; Neoplasms; Neoplasms, Experimental; Oncogenes; Rats; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Signal Transduction; Urogenital Neoplasms

The processes of cellular proliferation and progressive acquisition of a specialized phenotype show a remarkable degree of coordination that involves both intracellular programming and intercellular communication. One of the major incentives for studying factors that regulate the processes of cellular proliferation and differentiation is the recognition of their potential contribution to tumorigenesis. In normal cells, stimulatory and inhibitory events are believed to be under the control of growth factors and growth inhibitory factors, which are known to be protooncogene products. Growth regulatory mechanisms usually involve the binding of a growth factor to a specific receptor on the cell surface, which then through an intracellular biochemical cascade leads to cell division. The cell regulation pathways initiated by growth factors may be subverted at several distinct levels in cancer cells. Studies of oncogenes have shown that they may function as abnormal growth factors or abnormal receptors, induce expression of potential signal regulators or encode proteins which modulate gene transcription. The purpose of the present paper is to examine the role of growth factors, growth factor receptors and intracellular proteins involved in signal transduction (with particular regard to the epidermal growth factor receptor system) in the control of normal growth and differentiation, and their contribution to transformation and tumorigenesis. We also review the classical theories of neoplasia and various other models. Chemical carcinogenesis and Vogelstein-Lane model are presented.

Topics: Amino Acid Sequence; Cell Division; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Genes, Tumor Suppressor; Growth; Growth Substances; Humans; Molecular Sequence Data; Neoplasm Metastasis; Neoplasms; Oncogenes; Phenotype; Receptors, Growth Factor; Signal Transduction; Transcription, Genetic; Transforming Growth Factor alpha

This review considers the biology of the type 1 growth factor receptor family which is increasingly recognised as important in the control of normal cell proliferation and in the pathogenesis of human cancer. The family currently comprises three closely related members: the epidermal growth factor (EGF) receptor, c-erbB-2 and c-erbB-3, all of which show abnormalities of expression in various human tumours. The family of factors related to EGF has also expanded recently and now includes transforming growth factor alpha, heparin-binding EGF, amphiregulin, cripto and heregulin, as well as several other potential ligands for the c-erbB2-2 receptor. The involvement of these receptors and growth factors in human cancer has implications for the design of novel forms of therapy for cancer, and we review recent advances and future avenues for investigation.

Topics: Amino Acid Sequence; Amphiregulin; Animals; Base Sequence; Biomarkers, Tumor; Caenorhabditis; Consensus Sequence; Drosophila; Drosophila Proteins; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Glycoproteins; GPI-Linked Proteins; Growth Substances; Heparin; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Ligands; Mammals; Membrane Glycoproteins; Molecular Sequence Data; Multigene Family; Neoplasm Proteins; Neoplasms; Proto-Oncogene Proteins; Proto-Oncogenes; Receptor, ErbB-2; Receptor, ErbB-3; Signal Transduction; Transforming Growth Factor alpha; Viral Proteins

Topics: Amino Acid Sequence; Animals; Epidermal Growth Factor; ErbB Receptors; Extracellular Matrix; Gene Expression Regulation; Humans; Molecular Sequence Data; Neoplasms; Neovascularization, Pathologic; Oncogenes

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Cell Division; Cell Transformation, Neoplastic; Embryonic and Fetal Development; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Growth Substances; Humans; Membrane Glycoproteins; Mice; Molecular Sequence Data; Multigene Family; Neoplasm Proteins; Neoplasms; Protein Precursors; Protein Processing, Post-Translational; Rats; Signal Transduction; Transforming Growth Factor alpha

The capacity of growth factors to activate receptors through autocrine and paracrine pathways continues to be a major focus of cancer biology research. This review of growth factors for solid tumor cells complements our summary of hematopoietic growth factors.

Topics: Amphiregulin; Cell Differentiation; Cell Division; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Glycoproteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Neoplasms; Platelet-Derived Growth Factor; Proto-Oncogene Proteins; Receptor, ErbB-2; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor; Sequence Homology, Nucleic Acid; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Immunohistochemical techniques are prerequisite for the classification and characterization of numerous diseases. The incubation with labelled ligands is an alternative to immunohistochemistry, and allows the recognition of receptors independent from their immunogenic properties. The biotinylation and immobilization of small molecules such as sugars can be performed under the conservation of active receptor-binding sites. Certain sugars bound to bovine serum albumin are highly specific markers for bronchial carcinomas and lung parenchyma altered by cytostatic drug regimes, infection or inflammation. Biotinylated growth factors such as epidermal growth factor or biotinylated hormones are useful tools for the demonstration of the corresponding receptors. These tools are called "neoligandoproteins". The number specific binding sites and the thermodynamic binding parameters can be measured with sugars bound to enzymes. The number of and the expression of specific sugar binding sites can be correlated to the differentiation and metastatic potency of malignant tumors. In addition, cytostatic drugs can be bound to carbohydrates. This technique offers new perspectives in the affinity and quantification of target-related cytostatic drug regimens.

Topics: Animals; Binding Sites; Biomarkers, Tumor; Biotin; Cytokines; Epidermal Growth Factor; Gangliosides; Glycoproteins; Histocytochemistry; Hormones; Humans; Ligands; Neoplasms

The ability of malignant cells to escape the constraint that normally regulate cell growth and differentiation has been a primary focus of attention for investigators of cancer cell biology. An outcome of this attention has been the discovery that the protein products of oncogenes play a role in the activation of growth signal pathways. A second outcome, possibly related to abnormal oncogene expression, has been the discovery that malignant cells frequently show an ability to regulate their own growth by the release of autocrine growth modulatory substances. Most important, the growth of certain malignant cell types has been shown to depend on autocrine growth circuits. A malignant tumor whose continued growth depends on the release of an autocrine growth factor may be vulnerable to treatment with specific receptor antagonists or immunoneutralizing antibodies designed to break the autocrine circuit. Information is rapidly emerging concerning autocrine growth factors in selected human solid tissue malignancy.

Topics: Cytokines; Epidermal Growth Factor; Growth Substances; Humans; Neoplasms; Oncogenes; Transforming Growth Factors

A Monoclonal Antibody Program was launched at the National Institute of Oncology and Radiobiology (INOR) in Cuba in 1985. Eleven MAbs have been obtained which recognize different leukocyte antigens. Four MAbs a series of reagents raised against different types of cytokeratins, carcino-embryonic antigen and the membrane receptor for Epidermal Growth Factor. These MAbs are especially useful in immunohistochemistry for the study of human tumours, leukemias and lymphomas. Three ultramicro-ELISA systems have being deviced using MAbs. First trials are being made with radioactive MAbs for immunoscintigraphy. The use of anti T-cell MAbs for the prevention of rejection crisis in patients receiving organ transplantation and for the treatment of patient with cutaneous T-cell lymphoma is still mostly a matter for research.

Topics: Academies and Institutes; Antibodies, Monoclonal; Carcinoembryonic Antigen; Cuba; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Humans; Immunoassay; Immunoglobulins; Immunohistochemistry; Keratins; Leukocytes; Medical Oncology; Neoplasms; Radiobiology; Research

Many types of cancer cells display aberrantly high numbers of EGF receptors on their surface. We have targeted these cells for elimination by combining the cell binding ability of either epidermal growth factor or transforming growth factor type alpha with the potent cell killing activity of Pseudomonas exotoxin. These chimeric molecules are formed either by chemical conjugation of the two proteins or by expression of a gene fusion into a product containing both proteins. In this review, we show that these chimeric toxins are extremely cytotoxic to a variety of cancer cell lines.

Topics: Cell Survival; Epidermal Growth Factor; Exotoxins; Humans; Immunotoxins; Neoplasms; Pseudomonas aeruginosa; Transforming Growth Factor alpha

The article is a review of recent knowledge of the structural relationships of epidermal growth factor (EGF) with other biological products occurring in a wide range of animal species and some viruses. The place of EGF and related products in the physiology of the whole organism is discussed against this background. Special attention is given to the role of EGF as a tissue growth factor and to the participation of related products in cancer.

Topics: Amino Acid Sequence; Animals; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Sequence Homology, Nucleic Acid; Structure-Activity Relationship; Transforming Growth Factors

Topics: Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Female; Growth Substances; Humans; Interferon Type I; Neoplasms; Proto-Oncogenes; Signal Transduction; Transforming Growth Factors

This review describes the discovery and initial characterization of the epidermal growth factors (EGFs) and transforming growth factors alpha (TGF alpha), the biosynthesis and tissue distribution of these molecules and a brief description of the effects of these molecules in culture and animals. An analysis of the similarities between the EGFs and TGF alpha s from different organisms is used to describe the features of the molecules which appear to be critical for their function. The review concludes with a discussion of the relationship between the production of the EGF or TGF alpha to cancer induction and/or metastasis.

Topics: Animals; Epidermal Growth Factor; Genes; Humans; Mice; Neoplasms; Transforming Growth Factors

Topics: Animals; Culture Techniques; Epidermal Growth Factor; Fibroblast Growth Factors; Growth Inhibitors; Growth Substances; Humans; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Neoplasms; Neoplasms, Experimental; Platelet-Derived Growth Factor

This article is aimed at reviewing and analyzing studies that are related to the possible therapeutic use of a potent and ubiquitously-distributed hormone--somato-statin (SS-14), and its analogues. Administration of these substances has provided beneficial effects in treating acromegaly, gastro-intestinal hemorrhagic and hypersecretory disorders, acute pancreatitis, diabetes mellitus, and certain types of cancer. Further studies with SS-14-analogues might provide new therapies for treating certain life-threatening disorders of man.

Topics: Acromegaly; Alzheimer Disease; Epidermal Growth Factor; Gastrointestinal Diseases; Humans; Neoplasms; Pancreatic Diseases; Somatostatin

Topics: Animals; Cell Division; Embryonic and Fetal Development; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Oncogene Proteins v-erbB; Phosphorylation; Protein-Tyrosine Kinases; Viral Proteins

Polypeptide growth factors modulate proliferation of nontransformed cells in vivo and in vitro, while cancer appears to reflect an alteration of growth-regulatory mechanisms found in normal cells. Some provocative clues for understanding the cellular biochemical events involved in growth regulation have come from the study of transforming retroviral oncogenes. Some of these oncogenes encode proteins similar to those implicated in growth factor-mediated growth control. Of particular interest is the study of growth factor receptors present on the cell surface, which are cellular homologs of members of the largest class of oncogenes, the tyrosine kinases. It is likely that the study of the interplay of growth factors, and the molecular basis of pleiotropic effects elicited by growth factors, will help to explain how growth factor-signaling pathways affect gene expression and cell division in normal and transformed states.

Topics: Animals; Cell Division; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Neoplasms; Receptors, Cell Surface

Cancer Research has changed substantially over the past several years since oncogenes were isolated from cancer cells. More than 40 oncogenes have been identified to date from tumor viruses and cancer cells. Many of the gene products seem related to signalling pathways that determine growth and differentiation of cells. This review attempts to summarize much of the currently available data and to gain future perspectives.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Cell Transformation, Neoplastic; Cell Transformation, Viral; Epidermal Growth Factor; Genes, Viral; Humans; Mammary Neoplasms, Experimental; Neoplasms; Platelet-Derived Growth Factor; Retroviridae; Transfection; Urinary Bladder Neoplasms

Topics: Biomarkers, Tumor; Bombesin; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Neoplasms; Peptides; Transforming Growth Factors

For cell growth and division to occur, a large variety of metabolic processes must be carefully coordinated in the cell. Through evolutionary pressures, specific hormones and growth factors have acquired the ability to trigger a complex coordinated "pleiotropic growth response" in their target cells. This complex response is mediated by specific cellular receptors and intracellular messengers. Teleologically then, it makes sense that in oncogenesis this growth regulating network is utilized by the production of proteins which mimic growth factors, the activated form of their receptors or, the messengers themselves. Several lines of evidence indicate that the epidermal growth factor-stimulated growth regulatory system is involved in cellular proliferation, both normal and neoplastic. Some of the effects of epidermal growth factor in carcinogenesis are separable from its direct, growth stimulatory effects. Thus, the role of epidermal growth factor in carcinogenesis is more complex than is its role in stimulating growth.

Topics: Animals; Carcinogens; Cell Transformation, Neoplastic; Cell Transformation, Viral; Epidermal Growth Factor; ErbB Receptors; Humans; Immune Tolerance; Neoplasms; Oncogene Proteins, Viral; Oncogenes; Peptides; Receptors, Cell Surface; Transforming Growth Factors

Growth factors, defined as polypeptides that stimulate cell proliferation, are major growth-regulatory molecules for cells in culture and probably also for cells in vivo. Nontransformed cells show an absolute requirement for growth factors for proliferation in culture and generally more than one growth factor is required. Under usual culture conditions, growth factors are more rapidly depleted than other media components and thus become rate limiting for proliferation. The loss of or decreased requirement for specific growth factors is a common occurrence in neoplastically transformed cells and may lead to a growth advantage, a cardinal feature of cancer cells. Recent work with transforming growth factors, the platelet-derived growth factor, and oncogenes has produced some insight into the mechanisms through which alterations in growth factor-receptor-response pathways could lead to a growth advantage. Evidence has been derived for autocrine secretion in which the cell produces its own growth factor. Many transformed mesenchymal cells produce PDGF (the product of the c-sis proto-oncogene) and certain transformed cells both produce and respond in a growth-stimulatory manner to TGF beta. With TGF beta, which is a growth inhibitor for certain epithelial and other cell types, the loss of the normal inhibitory response in transformed cells could have the same result as the activation of a growth-stimulatory response. Two proto-oncogenes, erbB and fms, encode growth factor receptors. In the erbB case, the viral erbB aberrant receptor produced is truncated and appears to be constitutively activated without the need for a growth factor. Recent studies suggest that the p21 product of the ras oncogene may be an obligatory intermediate in transducing the growth factor signal. Activation of ras may, therefore, activate the growth factor pathway without the need for either a growth factor or its receptor. The transcription of myc and fos is induced by growth factor stimulation of quiescent cells. The protein products of both are nuclear associated and conceivably could be involved in regulating other genes important in the control of cell proliferation. Activation or inappropriate expression of either myc or fos could produce the same end result as stimulation of a growth factor pathway leading to a growth advantage. Study of the molecular mechanism(s) of growth factor action has just begun. The excitement and attention focused on cellular oncogenes in recent years is

Topics: Animals; Cell Cycle; Colony-Stimulating Factors; Epidermal Growth Factor; Fibroblast Growth Factors; Growth Substances; Humans; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Interleukin-2; Neoplasms; Nerve Growth Factors; Peptides; Platelet-Derived Growth Factor; Proto-Oncogene Mas; Proto-Oncogenes; Receptors, Cell Surface; Transforming Growth Factors

Topics: Bombesin; Cell Line; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Gastrins; Growth Substances; Humans; Neoplasms; Nerve Growth Factors; Oncogenes; Platelet-Derived Growth Factor; Receptors, Cell Surface; Somatomedins; Transferrin

Polypeptide growth factors form a class of regulatory molecules which exert their effects by binding to specific receptors present on the cell surface. Most of the time the exact role of these factors in the healthy body is unknown. Some, like PDGF and TGF beta, seem to be involved in wound healing. Others, like EGF, promote epithelial cell growth and differentiation. The site of synthesis of most polypeptide growth factors is unknown. Their target can be identified by detecting the cells which present the specific receptors at their surface. It is though that polypeptide growth factors have a paracrine mode of action. Many different cancerous cells produce polypeptide growth factors and the appropriate receptors. Thus, they are able to stimulate their own growth in an autocrine fashion. Recently, some polypeptide growth factors and receptor genes or cDNAs have been molecularly cloned. Growth factor genes and messengers are much more complex than would be expected from the size of the polypeptide. Some cDNAs have been introduced into bacterial expression vectors and large amounts of the factors have been produced by bacteria. New tools, such as molecular probes and specific antibodies, are thus now available to investigate the production of the growth factors and their receptors. The same tools will facilitate the identification and understanding of the molecular mechanism whereby cancerous cells produce the growth factors and the appropriate receptors simultaneously. The importance of growth factors and receptors in cancer is stressed by the finding that three oncogenes are in fact the genes coding for one growth factor and two receptors. Finally, the molecular probes and the specific antibodies raised against these molecules can be used to identify precisely the growth factor(s) and receptor(s) produced abnormally in cancers. Antibodies that inhibit specifically the interaction of this very growth factor with its receptor could then be developed, thus allowing human tumour cell growth to be controlled.

Topics: Animals; Cell Transformation, Neoplastic; Cloning, Molecular; Endocrine Glands; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Neoplasms; Oncogenes; Peptides; Platelet-Derived Growth Factor; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor; Transforming Growth Factors

Topics: Adult; Aged; Endocrine System Diseases; Epidermal Growth Factor; Female; Humans; Kidney Diseases; Liver Diseases; Male; Middle Aged; Neoplasms; Radioimmunoassay; Radioligand Assay

Topics: Aminoacridines; Amsacrine; Animals; Anthracenes; Antineoplastic Agents; Blood Physiological Phenomena; Bone Marrow; Breast Neoplasms; Colony-Forming Units Assay; Drug Evaluation; Drug Evaluation, Preclinical; Drug Stability; Epidermal Growth Factor; Female; Head and Neck Neoplasms; Humans; Interferons; Karyotyping; Leukemia; Lung Neoplasms; Melanoma; Mice; Mice, Nude; Multiple Myeloma; Neoplasm Transplantation; Neoplasms; Ovarian Neoplasms; Platelet-Derived Growth Factor; Suspensions; Transplantation, Heterologous; Tumor Stem Cell Assay

Topics: Biological Assay; Blood Platelets; Cell Differentiation; Connective Tissue Cells; Epidermal Growth Factor; Extracellular Matrix; Growth Substances; Humans; Inflammation; Insulin; Interleukin-1; Neoplasms; Nerve Growth Factors; Peptides; Platelet-Derived Growth Factor; Somatomedins

Cellular oncogenes and their activated and retrovirus-coded counterparts play an important role in cellular regulation. Here the relationship between such oncogenes and the genes coding for the transforming proteins of the papovaviruses, polyoma viruses, and simian virus 40 (SV40) is discussed. It is concluded that polyoma virus may transform established cells by a mechanism involving activation of a cellular oncogene product, whereas SV40 may transform by a mechanism involving a previously little studied cytoplasmic form of the transforming protein.

Topics: Animals; Antigens, Polyomavirus Transforming; Antigens, Viral, Tumor; Base Sequence; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA; DNA-Binding Proteins; DNA, Viral; Epidermal Growth Factor; Humans; Neoplasms; Oncogenes; Papillomaviridae; Platelet-Derived Growth Factor; Polyomaviridae; Polyomavirus; Protein Kinases; Protein-Tyrosine Kinases; Simian virus 40; Transcription, Genetic; Viral Proteins

In the preceding sections we have shown evidence that growth-promoting factors are involved in three basic situations. In normal embryonic development and function of mature organisms, growth factors such as NGF and EGF are of prime importance in supporting the necessary embryonic cell proliferation and the development of specific cell types. Other factors operate on subsets of mature cells during specialized functions such as inflammation. Included in this set would be factors such as CSF/MGF and Interleukin-2. Another basic function of growth factors has been shown to be wound repair and organ regeneration. This includes the well characterized PDGF and FGF as well as the various renotropic factors and liver growth factors. As these factors must operate in mature organisms with many different cell types and similar cell types in many locations, more specificity is needed than in embryonic growth. This has resulted in the organ specific factors such as the renotropins and in the unique delivery system of the PDGF. The recent discovery and characterization of the transforming growth factors has provided a possible connection between embryonic and normal developmental growth and the rapid cellular proliferation characteristic of tumor cells. The TGF not only interacts with receptors for normal growth factors such as EGF but are also detectable in low levels in normal tissue and embryos. The exact relationships between these various factors will have to await the determinations of more amino acid sequences for comparisons. The other tumor-related product, tumor angiogenesis factor, is also found in normal tissue and inflammatory reaction sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Angiogenesis Inducing Agents; Animals; Cell Division; Cell Transformation, Neoplastic; Epidermal Growth Factor; Fibroblast Growth Factors; Growth Substances; Humans; Hypertrophy; Interleukin-2; Interleukin-3; Neoplasms; Nerve Growth Factors; Peptides; Platelet-Derived Growth Factor; Transforming Growth Factors; Wound Healing

Topics: Animals; Blood Platelets; Cell Cycle; Cell Transformation, Neoplastic; Cells, Cultured; Chickens; Embryo, Mammalian; Embryo, Nonmammalian; Epidermal Growth Factor; Female; Fibroblasts; Growth Substances; Humans; In Vitro Techniques; Insulin; Kidney; Liver; Mice; Neoplasms; Nerve Growth Factors; Peptides; Pituitary Gland; Pregnancy; Rats; Somatomedins

Topics: Animals; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Cells, Cultured; Enzyme Activation; Epidermal Growth Factor; Epithelial Cells; Fetal Hemoglobin; Fibroblasts; Gene Expression Regulation; Glycopeptides; Growth Inhibitors; Growth Substances; Hemoglobins; Humans; Isoenzymes; Liver; Models, Biological; Mutation; Neoplasms; Peptides; Protein Kinases; RNA, Messenger; Thalassemia; Transcription, Genetic; Transforming Growth Factors

The genetic conservation and evolution of virogenes in mammalian species are described in relation to the horizontal, vertical, and congenital transmission of type C RNA oncogenic viruses within members of a given species, or among members of near and/or distantly related species. Examples of oncogenic virus infection between ancestors of the mouse, cat, pig, and primates as well as the integration of the virus into the host's genome are documented. The possible normal functions of the virogenes as a part of the cellular genome of many species are explored. Recent evidence of genetic recombination (genetic mixing) among distinct type C viruses is also reviewed, thus completing an overview of the evolutionary past, present, and complex future relationships of oncogenic RNA viruses and mammalian species.

Topics: Animals; Biological Evolution; DNA, Recombinant; Epidermal Growth Factor; Genes, Viral; Humans; Mice; Neoplasms; Primates; Retroviridae; Sarcoma, Experimental; Transformation, Genetic

To evaluate the effectiveness of an educational program for cancer patients who developed pruritus, rash or photosensitivity induced by chemotherapy, epidermal growth factor inhibitor (EGFRI) treatments, or immunotherapy.. This study is a pilot randomized controlled study. The patients in the experimental pool were assigned to attend the educational program once weekly, for 4 weeks. For the patients in the control group the usual information was provided to them, as with any cancer patient who initiates treatment. Each participant's induction day to the program (symptoms initiation) was considered part of week 0. For the Primary endpoint repeated measurements were taken weekly regarding the grade of skin reaction. For the Secondary endpoint the 36-Item Short Form Survey questionnaire (SF-36) (since week 0) and the Dermatology Life Quality Index (DLQI) questionnaire (since week 1) were recorded.. The study was conducted between January 2019 and December 2020 and included 40 patients. The grades of skin reactions, showed a statistically significant improvement in the intervention group compared to the control (Walds X. Further validation of the effectiveness of the educational program over longer periods of time will be required.

Topics: Epidermal Growth Factor; ErbB Receptors; Humans; Immunotherapy; Neoplasms; Pilot Projects; Quality of Life

Novel direct-acting antivirals (DAAs) have completely changed the panorama of hepatitis C due to their high efficacy and optimal safety profile. Unfortunately, an unexpectedly high rate of early recurrence of hepatocellular carcinoma has been reported within weeks of starting treatment, but the mechanism is not known.. We monitored the serum level of vascular endothelial growth factor (VEGF) and changes in the pattern of circulating interleukins in 103 chronic hepatitis C patients during antiviral treatment with DAA-regimens. VEGF, epidermal growth factor (EGF), and several interleukins were assessed at baseline, during treatment, and after treatment. The biological effect of DAA-treated patient serum on human umbilical vein endothelial cell (HUVEC) proliferation was also confirmed.. After 4 weeks of therapy, VEGF increased approximately 4-fold compared to baseline, remained elevated up to the end of treatment, and returned to the pre-treatment level after the end of therapy. In contrast, interleukin-10 and tumor necrosis factor-alpha significantly decreased during therapy, which was coincident with HCV clearance. The levels of both remained low after treatment. The addition of serum from patients collected during therapy induced HUVEC proliferation; however, this disappeared after the end of therapy.. DAA administration induces an early increase in serum VEGF and a change in the inflammatory pattern, coinciding with HCV clearance. This may alter the balance between inflammatory and anti-inflammatory processes and modify the antitumor surveillance of the host. Fortunately, such modifications return reverse to normal after the end of treatment.

Topics: Adult; Aged; Aged, 80 and over; Antiviral Agents; Cell Proliferation; Epidermal Growth Factor; Female; Hepacivirus; Hepatitis C; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Interleukin-10; Male; Middle Aged; Neoplasms; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Inhibition of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) pathways may result in synergistic antitumour activity. We designed a phase I study to evaluate the combination of vandetanib, an investigational agent with activity against EGF receptor and VEGF receptor 2, and bevacizumab, a monoclonal antibody against VEGF.. Patients with advanced solid tumours and lymphomas were enrolled. Objectives were to determine the safety and maximum tolerated dose of the combination, characterise pharmacokinetics, measure angiogenic marker changes in blood, and assess tumour blood flow using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Vandetanib was given orally once daily and bevacizumab intravenously once in every 3 weeks in 21-day cycles utilising a standard dose-escalation design.. Fifteen patients were enrolled, and a total of 94 cycles of therapy were administered. No protocol-defined dose-limiting toxicities were observed; due to toxicities associated with chronic dosing, hypertension, proteinuria, diarrhoea and anorexia, dose escalation was stopped at the second dose level. We observed one partial response and one minor response; 9 patients experienced stable disease. There were significant changes in plasma VEGF and placental-derived growth factor levels, and decreases in K(trans) and k(ep) were observed by DCE-MRI.. In this trial, we safely combined two targeted agents that cause dual blockade of the VEGF pathway, demonstrated preliminary evidence of clinical activity, and conducted correlative studies demonstrating anti-angiogenic effect. The recommended phase II dose was established as vandetanib 200 mg daily and bevacizumab 7.5 mg/kg every 3 weeks.

Topics: Adult; Aged; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Bevacizumab; Biomarkers, Tumor; Contrast Media; Epidermal Growth Factor; Female; Humans; Lymphoma; Magnetic Resonance Imaging; Male; Middle Aged; Neoplasms; Piperidines; Quinazolines; Signal Transduction; Time Factors; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

The Notch signaling pathway is an important therapeutic target for the treatment of inflammatory diseases and cancer. We previously created ligand-specific inhibitors of Notch signaling comprised of Fc fusions to specific EGF-like repeats of the Notch1 extracellular domain, called Notch decoys, which bound ligands, blocked Notch signaling, and showed anti-tumor activity with low toxicity. However, the study of their function depended on virally mediated expression, which precluded dosage control and limited clinical applicability. We have refined the decoy design to create peptibody-based Notch inhibitors comprising the core binding domains, EGF-like repeats 10-14, of either Notch1 or Notch4. These Notch peptibodies showed high secretion properties and production yields that were improved by nearly 100-fold compared to previous Notch decoys. Using surface plasmon resonance spectroscopy coupled with co-immunoprecipitation assays, we observed that Notch1 and Notch4 peptibodies demonstrate strong but distinct binding properties to Notch ligands DLL4 and JAG1. Both Notch1 and Notch4 peptibodies interfere with Notch signaling in endothelial cells and reduce expression of canonical Notch targets after treatment. While prior DLL4 inhibitors cause hyper-sprouting, the Notch1 peptibody reduced angiogenesis in a 3-dimensional in vitro sprouting assay. Administration of Notch1 peptibodies to neonate mice resulted in reduced radial outgrowth of retinal vasculature, confirming anti-angiogenic properties. We conclude that purified Notch peptibodies comprising EGF-like repeats 10-14 bind to both DLL4 and JAG1 ligands and exhibit anti-angiogenic properties. Based on their secretion profile, unique Notch inhibitory activities, and anti-angiogenic properties, Notch peptibodies present new opportunities for therapeutic Notch inhibition.

Topics: Angiogenesis Inhibitors; Animals; Endothelial Cells; Epidermal Growth Factor; Immunoprecipitation; Inflammation; Ligands; Mice; Neoplasms; Receptor, Notch1; Receptor, Notch4; Recombinant Fusion Proteins; Retinal Vessels; Surface Plasmon Resonance

Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene (Cbl) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. Here, we used stable isotope labeling with amino acids in cell culture mass spectrometry to compare Cbl complexes with or without epidermal growth factor (EGF) stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In PC9 and H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and endosomal trafficking. Furthermore, we determined that FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl dependent, as coknockdown of Cbl and Cbl-b restored EGFR levels. In addition, FLOT2 overexpression decreased EGFR signaling and growth. Overexpression of wildtype (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. Lastly, FLOT2 KO increased tumor formation and tumor volume in nude mice and NSG mice, respectively. Together, these data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferation in vitro and in vivo.

Topics: Animals; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; HEK293 Cells; HeLa Cells; Humans; Membrane Proteins; Mice; Mice, Nude; Neoplasms; Phosphorylation; Proteolysis; Proto-Oncogene Proteins c-cbl; Ubiquitination

Exogenous epidermal growth factor (EGF) causes apoptosis in EGF receptor (EGFR)-overexpressing cell lines. The apoptosis-inducing factors could be a therapeutic target. We aimed to determine the mechanism of EGF-induced apoptosis using a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-based knockout screen.. Two-vector system of the human genome-scale CRISPR knockout library v2 was used to target 19,050 genes using 123,411 single guide RNAs (sgRNAs). Recombinant human EGF (100 nM) or distilled water four times was administered to the experimental and control groups, respectively. The read counts of each sgRNA obtained from next-generation sequencing were analyzed using the edgeR algorithm. We used another EGFR-overexpressing cell line (A549) and short hairpin RNAs (shRNAs) targeting five EGF-resistance genes for validation. DUSP1 expression in A431, A549, and HEK293FT cells was calculated using reverse transcription-quantitative polymerase chain reaction.. We found 77 enriched and 189 depleted genes in the experimental group using the CRISPR-based knockout screen and identified the top five EGF-resistance genes: DDX20, LHFP, REPS1, DUSP1,<.i> and KRTAP10-12. Transfecting shRNAs targeting these genes into A549 cells significantly increased the surviving fractions after EGF treatment, compared with those observed in the control shRNA-transfected cells. The expression ratio of DUSP1 (inhibits ERK signaling) increased in A431 and A549 cells after EGF treatment. However, DUSP1 expression remained unchanged in HEK293FT cells after EGF treatment.. The CRISPR-based knockout screen revealed 266 genes possibly responsible for EGF-induced apoptosis. DUSP1 might be a critical component of EGF-induced apoptosis and a novel target for EGFR-overexpressing cancers.

Topics: Apoptosis; Calcium-Binding Proteins; CRISPR-Cas Systems; Early Detection of Cancer; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; RNA, Guide, CRISPR-Cas Systems

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) commonly targeted for inhibition by anticancer therapeutics. Current therapeutics target EGFR's kinase domain or extracellular region. However, these types of inhibitors are not specific for tumors over healthy tissue and therefore cause undesirable side effects. Our lab has recently developed a new strategy to regulate RTK activity by designing a peptide that specifically binds to the transmembrane (TM) region of the RTK to allosterically modify kinase activity. These peptides are acidity-responsive, allowing them to preferentially target acidic environments like tumors. We have applied this strategy to EGFR and created the PET1 peptide. We observed that PET1 behaves as a pH-responsive peptide that modulates the configuration of the EGFR TM through a direct interaction. Our data indicated that PET1 inhibits EGFR-mediated cell migration. Finally, we investigated the mechanism of inhibition through molecular dynamics simulations, which showed that PET1 sits between the two EGFR TM helices; this molecular mechanism was additionally supported by AlphaFold-Multimer predictions. We propose that the PET1-induced disruption of native TM interactions disturbs the conformation of the kinase domain in such a way that it inhibits EGFR's ability to send migratory cell signals. This study is a proof-of-concept that acidity-responsive membrane peptide ligands can be generally applied to RTKs. In addition, PET1 constitutes a viable approach to therapeutically target the TM of EGFR.

Topics: Allosteric Regulation; Antineoplastic Agents; Cell Membrane; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Humans; Hydrogen-Ion Concentration; Neoplasms; Peptides; Phosphorylation; Protein Domains; Protein Structure, Secondary; Receptor Protein-Tyrosine Kinases

The epidermal growth factor receptor (EGFR) is one of the main tumor drivers and is an important therapeutic target for many cancers. Calcium is important in EGFR signaling pathways. Sorcin is one of the most important calcium sensor proteins, overexpressed in many tumors, that promotes cell proliferation, migration, invasion, epithelial-to-mesenchymal transition, malignant progression and resistance to chemotherapeutic drugs. The present work elucidates a functional mechanism that links calcium homeostasis to EGFR signaling in cancer. Sorcin and EGFR expression are significantly correlated and associated with reduced overall survival in cancer patients. Mechanistically, Sorcin directly binds EGFR protein in a calcium-dependent fashion and regulates calcium (dys)homeostasis linked to EGF-dependent EGFR signaling. Moreover, Sorcin controls EGFR proteostasis and signaling and increases its phosphorylation, leading to increased EGF-dependent migration and invasion. Of note, silencing of Sorcin cooperates with EGFR inhibitors in the regulation of migration, highlighting calcium signaling pathway as an exploitable target to enhance the effectiveness of EGFR-targeting therapies.

Topics: Calcium; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Signal Transduction

CaMKK2-mediation of EGFR action may enable cancer cells to use intracellular calcium elevation as a signal for growth and survival.

Topics: Calcium; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Epidermal Growth Factor; ErbB Receptors; Neoplasms; Proto-Oncogene Proteins c-akt

The ErbB receptor family, including the epidermal growth factor receptor (EGFR) and ErbB2/3/4, regulate cell proliferation, differentiation, apoptosis, motility, etc., and their abnormalities can cause cancer and other diseases. Ligand-induced endocytosis of ErbB receptors is the key to various cancer treatment strategies, and different techniques have been developed to study this important process. Among them, single particle tracking (SPT) can reveal the spatiotemporal heterogeneity of ErbB receptors on the live cell membrane and has been used to characterize the EGFR dimerization process. Herein, we studied the endocytosis dynamics of two different ErbB receptors using dark-field microscopy. With anti-ErbB modified plasmonic gold nanorods (AuNRs) as probes, we compared the trajectories of individual anti-EGFR AuNRs (cAuNRs) and anti-ErbB AuNRs (tAuNRs) interacting with MCF-7 cells in situ in real time. The results revealed that the internalization rate of cAuNRs was faster than that of tAuNRs. Detailed SPT analysis suggests that cAuNRs enter cells through EGFR endocytosis pathway, and multiple intracellular transport modes sort the cAuNRs away from the transmembrane site. In contrast, the endocytosis resistance of ErbB2 slows down the cellular uptake rate of tAuNRs and causes some tAuNRs-ErbB2 complexes to be confined on the membrane with "circular" and "rolling circle" motions for a much longer time. Our results provide insights into the endocytosis process of the ErbB receptor family at the nanometer scale and could be potentially useful to develop cancer treatment strategies.

Topics: Endocytosis; Epidermal Growth Factor; Gold; Humans; Ligands; Neoplasms; Receptor, ErbB-2

Silver nanoparticles (AgNPs) are the most toxic nanostructures for both cancer and healthy cells. Thus, their usefulness in the anticancer therapy is limited. Interestingly, the epidermal growth factor receptor (EGFR) is overexpressed in many cancer cells, e.g. in lung and tongue cancers. Therefore, the aim of this study was to develop a way to direct the cytotoxic effect of AgNPs against cancer cells, saving healthy ones by entrapping these NPs inside liposomes labeled with the epidermal growth factor (EGF-LipoAgNPs) and directing these structures into EGFR-overexpressing cancer cells. The obtained results showed spherical structures with a 107.9 nm size and - 16.60 mV zeta-potential. The UV-Vis scan and TEM images did not show free AgNPs in the solution. The obtained complexes were able to decrease the metabolic activity in the A549 and SCC-15 cells more effectively than native AgNPs. Furthermore, the ROS production, lactate dehydrogenase release, and caspase-9 and -3 activity were significantly increased after the treatment with EGF-LipoAgNPs for 24 and 48 h. The expression of genes encoding catalase, superoxide dismutase, and p53 protein increased significantly, while the KI67 gene expression decreased, especially in the A549 and SCC-15 cells. Moreover, the KI67 protein expression was lower than in the cells treated with native AgNPs, while catalase activity was decreased significantly after the treatment with the obtained complexes. In turn, SOD activity increased more efficiently in the EGFR-overexpressing cancer cells. In all tested parameters, EGF-LipoAgNPs exerted a lower toxic effect on the BJ cells than native AgNPs. Summarizing, the created liposomal system reduces the toxicity of AgNPs against normal human fibroblasts and enhances the toxic and proapoptotic effect of these NPs, which may be caused by improvement of their uptake by EGFR-overexpressing cancer cells.

Topics: Catalase; Epidermal Growth Factor; ErbB Receptors; Humans; Ki-67 Antigen; Liposomes; Metal Nanoparticles; Neoplasms; Silver

Programmed death ligand 1 (PD-L1) is an immune checkpoint protein that suppresses cytotoxic T lymphocytes and is often overexpressed in cancers. Due to favorable clinical trial results, immune checkpoint inhibition (ICI) is part of Food and Drug Administration approved immuno-oncology therapies; however, not all patients benefit from ICI therapy. High blood platelet-to-lymphocyte ratio has been associated with failure of ICI treatment, but whether platelets have a role in hindering ICI response is unclear. Here, we report that coculturing platelets with cancer cell lines increased protein and gene expression of tumor cell PD-L1, which was reduced by antiplatelet agents, such as aspirin and ticagrelor. Platelet cytokine arrays revealed that the well-established cytokines, including interferon-γ, were not the main regulators of platelet-mediated PD-L1 upregulation. Instead, the high molecular weight epidermal growth factor (EGF) is abundant in platelets, which caused an upregulation of tumor cell PD-L1. Both an EGF-neutralizing antibody and cetuximab (EGF receptor [EGFR] monoclonal antibody) inhibited platelet-induced increases in tumor cell PD-L1, suggesting that platelets induce tumor cell PD-L1 in an EGFR-dependent manner. Our data reveal a novel mechanism for platelets in tumor immune escape and warrant further investigation to determine if targeting platelets improves ICI therapeutic responses.

Topics: Antibodies, Neutralizing; Aspirin; B7-H1 Antigen; Blood Platelets; Cetuximab; Epidermal Growth Factor; ErbB Receptors; Humans; Immune Checkpoint Inhibitors; Immune Checkpoint Proteins; Interferon-gamma; Neoplasms; Platelet Aggregation Inhibitors; Ticagrelor; United States

Human Cripto-1 is a member of the epidermal growth factor (EGF)-Cripto-FRL-1-Cryptic (CFC) family family and performs critical roles in cancer and various pathological and developmental processes. Recently we demonstrated that a soluble form of Cripto-1 suppresses the self-renewal and enhances the differentiation of cancer stem cells (CSCs). A functional form of soluble Cripto-1 was found to be difficult to obtain because of the 12 cysteine residues in the protein which impairs the folding process. Here, we optimized the protocol for a T7 expression system, purification from inclusion bodies under denatured conditions refolding of a His-tagged Cripto-1 protein. A concentrations of 0.2-0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37°C was found to be the optimal concentration for Cripto-1 expression while imidazole at 0.5 M was the optimum concentration to elute the Cripto-1 protein from a Ni-column in the smallest volume. Cation exchange column chromatography of the Cripto-1 protein in the presence of 8 M urea exhibited sufficient elution profile at pH 5, which was more efficient at recovery. The recovery of the protein reached to more than 26.6% after refolding with arginine. The purified Cripto-1 exhibited high affinity to the anti-ALK-4 antibody and suppressed sphere forming ability of CSCs at high dose and induced cell differentiation.

Topics: Cell Differentiation; Epidermal Growth Factor; GPI-Linked Proteins; Humans; Neoplasms; Neoplastic Stem Cells

The surface of silver nanoparticles (AgNPs) is characterized by high reactivity resulting in prooxidative and cytotoxic properties. These effects are observed both in normal and in cancer cells, which overexpress the Epidermal Growth Factor Receptor (EGFR). In our previous paper, we have demonstrated that, with the use of liposomes labeled with the Epidermal Growth Factor (EGF), it is possible to direct the toxic effect of AgNPs in EGFR-overexpressing cells. Unfortunately, the mechanism of uptake and toxicity induction by such liposomes is still unknown. Therefore, the aim of this study was to determine the impact of EGF-LipoAgNPs on certain genes related to endocytosis and toxicity induction by such liposomes in human lung (A549) and tongue (SCC-15) cancer cells. The siRNA knock-out gene method was used in this study to determine the engagement of EGFR in this process. The confocal microscopy study revealed that the number of liposomes in the cytoplasm of the A549

Topics: Cell Line, Tumor; Clathrin; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Humans; Liposomes; Metal Nanoparticles; Neoplasms; Silver

Topics: Epidermal Growth Factor; ErbB Receptors; Humans; Ligands; Neoplasms

Epidermal growth factor (EGF) is one of the most well-characterized growth factors and plays a crucial role in cell proliferation and differentiation. Its receptor EGFR has been extensively explored as a therapeutic target against multiple types of cancers, such as lung cancer and glioblastoma. Recent studies have established a connection between deregulated EGF signaling and metabolic reprogramming, especially rewiring in aerobic glycolysis, which is also known as the Warburg effect and recognized as a hallmark in cancer. Pyruvate kinase M2 (PKM2) is a rate-limiting enzyme controlling the final step of glycolysis and serves as a major regulator of the Warburg effect. We previously showed that PKM2 T405/S406 O-GlcNAcylation, a critical mark important for PKM2 detetramerization and activity, was markedly upregulated by EGF. However, the mechanism by which EGF regulates PKM2 O-GlcNAcylation still remains uncharacterized. Here, we demonstrated that EGF promoted O-GlcNAc transferase (OGT) binding to PKM2 by stimulating OGT Y976 phosphorylation. As a consequence, we found PKM2 O-GlcNAcylation and detetramerization were upregulated, leading to a significant decrease in PKM2 activity. Moreover, distinct from PKM2, we observed that the association of additional phosphotyrosine-binding proteins with OGT was also enhanced when Y976 was phosphorylated. These proteins included STAT1, STAT3, STAT5, PKCδ, and p85, which are reported to be O-GlcNAcylated. Together, we show EGF-dependent Y976 phosphorylation is critical for OGT-PKM2 interaction and propose that this posttranslational modification might be important for substrate selection by OGT.

Topics: Epidermal Growth Factor; ErbB Receptors; Humans; N-Acetylglucosaminyltransferases; Neoplasms; Phosphorylation; Phosphotyrosine; Pyruvate Kinase; STAT5 Transcription Factor; Tyrosine

Epidermal growth factor receptor (EGFR) nuclear translocation correlates with the abnormal proliferation, migration, and anti-apoptosis of tumor cells. Monitoring EGFR nuclear translocation provides insights into the molecular mechanisms underlying cancers. EGFR nuclear translocation includes two processes, EGFR phosphorylation and phosphorylated EGFR translocation to the nucleus. With the help of aptamers, probes that can achieve the first step of anchoring phosphorylated EGFR have been developed. However, the EGFR nuclear translocation can last for hours, posing a challenge to monitor the entire nuclear translocation in living cells. Herein, we designed a circular bivalent aptamer-functionalized optical probe with greatly enhanced stability for long-term visualization of EGFR nuclear translocation in situ. The results of cell experiments show that the probe could monitor the entire nuclear translocation of EGFR. The findings of tissue and

Topics: Cell Line, Tumor; Cell Nucleus; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Oligonucleotides; Phosphorylation; Protein Transport

Neuregulins, members of the largest subclass of growth factors of the epidermal growth factor family, mediate a myriad of cellular functions including survival, proliferation, and differentiation in normal tissues through binding to receptor tyrosine kinases of the ErbB family. However, aberrant neuregulin signaling in the tumor microenvironment is increasingly recognized as a key player in initiation and malignant progression of human cancers. In this chapter, we focus on the role of neuregulin signaling in the hallmarks of cancer, including cancer initiation and development, metastasis, as well as therapeutic resistance. Moreover, role of neuregulin signaling in the regulation of tumor microenvironment and targeting of neuregulin signaling in cancer from the therapeutic perspective are also briefly discussed.

Topics: Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Neuregulins; Signal Transduction; Tumor Microenvironment

Local basement membrane (BM) disruption marks the initial step of breast cancer invasion. The activation mechanisms of force-driven BM-weakening remain elusive. We studied the mechanical response of MCF10A-derived human breast cell acini with BMs of tuneable maturation to physical and soluble tumour-like extracellular matrix (ECM) cues. Traction force microscopy (TFM) and elastic resonator interference stress microscopy (ERISM) were used to quantify pro-invasive BM stress and protrusive forces. Substrate stiffening and mechanically impaired BM scaffolds induced the invasive transition of benign acini synergistically. Robust BM scaffolds attenuated this invasive response. Additional oncogenic EGFR activation compromised the BMs' barrier function, fuelling invasion speed and incidence. Mechanistically, EGFR-PI3-Kinase downstream signalling modulated both MMP- and force-driven BM-weakening processes. We show that breast acini form non-proteolytic and BM-piercing filopodia for continuous matrix mechanosensation, which significantly push and pull on the BM and ECM under pro-invasive conditions. Invasion-triggered acini further shear and compress their BM by contractility-based stresses that were significantly increased (3.7-fold) compared to non-invasive conditions. Overall, the highest amplitudes of protrusive and contractile forces accompanied the highest invasiveness. This work provides a mechanistic concept for tumour ECM-induced mechanically misbalanced breast glands fuelling force-driven BM disruption. Finally, this could facilitate early cell dissemination from pre-invasive lesions to metastasize eventually.

Topics: Acinar Cells; Basement Membrane; Breast; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Extracellular Matrix; Female; Humans; Mammary Glands, Human; Mechanical Phenomena; Neoplasm Invasiveness; Neoplasms; Pseudopodia

Human ELTD1 (Epidermal growth factor, latrophilin and seven-transmembrane domain-containing 1), an orphan G-protein-coupled receptor (GPCR) belonging to the adhesion GPCR family, has been reported as a novel regulator of angiogenesis and a potential anti-cancer therapeutic target. However, little is known about the function of ELTD1, especially its undiscovered ligands. In this experiment, an ELTD1 homozygous knockout human embryonic stem cell line, FAHZUe001-A, was generated by the iCRISPR/Cas9 system to achieve a deeper understanding of ELTD1. The FAHZUe001-A was confirmed with normal karyotype, typical undifferentiated morphology, pluripotency and trilineage differentiation potential in vitro.

Topics: CRISPR-Cas Systems; Epidermal Growth Factor; Human Embryonic Stem Cells; Humans; Neoplasms; Neovascularization, Pathologic

The enhanced productive folding of translated polypeptides by heat shock protein 70 (HSP70) is often required for the survival of cancer cells. Although the folding activity of HSP70 is considered a significant determinant of the progression of cancer cells, it is still unknown how this activity could be regulated. Here, we report that the phosphorylation of HSP70 facilitates its folding activity, enhancing cell proliferation. Mass spectrometry identified the serine residues at positions 385 and 400 in the linker and substrate-binding domains of HSP70, respectively, as sites of phosphorylation mediated by EGF signaling, and this result was further confirmed by site-directed mutagenesis. ERK is known to be a specific kinase. The phosphorylation of the two sites induces the extended conformation of HSP70 via the regulation of the binding of the linker to the nucleotide- and substrate-binding domains, augmenting the binding affinity of HSP70 to substrates and enhancing its folding activity; this ultimately results in pro-proliferative effects. Cell lines harboring activated ERK showed increased phosphorylation of HSP70, and a positive correlation between the phosphorylation of HSP70 and the activity of ERK was observed. Thus, this study demonstrated that the ERK-dependent phosphorylation of HSP70 facilitated its folding activity and cellular proliferative function.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Heterografts; HSP70 Heat-Shock Proteins; Humans; MAP Kinase Signaling System; Mice; Molecular Chaperones; Mutagenesis, Site-Directed; Neoplasms; Phosphorylation; Protein Folding

Type III epithelial-mesenchymal transition (EMT) has been previously associated with increased cell migration, invasion, metastasis, and therefore cancer aggressiveness. This reversible process is associated with an important gene expression reprogramming mainly due to epigenetic plasticity. Nevertheless, most of the studies describing the central role of epigenetic modifications during EMT were performed in a single-cell model and using only one mode of EMT induction. In our study, we studied the overall modulations of gene expression and epigenetic modifications in four different EMT-induced cell models issued from different tissues and using different inducers of EMT. Pangenomic analysis (transcriptome and ChIP-sequencing) validated our hypothesis that gene expression reprogramming during EMT is largely regulated by epigenetic modifications of a wide range of genes. Indeed, our results confirmed that each EMT model is unique and can be associated with a specific transcriptome profile and epigenetic program. However, we could select some genes or pathways that are similarly regulated in the different models and that could therefore be used as a common signature of all EMT models and become new biomarkers of the EMT phenotype. As an example, we can cite the regulation of gene-coding proteins involved in the degradation of the extracellular matrix (ECM), which are highly induced in all EMT models. Based on our investigations and results, we identified ADAM19 as a new biomarker of in vitro and in vivo EMT and we validated this biological new marker in a cohort of non-small lung carcinomas.

Topics: A549 Cells; Epidermal Growth Factor; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; Retrospective Studies; Tumor Necrosis Factor-alpha

Targeting of specific tissues and cells by viruses is one of the challenges faced by researchers. Lentiviral vectors (LVs) are one of the most promising gene delivery systems in cancer gene therapy. Therefore, we aimed to design a novel lentiviral delivery system that expresses anti- human epidermal growth factor 2 (HER2) designed anykrin repeat protein (DARPin) on the vector envelope to create a pseudotyped lentivirus for targeting HER2-positive cancer cells.. A helper plasmid producing the viral vector envelope containing anti-HER2 DARPin-G3 was constructed. LV was produced by transfer vector containing green fluorescent protein (GFP) gene and helper plasmids in human embryonic kidney 293 cells. The human breast cancer cell lines SKBR3 (normal and with inhibited endocytosis) (HER2-positive) and MDA-MB-231 (HER2-negative) were transduced by the recombinant viral vector. The GFP-based transduction rate was determined by flow cytometry and fluorescence microscopy.. The anti-HER2 DARPin concentration in DARPin-LVs was significantly higher than the envelope G glycoprotein of the vesicular stomatitis virus-LVs (non-anti-HER2 control) (p < 0.0001). In flow cytometry assays, the percentage of transduction by recombinant LV was significantly higher in SKBR3 cells than in SKBR3 cells with inhibited endocytosis (p = 0.0074) and MDA-MB-231 cells (p = 0.0037). In fluorescence microscopy assays, the percentage of transduction by new LV was significantly higher in SKBR3 cells than in SKBR3 cells with inhibited endocytosis (p = 0.0026) and MDA-MB-231 cells (p = 0.0014).. We constructed a new recombinant LV with a defect in cell entry directly, containing an anti-HER2 DARPin on the vector envelope with specific tropism to HER2 receptor on HER2-positive cancer cells. We assumed that this viral vector transduces cells via an endocytosis-dependent process.

Topics: Cell Line, Tumor; Epidermal Growth Factor; Gene Expression; Gene Transfer Techniques; Genes, Reporter; Genetic Engineering; Genetic Vectors; Host Specificity; Humans; Lentivirus; Neoplasms; Transduction, Genetic; Transgenes

Tight regulation of signaling from receptor tyrosine kinases is required for normal cellular functions and uncontrolled signaling can lead to cancer. Fibroblast growth factor receptor 2 (FGFR2) is a receptor tyrosine kinase that induces proliferation and migration. Deregulation of FGFR2 contributes to tumor progression and activating mutations in FGFR2 are found in several types of cancer. Here, we identified a negative feedback loop regulating FGFR2 signaling. FGFR2 stimulates the Ras/MAPK signaling pathway consisting of Ras-Raf-MEK1/2-ERK1/2. Inhibition of this pathway using a MEK1/2 inhibitor increased FGFR2 signaling. The putative ERK1/2 phosphorylation site at serine 780 (S780) in FGFR2 corresponds to serine 777 in FGFR1 which is directly phosphorylated by ERK1/2. Substitution of S780 in FGFR2 to an alanine also increased signaling. Truncated forms of FGFR2 lacking the C-terminal tail, including S780, have been identified in cancer and S780 has been found mutated to leucine in bladder cancer. Substituting S780 in FGFR2 with leucine increased FGFR2 signaling. Importantly, cells expressing these mutated versions of S780 migrated faster than cells expressing wild-type FGFR2. Thus, ERK1/2-mediated phosphorylation of S780 in FGFR2 constitutes a negative feedback loop and inactivation of this feedback loop in cancer cells causes hyperactivation of FGFR2 signaling, which may result in increased invasive properties.

Topics: Amino Acid Sequence; Cell Line, Tumor; Disease Progression; Epidermal Growth Factor; Feedback, Physiological; Fibroblast Growth Factor 1; Humans; MAP Kinase Signaling System; Mutation; Neoplasms; Phosphorylation; Receptor, Fibroblast Growth Factor, Type 2; Serine; Signal Transduction

Extremely low numbers of active epidermal growth factor receptors are sufficient to drive tumor growth.

Topics: Endocytosis; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Phosphorylation; Ubiquitination

To assess changes in metabolic risk factors and cancer-related growth factors associated with short-term abstinence from alcohol.. Prospective, observational study.. Single tertiary centre.. Healthy subjects were recruited based on intention to: (1) abstain from alcohol for 1 month (abstinence group), or (2) continue to drink alcohol (control group). Inclusion criteria were baseline alcohol consumption >64 g/week (men) or >48 g/week (women). Exclusion criteria were known liver disease or alcohol dependence.. The primary outcome was change in insulin resistance (homeostatic model assessment (HOMA) score). Secondary outcomes were changes in weight, blood pressure (BP), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and liver function tests. Primary and secondary outcomes were adjusted for changes in diet, exercise and cigarette smoking.. The abstinence group comprised 94 participants (mean age 45.5 years, SD ±1.2) and the control group 47 participants (mean age 48.7 years, SD ±1.8). Baseline alcohol consumption in the abstinence group was 258.2 g/week, SD ±9.4, and in the control group 233.8 g, SD ±19.0. Significant reductions from baseline in the abstinence group (all p<0.001) were found in: HOMA score (-25.9%, IQR -48.6% to +0.3%), systolic BP (-6.6%, IQR -11.8% to 0.0%), diastolic BP (-6.3%, IQR -14.1% to +1.3%), weight (-1.5%, IQR -2.9% to -0.4%), VEGF (-41.8%, IQR -64.9% to -17.9%) and EGF (-73.9%, IQR -86.1% to -36.4%). None of these changes were associated with changes in diet, exercise or cigarette smoking. No significant changes from baseline in primary or secondary outcomes were noted in the control group.. These findings demonstrate that abstinence from alcohol in moderate-heavy drinkers improves insulin resistance, weight, BP and cancer-related growth factors. These data support an independent association of alcohol consumption with cancer risk, and suggest an increased risk of metabolic diseases such as type 2 diabetes and fatty liver disease.

Topics: Adult; Alcohol Drinking; Alcoholism; Blood Pressure; Body Weight; Cardiovascular Diseases; Diabetes Mellitus, Type 2; Epidermal Growth Factor; Ethanol; Fatty Liver; Female; Humans; Insulin Resistance; Liver; Liver Function Tests; Male; Middle Aged; Neoplasms; Prospective Studies; Risk Factors; Vascular Endothelial Growth Factor A

The epidermal growth factor (EGF) receptor (EGFR) overexpressed in many cancers, including lung and head and neck cancers, and is involved in cancer cell progression and survival. PD-L1, increases in tumor cells to evade and inhibit CD8+ T cells, is a clinical immunotherapeutic target. This study investigated the molecular mechanism of EGF on regulating PD-L1 in EGFR-positive cancers and determined potential agents to reduce PD-L1 expression. RNA sequencing (RNAseq) and bioinformatics analysis were performed to determine potential driver genes that regulate PD-L1 in tumor cells-derived tumorspheres which mimicking cancer stem cells. Then, the specific inhibitors targeting EGFR were applied to reduce the expression of PD-L1 in vitro and in vivo. We validated that EGF could induce PD-L1 expression in the selected EGFR-positive cancers. RNAseq results revealed that STAT1 increased as a driver gene in KOSC-3-derived tumorspheres; these data were analyzed using PANTHER followed by NetworkAnalyst. The blockade of EGFR by afatinib resulted in decreased STAT1 and IRF-1 levels, both are transcriptional factors of PD-L1, and disabled the IFNr-STAT1-mediated PD-L1 axis in vitro and in vivo. Moreover, STAT1 knockdown significantly reduced EGF-mediated PD-L1 expression, and ruxolitinib, a JAK1/JAK2 inhibitor, significantly inhibited STAT1 phosphorylation to reduce the IFNr-mediated PD-L1 axis. These results indicate that EGF exacerbates PD-L1 by increasing the protein levels of STAT1 to enforce the IFNr-JAK1/2-mediated signaling axis in selected EGFR-positive cancers. The inhibition of EGFR by afatinib significantly reduced PD-L1 and may be a potential strategy for enhancing immunotherapeutic efficacy.

Topics: Afatinib; Animals; B7-H1 Antigen; Biomarkers; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Immunophenotyping; Interferon-gamma; Male; Mice; Neoplasms; Protein Kinase Inhibitors; STAT1 Transcription Factor

Inhibition of epidermal growth factor receptor (EGFR) signaling by small molecule kinase inhibitors and monoclonal antibodies has proven effective in the treatment of multiple cancers. In contrast, metastatic breast cancers (BC) derived from EGFR-expressing mammary tumors are inherently resistant to EGFR-targeted therapies. Mechanisms that contribute to this inherent resistance remain poorly defined. Here, we show that in contrast to primary tumors, ligand-mediated activation of EGFR in metastatic BC is dominated by STAT1 signaling. This change in downstream signaling leads to apoptosis and growth inhibition in response to epidermal growth factor (EGF) in metastatic BC cells. Mechanistically, these changes in downstream signaling result from an increase in the internalized pool of EGFR in metastatic cells, increasing physical access to the nuclear pool of STAT1. Along these lines, an EGFR mutant that is defective in endocytosis is unable to elicit STAT1 phosphorylation and apoptosis. Additionally, inhibition of endosomal signaling using an EGFR inhibitor linked to a nuclear localization signal specifically prevents EGF-induced STAT1 phosphorylation and cell death, without affecting EGFR:ERK1/2 signaling. Pharmacologic blockade of ERK1/2 signaling through the use of the allosteric MEK1/2 inhibitor, trametinib, dramatically biases downstream EGFR signaling toward a STAT1-dominated event, resulting in enhanced EGF-induced apoptosis in metastatic BC cells. Importantly, combined administration of trametinib and EGF also facilitated an apoptotic switch in EGFR-transformed primary tumor cells, but not normal mammary epithelial cells. These studies reveal a fundamental distinction for EGFR function in metastatic BC. Furthermore, the data demonstrate that pharmacological biasing of EGFR signaling toward STAT1 activation is capable of revealing the apoptotic function of this critical pathway.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Ligands; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Signaling System; Mice; Neoplasms; Phosphorylation; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Signal Transduction; STAT1 Transcription Factor

Lysyl oxidase (LOX) remodels the tumour microenvironment by cross-linking the extracellular matrix. LOX overexpression is associated with poor cancer outcomes. Here, we find that LOX regulates the epidermal growth factor receptor (EGFR) to drive tumour progression. We show that LOX regulates EGFR by suppressing TGFβ1 signalling through the secreted protease HTRA1. This increases the expression of Matrilin2 (MATN2), an EGF-like domain-containing protein that traps EGFR at the cell surface to facilitate its activation by EGF. We describe a pharmacological inhibitor of LOX, CCT365623, which disrupts EGFR cell surface retention and delays the growth of primary and metastatic tumour cells in vivo. Thus, we show that LOX regulates EGFR cell surface retention to drive tumour progression, and we validate the therapeutic potential of inhibiting this pathway with the small molecule inhibitor CCT365623.

Topics: Aminopropionitrile; Animals; Biosensing Techniques; Cell Line, Tumor; Cell Membrane; Cell Proliferation; Disease Progression; Dogs; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; High-Temperature Requirement A Serine Peptidase 1; Humans; Matrilin Proteins; Mice; Models, Biological; Neoplasm Metastasis; Neoplasms; Protein-Lysine 6-Oxidase; Rats; Signal Transduction; Transforming Growth Factor beta1

The fight against cancer is hindered by its highly heterogeneous nature. Genome-wide sequencing studies have shown that individual malignancies contain many mutations that range from those commonly found in tumor genomes to rare somatic variants present only in a small fraction of lesions. Such rare somatic variants dominate the landscape of genomic mutations in cancer, yet efforts to correlate somatic mutations found in one or few individuals with functional roles have been largely unsuccessful. Traditional methods for identifying somatic variants that drive cancer are 'gene-centric' in that they consider only somatic variants within a particular gene and make no comparison to other similar genes in the same family that may play a similar role in cancer. In this work, we present oncodomain hotspots, a new 'domain-centric' method for identifying clusters of somatic mutations across entire gene families using protein domain models. Our analysis confirms that our approach creates a framework for leveraging structural and functional information encapsulated by protein domains into the analysis of somatic variants in cancer, enabling the assessment of even rare somatic variants by comparison to similar genes. Our results reveal a vast landscape of somatic variants that act at the level of domain families altering pathways known to be involved with cancer such as protein phosphorylation, signaling, gene regulation, and cell metabolism. Due to oncodomain hotspots' unique ability to assess rare variants, we expect our method to become an important tool for the analysis of sequenced tumor genomes, complementing existing methods.

Topics: Computational Biology; Databases, Protein; Epidermal Growth Factor; Humans; Mitochondrial Proteins; Models, Molecular; Mutation; Neoplasms; Oncogene Proteins; Protein Binding; Protein Domains; ras Proteins

Epidermal growth factor receptor (EGFR) is an important regulator of epithelial cell growth and survival in normal and cancerous tissues and is a principal therapeutic target for cancer treatment. EGFR is associated in epithelial cells with the heavily glycosylated transmembrane mucin protein MUC1, a natural ligand of galectin-3 that is overexpressed in cancer. This study reveals that the expression of cell surface MUC1 is a critical enhancer of EGF-induced EGFR activation in human breast and colon cancer cells. Both the MUC1 extracellular and intracellular domains are involved in EGFR activation but the predominant influence comes from its extracellular domain. Binding of galectin-3 to the MUC1 extracellular domain induces MUC1 cell surface polarization and increases MUC1-EGFR association. This leads to a rapid increase of EGFR homo-/hetero-dimerization and subsequently increased, and also prolonged, EGFR activation and signalling. This effect requires both the galectin-3 C-terminal carbohydrate recognition domain and its N-terminal ligand multi-merization domain. Thus, interaction of galectin-3 with MUC1 on cell surface promotes EGFR dimerization and activation in epithelial cancer cells. As MUC1 and galectin-3 are both commonly overexpressed in most types of epithelial cancers, their interaction and impact on EGFR activation likely makes important contribution to EGFR-associated tumorigenesis and cancer progression and may also influence the effectiveness of EGFR-targeted cancer therapy.

Topics: Blood Proteins; Cell Line, Tumor; Cell Membrane; Endocytosis; Enzyme Activation; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Galectin 3; Galectins; Humans; Lapatinib; MAP Kinase Signaling System; Mucin-1; Mutation; Neoplasms; Protein Binding; Protein Domains; Protein Kinase Inhibitors; Protein Multimerization; Quinazolines

Previous studies have indicated that type II cyclic guanosine monophosphate (cGMP)‑dependent protein kinase (PKG II) could inhibit the proliferation and migration of gastric cancer cells. However, the effects of PKG II on the biological functions of other types of cancer cells remain to be elucidated. Therefore, the aim of the present study was to investigate the effects of PKG II on cancer cells derived from various types of human tissues, including A549 lung, HepG2 hepatic, OS‑RC‑2 renal, SW480 colon cancer cells and U251 glioma cells. Cancer cells were infected with adenoviral constructs coding PKG II (Ad‑PKG II) to up‑regulate PKG II expression, and treated with 8‑(4‑chlorophenylthio) (8‑pCPT)‑cGMP to activate the kinase. A Cell Counting kit 8 assay was used to detect cell proliferation. Cell migration was measured using a Transwell assay, whereas a terminal deoxynucleotidyl transferase 2'‑deoxyuridine, 5'‑triphosphate nick‑end labeling assay was used to detect cell apoptosis. A pull‑down assay was used to investigate the activation of Ras‑related C3 botulinum toxin substrate (Rac) 1 and western blotting was used to detect the expression of proteins of interest. The present results demonstrated that EGF (100 ng/ml, 24 h) promoted the proliferation and migration of cancer cells, and it suppressed their apoptosis. In addition, treatment with EGF enhanced the activation of Rac1, and up‑regulated the protein expression of proliferating cell nuclear antigen, matrix metalloproteinase (MMP)2, MMP7 and B‑cell lymphoma (Bcl)‑2, whereas it down‑regulated the expression of Bcl‑2‑associated X protein. Transfection of cancer cells with Ad‑PKG II, and PKG II activation with 8‑pCPT‑cGMP, was identified to counteract the effects triggered by EGF. The present results suggested that PKG II may exert inhibitory effects on the proliferation and migration of various types of cancer cells.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclic GMP-Dependent Protein Kinase Type II; Epidermal Growth Factor; Gene Expression; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 7; Neoplasms; rac1 GTP-Binding Protein; Signal Transduction

Tumor invasion through a basement membrane is one of the earliest steps in metastasis, and growth factors, such as Epidermal Growth Factor (EGF) and Hepatocyte Growth Factor (HGF), stimulate this process in a majority of solid tumors. Basement membrane breakdown is one of the hallmarks of invasion; therefore, tumor cells secrete a variety of proteases to aid in this process, including lysosomal proteases. Previous studies demonstrated that peripheral lysosome distribution coincides with the release of lysosomal cathepsins.. Immunofluorescence microscopy, western blot, and 2D and 3D cell culture techniques were performed to evaluate the effects of EGF on lysosome trafficking and cell motility and invasion.. EGF-mediated lysosome trafficking, protease secretion, and invasion is regulated by the activity of p38 mitogen activated protein kinase (MAPK) and sodium hydrogen exchangers (NHEs). Interestingly, EGF stimulates anterograde lysosome trafficking through a different mechanism than previously reported for HGF, suggesting that there are redundant signaling pathways that control lysosome positioning and trafficking in tumor cells.. These data suggest that EGF stimulation induces peripheral (anterograde) lysosome trafficking, which is critical for EGF-mediated invasion and protease release, through the activation of p38 MAPK and NHEs. Taken together, this report demonstrates that anterograde lysosome trafficking is necessary for EGF-mediated tumor invasion and begins to characterize the molecular mechanisms required for EGF-stimulated lysosome trafficking.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Humans; Lysosomes; Microscopy, Fluorescence; Neoplasm Invasiveness; Neoplasms; p38 Mitogen-Activated Protein Kinases; Protein Transport; Signal Transduction; Sodium-Hydrogen Exchangers

Epidermal growth factor (EGF) activates the EGF receptor (EGFR) and stimulates its internalization and trafficking to lysosomes for degradation. However, a percentage of EGFR undergoes ligand-independent endocytosis and is rapidly recycled back to the plasma membrane. Importantly, alterations in EGFR recycling are a common hallmark of cancer, and yet, our understanding of the machineries controlling the fate of endocytosed EGFR is incomplete. Intersectin-s is a multi-domain adaptor protein that is required for internalization of EGFR Here, we discover that intersectin-s binds DENND2B, a guanine nucleotide exchange factor for the exocytic GTPase Rab13, and this interaction promotes recycling of ligand-free EGFR to the cell surface. Intriguingly, upon EGF treatment, DENND2B is phosphorylated by protein kinase D and dissociates from intersectin-s, allowing for receptor targeting to degradation. Our study thus reveals a novel mechanism controlling the fate of internalized EGFR with important implications for cancer.

Topics: Adaptor Proteins, Vesicular Transport; Cell Membrane; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Guanine Nucleotide Exchange Factors; HEK293 Cells; Humans; Neoplasms; Phosphorylation; Protein Binding; Protein Kinase C; Protein Transport; rab GTP-Binding Proteins; Signal Transduction; Tumor Suppressor Proteins

Huntington-interacting protein 1 (HIP1) is associated with various tumor types; however, its precise functions in tumor cells are unclear. In this study, the effects of HIP1 on the degradation of EGFR, which have important roles in carcinogenesis after EGF stimulation, were examined. After screening 17 cell lines, the coexpression of HIP1 and EGFR was detected in HeLa cells. Accordingly, the expression of HIP1 was knocked down in HeLa cells using various HIP1 siRNA sequences. The endocytosis of EGFR and localization of clathrin in HeLa cells were examined after stimulation by EGF at various concentrations (i.e., 1.5 and 100 ng/ml). After HIP1 expression was blocked by siRNAs, EGFR endocytosis was accelerated and this effect was dependent on the EGF concentration. This endocytosis was colocalized with clathrin expression. These findings indicate that the inhibition of HIP1 can accelerate the endocytosis and degradation of EGFR. Furthermore, they suggest that HIP1 is a potential therapeutic target for various cancer types, particularly those with high EGFR expression, but further research is needed to examine this hypothesis.

Topics: DNA-Binding Proteins; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; HeLa Cells; Humans; Ligands; Neoplasms; RNA, Small Interfering

Necitumumab is a second-generation, recombinant, human immunoglobulin G1, epidermal growth factor (EGFR) receptor antibody that specifically blocks the ligand binding site of EGFR. Necitumumab potentially acts by blocking ligand epidermal growth factor (EGF) binding-mediated activation of the EGFR signaling pathway, inhibiting tumor growth, angiogenesis, and anti-apoptotic mechanisms. Necitumumab inhibited the interaction of EGF and EGFR with a concentration that inhibits binding by 50 % of approximately 0.9 nM (0.13 mg/L) and demonstrated antitumor activity during in vivo experiments associated with trough plasma concentrations of approximately 40 mg/L. This work describes the population pharmacokinetics of necitumumab in cancer patients when administered with or without concomitant chemotherapy and evaluates patient characteristics that may guide dosing. Nonlinear mixed-effects modeling of serum concentration data across five clinical studies (phases I-III) indicated that necitumumab exhibited target-mediated drug disposition, commonly observed with monoclonal antibodies, and that pharmacokinetics were expected to be linear in the studied dose ranges when administered as repeated infusions. No age, sex, race, or concomitant medication factors were found influential, while weight was a statistically significant factor for both distribution and elimination. Simulations from the final model indicated that only a limited reduction in patient drug exposure variability would be achieved by weight- or body surface area-based dosing. Necitumumab effective half-life was estimated to approximately 2 weeks, and steady state was achieved within three to four cycles of treatment. The phase III dosing schedule of 800 mg dosed on days 1 and 8 of a 21-day schedule resulted in serum concentrations that exceeded the 40-mg/L threshold indicated by preclinical experiments.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunoglobulin G; Male; Middle Aged; Models, Biological; Neoplasms; Protein Binding; Young Adult

Autophagy, the mechanism by which cells deliver material to the lysosome, has been associated with resistance to anticancer drugs, leading autophagy inhibition to be widely studied as a potential chemosensitization strategy for cancer cells. This strategy is based on the idea that inhibition of autophagy will increase drug sensitivity and kill more cancer cells. Here we report an unintended negative effect of this strategy. When modeling the effect of drug resistance in a heterogeneous cancer cell population, we found that autophagy inhibition in drug-sensitive tumor cells causes increased growth of drug-resistant cells in the population through a mechanism involving caspase activation and prostaglandin E

Topics: Animals; Autophagy; Autophagy-Related Proteins; Cell Line, Tumor; Cell Proliferation; Diphtheria Toxin; Drug Resistance, Neoplasm; Epidermal Growth Factor; Gene Knockdown Techniques; Green Fluorescent Proteins; Humans; Mice; Neoplasms; Recombinant Fusion Proteins; RNA, Small Interfering; TNF-Related Apoptosis-Inducing Ligand

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF.

Topics: Animals; Antibodies, Monoclonal; Cell Line, Tumor; Epidermal Growth Factor; Epitope Mapping; ErbB Receptors; Heparin-binding EGF-like Growth Factor; Humans; Immunohistochemistry; Mice; Neoplasms; Paraffin

The tumor suppressor p53 functions by inducing the transcription of a collection of target genes. We previously attempted to identify p53 target genes by microarray expression and ChIP-sequencing analyses. In this study, we describe a novel p53 target gene, FUCA1, which encodes a fucosidase. Although fucosidase, α-l-1 (FUCA1) has been reported to be a lysosomal protein, we detected it outside of lysosomes and observed that its activity is highest at physiological pH. As there is a reported association between fucosylation and tumorigenesis, we investigated the potential role of FUCA1 in cancer. We found that overexpression of FUCA1, but not a mutant defective in enzyme activity, suppressed the growth of cancer cells and induced cell death. Furthermore, we showed that FUCA1 reduced fucosylation and activation of epidermal growth factor receptor, and concomitantly suppressed epidermal growth factor signaling pathways. FUCA1 loss-of-function mutations are found in several cancers, its expression is reduced in cancers of the large intestine, and low FUCA1 expression is associated with poorer prognosis in several cancers. These results show that protein defucosylation mediated by FUCA1 is involved in tumor suppression.

Topics: alpha-L-Fucosidase; Cell Death; Cell Division; Cell Line; Cell Survival; Epidermal Growth Factor; ErbB Receptors; Fucose; Humans; Mutant Proteins; Neoplasms; Signal Transduction; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

As targeted cancer therapies and molecular profiling become widespread, the era of "precision oncology" is at hand. However, cancer genomes are complex, making mutation-specific outcomes difficult to track. We created a proof-of-principle, CUSTOM-SEQ: Continuously Updating System for Tracking Outcome by Mutation, to Support Evidence-based Querying, to automatically calculate and display mutation-specific survival statistics from electronic health record data.. Patients with cancer genotyping were included, and clinical data was extracted through a variety of algorithms. Results were refreshed regularly and injected into a standard reporting platform. Significant results were highlighted for visual cueing. A subset was additionally stratified by stage, smoking status, and treatment exposure.. By August 2015, 4310 patients with a median follow-up of 17 months had sufficient data for survival calculation. As expected, epidermal growth factor receptor (EGFR) mutations in lung cancer were associated with superior overall survival, hazard ratio (HR) = 0.53 (P < .001), validating the approach. Guanine nucleotide binding protein (G protein), q polypeptide (GNAQ) mutations in melanoma were associated with inferior overall survival, a novel finding (HR = 3.42, P < .001). Smoking status was not prognostic for epidermal growth factor receptor-mutated lung cancer patients, who also lived significantly longer than their counterparts, even with advanced disease (HR = 0.54, P = .001).. CUSTOM-SEQ represents a novel rapid learning system for a precision oncology environment. Retrospective studies are often limited by study of specific time periods and can lead to incomplete conclusions. Because data is continuously updated in CUSTOM-SEQ, the evidence base is constantly growing. Future work will allow users to interactively explore populations by demographics and treatment exposure, in order to further investigate significant mutation-specific signals.

Topics: Algorithms; Cohort Studies; Computational Biology; DNA, Neoplasm; Electronic Health Records; Epidermal Growth Factor; Follow-Up Studies; Genotype; Humans; Information Storage and Retrieval; Kaplan-Meier Estimate; Lung Neoplasms; Mutation; Neoplasms; Precision Medicine; Proportional Hazards Models; Tobacco Smoking

The Rb tumor suppressor is conserved in Drosophila, and its inactivation can lead to cell proliferation or death depending on the specific cellular context. Therefore, identifying genes that affect the survival of Rb-mutant cells can potentially identify novel targets for therapeutic intervention in cancer. From a genetic screen in Drosophila, we identified synthetic lethal interactions between mutations of fly Rb (rbf) and the ESCRT-0 components stam and hrs We show that inactivation of ESCRT-0 sensitizes rbf-mutant cells to undergo apoptosis through inhibition of EGFR signaling and accumulation of Hid protein. Mutation of stam inhibits EGFR signaling upstream of secreted Spi and downstream of Rhomboid expression, and causes Rhomboid protein to accumulate in the abnormal endosomes labeled with both the early and late endosomal markers Rab5 and Rab7. These results reveal that ESCRT-0 mutants inhibit EGFR signaling by disrupting Rhomboid endosomal trafficking in the ligand-producing cells. Because ESCRT-0 also plays crucial roles in EGFR downregulation after ligand binding, this study provides new insights into how loss of ESCRT-0 function can either increase or decrease EGFR signaling.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Animals; Apoptosis; Cell Proliferation; Cell Survival; Drosophila; Drosophila Proteins; Endosomal Sorting Complexes Required for Transport; Endosomes; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Membrane Proteins; Mutation; Neoplasms; Phosphoproteins; Receptors, Invertebrate Peptide; Signal Transduction

The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, β-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and β-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and β-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells.

Topics: Active Transport, Cell Nucleus; Betacellulin; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Heparin-binding EGF-like Growth Factor; Humans; Neoplasms; Phosphorylation; Transforming Growth Factor alpha

Mitochondrial acetyl-CoA acetyltransferase 1 (ACAT1) regulates pyruvate dehydrogenase complex (PDC) by acetylating pyruvate dehydrogenase (PDH) and PDH phosphatase. How ACAT1 is "hijacked" to contribute to the Warburg effect in human cancer remains unclear. We found that active, tetrameric ACAT1 is commonly upregulated in cells stimulated by EGF and in diverse human cancer cells, where ACAT1 tetramers, but not monomers, are phosphorylated and stabilized by enhanced Y407 phosphorylation. Moreover, we identified arecoline hydrobromide (AH) as a covalent ACAT1 inhibitor that binds to and disrupts only ACAT1 tetramers. The resultant AH-bound ACAT1 monomers cannot reform tetramers. Inhibition of tetrameric ACAT1 by abolishing Y407 phosphorylation or AH treatment results in decreased ACAT1 activity, leading to increased PDC flux and oxidative phosphorylation with attenuated cancer cell proliferation and tumor growth. These findings provide a mechanistic understanding of how oncogenic events signal through distinct acetyltransferases to regulate cancer metabolism and suggest ACAT1 as an anti-cancer target.

Topics: Acetyl-CoA C-Acetyltransferase; Animals; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Mitochondria; Neoplasms; NIH 3T3 Cells; Oligopeptides; Phosphorylation; Protein-Tyrosine Kinases; Pyruvate Dehydrogenase Complex; Receptor, Fibroblast Growth Factor, Type 1

Epidermal growth factor (EGF) is a 53 amino acid polypeptide and its receptor EGFR is an established therapeutic target for anti-tumor therapy. Two major categories of EGFR-targeted drugs include monoclonal antibodies (mAbs) and small molecular tyrosine kinase inhibitors (TKIs). However, drug resistance occurs in a significant proportion of patients due to EGFR mutations. Since EGFR can maintain activation while abrogating the activity of mAbs or TKIs, or bypass signaling functions while successfully circumventing the EGF-EGFR switch, developing new mechanism-based inhibitors is necessary.. In this study, based on the principle of tumor immunotherapy, a recombinant protein pLLO-hEGF was constructed. The N-terminal portion contains three immunodominant epitopes from listeriolysin O (LLO) and the C-terminal includes EGF. To use EGF as a target vector to recognize EGFR-expressing cancer cells, immunodominant epitopes could enhance immunogenicity of tumor cells for immune cell activation and attack.. Recombinant protein pLLO-hEGF was successfully expressed and showed strong affinity to cancer cells. Also, pLLO-hEGF could significantly stimulate human lymphocyte proliferation and the lymphocytes demonstrated enhanced killing potency in EGFR-expressing cancer cells in vitro and in vivo.. This study can provide novel strategies and directions in tumor biotherapy.

Topics: Animals; Antineoplastic Agents; Bacterial Toxins; Cell Proliferation; Cells, Cultured; Cloning, Molecular; Epidermal Growth Factor; ErbB Receptors; HCT116 Cells; Heat-Shock Proteins; Hemolysin Proteins; HT29 Cells; Humans; Leukocytes, Mononuclear; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms; Recombinant Fusion Proteins; T-Lymphocytes; Transplantation, Heterologous

Inverse docking technology has been a trend of drug discovery, and bioinformatics approaches have been used to predict target proteins, biological activities, signal pathways and molecular regulating networks affected by drugs for further pharmacodynamic and mechanism studies.. In the present paper, inverse docking technology was applied to screen potential targets from potential drug target database (PDTD). Then, the corresponding gene information of the obtained drug-targets was applied to predict the related biological activities, signal pathways and processes networks of the compound by using MetaCore platform. After that, some most relevant regulating networks were considered, which included the nodes and relevant pathways of dioscin.. 71 potential targets of dioscin from humans, 7 from rats and 8 from mice were screened, and the prediction results showed that the most likely targets of dioscin were cyclin A2, calmodulin, hemoglobin subunit beta, DNA topoisomerase I, DNA polymerase lambda, nitric oxide synthase and UDP-N-acetylhexosamine pyrophosphorylase, etc. Many diseases including experimental autoimmune encephalomyelitis of human, temporal lobe epilepsy of rat and ankylosing spondylitis of mouse, may be inhibited by dioscin through regulating immune response alternative complement pathway, G-protein signaling RhoB regulation pathway and immune response antiviral actions of interferons, etc. The most relevant networks (5 from human, 3 from rat and 5 from mouse) indicated that dioscin may be a TOP1 inhibitor, which can treat cancer though the cell cycle- transition and termination of DNA replication pathway. Dioscin can down regulate EGFR and EGF to inhibit cancer, and also has anti-inflammation activity by regulating JNK signaling pathway.. The predictions of the possible targets, biological activities, signal pathways and relevant regulating networks of dioscin provide valuable information to guide further investigation of dioscin on pharmacodynamics and molecular mechanisms, which also suggests a practical and effective method for studies on the mechanism of other chemicals.

Topics: Animals; Anti-Inflammatory Agents; Antineoplastic Agents, Phytogenic; Cell Cycle; Computational Biology; Dioscorea; Diosgenin; DNA Topoisomerases, Type I; Down-Regulation; Drugs, Chinese Herbal; Epidermal Growth Factor; ErbB Receptors; Gene Regulatory Networks; Humans; Male; MAP Kinase Signaling System; Mice; Neoplasms; Phytotherapy; Protein Binding; Rats; Signal Transduction

The bioactive sphingolipid sphingosine-1-phosphate (S1P) mediates cellular proliferation, mitogenesis, inflammation, and angiogenesis. These biologies are mediated through S1P binding to specific GPCRs [sphingosine-1-phosphate receptor (S1PR)1-5] and some other less well-characterized intracellular targets. Ezrin-radixin-moesin (ERM) proteins, a family of adaptor molecules linking the cortical actin cytoskeleton to the plasma membrane, are emerging as critical regulators of cancer invasion via regulation of cell morphology and motility. Recently, we identified S1P as an acute ERM activator (via phosphorylation) through its action on S1PR2. In this work, we dissect the mechanism of S1P generation downstream of epidermal growth factor (EGF) leading to ERM phosphorylation and cancer invasion. Using pharmacologic inhibitors, small interfering RNA technologies, and genetic approaches, we demonstrate that sphingosine kinase (SK)2, and not SK1, is essential and sufficient in EGF-mediated ERM phosphorylation in HeLa cells. In fact, knocking down SK2 decreased ERM activation 2.5-fold. Furthermore, we provide evidence that SK2 is necessary to mediate EGF-induced invasion. In addition, overexpressing SK2 causes a 2-fold increase in HeLa cell invasion. Surprisingly, and for the first time, we find that this event, although dependent on S1PR2 activation, does not generate and does not require extracellular S1P secretion, therefore introducing a potential novel model of autocrine/intracrine action of S1P that still involves its GPCRs. These results define new mechanistic insights for EGF-mediated invasion and novel actions of SK2, therefore setting the stage for novel targets in the treatment of growth factor-driven malignancies.

Topics: Autocrine Communication; Cytoskeletal Proteins; Epidermal Growth Factor; HeLa Cells; Humans; Lysophospholipids; Membrane Proteins; Microfilament Proteins; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasms; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors

Proteases contribute to cancer in many ways, including tumor vascularization and metastasis, and their pharmacological inhibition is a potential anticancer strategy. We report that human endothelial cells (EC) express the trypsinogen 4 isoform of the serine protease 3 (PRSS3), and lack both PRSS2 and PRSS1. Trypsinogen 4 expression was upregulated by the combined action of VEGF-A, FGF-2 and EGF, angiogenic factors representative of the tumor microenvironment. Suppression of trypsinogen 4 expression by siRNA inhibited the angiogenic milieu-induced migration of EC from cancer specimens (tumor-EC), but did not affect EC from normal tissues. We identified tissue factor pathway inhibitor-2 (TFPI-2), a matrix associated inhibitor of cell motility, as the functional target of trypsinogen 4, which cleaved TFPI-2 and removed it from the matrix put down by tumor-EC. Silencing tumor-EC for trypsinogen 4 accumulated TFPI2 in the matrix. Showing that angiogenic factors stimulate trypsinogen 4 expression, which hydrolyses TFPI-2 favoring a pro-migratory situation, our study suggests a new pathway linking tumor microenvironment signals to endothelial cell migration, which is essential for angiogenesis and blood vessel remodeling. Abolishing trypsinogen 4 functions might be an exploitable strategy as anticancer, particularly anti-vascular, therapy.

Topics: Blotting, Western; Cell Movement; Cells, Cultured; Endothelial Cells; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Neoplasms; Proteolysis; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Trypsin; Tumor Microenvironment; Vascular Endothelial Growth Factor A

The design, preparation, as well as structural and functional characterizations of the recombinant fusion protein hVEGF-EGF as a dual-functional agent that may target both EGFR (R: receptor) and angiogenesis are reported. hVEGF-EGF was found to bind to EGFR more strongly than did EGF, and to bind to VEGFR similarly to VEGF. Mass spectrometry measurements showed that the sites of DTPA (diethylenetriaminepentaacetic acid) conjugated hVEGF-EGF (for radiolabeling) were the same as those of its parent hEGF and hVEGF proteins. All DTPA-conjugated proteins retained similar binding capacities to their respective receptors as compared to their respective parent proteins. In vitro cell binding studies using BAEC (a bovine aortic endothelial cell) and MDA-MB-231 (a human breast cancer) cells expressing both EGFR and VEGFR confirmed similar results. Treating BAEC cells with hVEGF-EGF induced remarkable phosphorylation of EGFR, VEGFR, and their downstream targets ERK1/2. Nevertheless, the radiolabeled (111)In-DTPA-hVEGF-EGF showed cytotoxicity against MDA-MB-231 cells. Pharmacokinetic studies using (111)In-DTPA-hVEGF-EGF in BALB/c nude mice showed that appreciable tracer activities were accumulated in liver and spleen. In all, this study demonstrated that the fusion protein hVEGF-EGF maintained the biological specificity toward both EGFR and VEGFR and may be a potential candidate as a dual-targeting moiety in developing anticancer drugs.

Topics: Animals; Antineoplastic Agents; Cattle; Cell Line; Cell Line, Tumor; Drug Carriers; Drug Delivery Systems; Epidermal Growth Factor; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms; Pentetic Acid; Recombinant Fusion Proteins; Vascular Endothelial Growth Factor A

The Ras-extracellular signal-regulated kinase (ERK) cascade is an important signaling module in cells. One regulator of the Ras-ERK cascade is phosphatidic acid (PA) generated by phospholipase D (PLD) and diacylglycerol kinase (DGK). Using a newly developed PA biosensor, PASS (phosphatidic acid biosensor with superior sensitivity), we found that PA was generated sequentially by PLD and DGK in epidermal growth factor (EGF)-stimulated HCC1806 breast cancer cells. Inhibition of PLD2, one of the two PLD members, was sufficient to eliminate most of the PA production, whereas inhibition of DGK decreased PA production only at the later stages of EGF stimulation, suggesting that PLD2 precedes DGK activation. The temporal production of PA by PLD2 is important for the nuclear activation of ERK. While inhibition of both PLD and DGK had no effect on the overall ERK activity, inhibition of PLD2 but not PLD1 or DGK blocked the nuclear ERK activity in several cancer cell lines. The decrease of active ERK in the nucleus inhibited the activation of Elk1, c-fos, and Fra1, the ERK nuclear targets, leading to decreased proliferation of HCC1806 cells. Together, these findings reveal that PA production by PLD2 determines the output of ERK in cancer cell growth factor signaling.

Topics: Animals; Binding Sites; Blotting, Western; Cell Line, Tumor; Cell Membrane; Cell Nucleus; Cell Proliferation; CHO Cells; Cricetinae; Cricetulus; Diacylglycerol Kinase; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Green Fluorescent Proteins; HEK293 Cells; Humans; Luminescent Proteins; Microscopy, Confocal; Neoplasms; Phosphatidic Acids; Phospholipase D; Qb-SNARE Proteins; Qc-SNARE Proteins; Red Fluorescent Protein; RNA Interference; Saccharomyces cerevisiae Proteins; Signal Transduction; Time Factors

Various types of cancer accounts for 10% of total death worldwide which necessitates better therapeutic strategies. Curcumin, a curcuminoid present in Curcuma longa, shown to exhibit antioxidant, anti-inflammatory and anticarcinogenic properties. Present study, we aimed to analyze inhibitory properties of curcumin towards virulent proteins for various cancers by computer aided virtual screening. Based on literature studies, twenty two receptors were selected which have critical virulent functions in various cancer. The binding efficiencies of curcumin towards selected targets were studied by molecular docking. Out of all, curcumin showed best results towards epidermal growth factor (EGF), virulent protein of gastric cancer; glutathione-S-transferase Pi gene (GST-PI), virulent protein for prostate cancer; platelet-derived growth factor alpha (PDGFA), virulent protein for mesothelioma and glioma compared with their natural ligands. The calculated binding energies of their docked conformations with curcumin found to be -7.59 kcal/mol, -7.98 kcal/mol and -7.93 kcal/mol respectively. Further, a comparative study was performed to screen binding efficiency of curcumin with two conventional antitumor agents, litreol and triterpene. Docking studies revealed that calculated binding energies of docked complex of litreol and EGF, GST-PI and PDGFA were found to be -5.08 kcal/mol, -3.69 kcal/mol and -1.86 kcal/mol respectively. The calculated binding energies of triterpene with EGF and PDGFA were found to be -4.02 kcal/mol and -3.11 kcal/mol respectively, whereas GST-PI showed +6.07 kcal/mol, indicate poor binding. The predicted pharmacological features of curcumin found to be better than litreol and triterpene. Our study concluded that curcumin has better interacting properties towards these cancer targets than their normal ligands and conventional antitumor agents. Our data pave insight for designing of curcumin as novel inhibitors against various types of cancer.

Topics: Administration, Oral; Algorithms; Animals; Antineoplastic Agents; Antioxidants; Caco-2 Cells; Catechols; Curcumin; Dogs; Drug Screening Assays, Antitumor; Epidermal Growth Factor; Glutathione S-Transferase pi; Humans; Ligands; Madin Darby Canine Kidney Cells; Molecular Conformation; Molecular Docking Simulation; Neoplasms; Platelet-Derived Growth Factor; Protein Binding; Triterpenes

The ubiquitin-proteasome system facilitates the degradation of ubiquitin-tagged proteins and performs a regulatory role in cells. Elevated proteasome activity and subunit expression are found in several cancers. However, the inherent molecular mechanisms responsible for increased proteasome function in cancers remain unclear despite the well investigated and defined role of the mammalian proteasome. This study was initiated to elucidate the mechanisms involved in the regulation of β subunits of the mammalian proteasome. Suppression of STAT3 tyrosine phosphorylation coordinately decreased the mRNA and protein levels of the β subunits of the 20 S core complex in DU145 cells. Notably, PSMB5, a molecular target of bortezomib, was shown to be a target of STAT3. Knockdown of STAT3 decreased PSMB5 protein. Inhibition of phospho-STAT3 substantially reduced PSMB5 protein levels in cells expressing constitutively active-STAT3. Accumulation of activated STAT3 resulted in the induction of PSMB5 promoter and protein levels. In addition, a direct correlation was observed between the endogenous levels of PSMB5 and constitutively active STAT3. PSMB5 and STAT3 protein levels remained unaltered following the inhibition of proteasome activity. The EGF-induced concerted increase of β subunits was blocked by inhibition of the EGF receptor or STAT3 but not by the PI3K/AKT or MEK/ERK pathways. Decreased proteasome activities were due to reduced protein levels of catalytic subunits of the proteasome in STAT3-inhibited cells. Combined treatments with bortezomib and inhibitor of STAT3 abrogated proteasome activity and enhanced cellular apoptosis. Overall, we demonstrate that aberrant activation of STAT3 regulates the expression of β subunits, in particular PSMB5, and the catalytic activity of the proteasome.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Boronic Acids; Bortezomib; Cell Line, Tumor; Epidermal Growth Factor; HEK293 Cells; HeLa Cells; Humans; Neoplasms; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Subunits; Pyrazines; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; STAT3 Transcription Factor; Tyrosine; Up-Regulation

Epidermal growth factor receptor (EGFR) plays a critical role in various types of human cancer such as lung, breast, brain and colon cancer. However, how EGFR activation is initiated and what type of interaction is linked to its transphosphorylation and autophosphorylation as well as the biological consequences in case of interruption and blockade of these two types of EGFR phosphorylation, remain elusive. In the present study, we provided evidence that EGFR transphosphorylation at Y845 indeed plays an important role in its autophosphorylation and kinase activity. Our results suggest that the modulation of EGFR transphosphorylation may influence its activity and function, indicating a potential target mechanism for treating EGFR-associated diseases and various types of cancer.

Topics: Amino Acid Substitution; Cell Line, Tumor; Cell Proliferation; DNA Replication; Epidermal Growth Factor; ErbB Receptors; HEK293 Cells; Humans; MCF-7 Cells; Mutation; Neoplasms; Phosphorylation; Phosphotransferases

RAS genes are among the most frequently mutated proto-oncogenes in cancer. However, how Ras stability is regulated remains largely unknown. Here, we report a regulatory loop involving the E3 ligase Nedd4-1, Ras, and PTEN. We found that Ras signaling stimulates the expression of Nedd4-1, which in turn acts as an E3 ubiquitin ligase that regulates Ras levels. Importantly, Ras activation, either by oncogenic mutations or by epidermal growth factor (EGF) signaling, prevents Nedd4-1-mediated Ras ubiquitination. This leads to Ras-induced Nedd4-1 overexpression, and subsequent degradation of the tumor suppressor PTEN in both human cancer samples and cancer cells. Our study thus unravels the molecular mechanisms underlying the interplay of Ras, Nedd4-1, and PTEN and suggests a basis for the high prevalence of Ras-activating mutations and EGF hypersignaling in cancer.

Topics: Animals; Carcinogenesis; Cell Line, Tumor; Endosomal Sorting Complexes Required for Transport; Epidermal Growth Factor; HEK293 Cells; HeLa Cells; Hep G2 Cells; Humans; Mice; Mice, Nude; Nedd4 Ubiquitin Protein Ligases; Neoplasms; NIH 3T3 Cells; Protein Binding; PTEN Phosphohydrolase; ras Proteins; Signal Transduction; Transplantation, Heterologous; Ubiquitin-Protein Ligases; Ubiquitination; Up-Regulation

Understanding the basic mechanism of the spatio-temporal self-control of genome-wide gene expression engaged with the complex epigenetic molecular assembly is one of major challenges in current biological science. In this study, the genome-wide dynamical profile of gene expression was analyzed for MCF-7 breast cancer cells induced by two distinct ErbB receptor ligands: epidermal growth factor (EGF) and heregulin (HRG), which drive cell proliferation and differentiation, respectively. We focused our attention to elucidate how global genetic responses emerge and to decipher what is an underlying principle for dynamic self-control of genome-wide gene expression. The whole mRNA expression was classified into about a hundred groups according to the root mean square fluctuation (rmsf). These expression groups showed characteristic time-dependent correlations, indicating the existence of collective behaviors on the ensemble of genes with respect to mRNA expression and also to temporal changes in expression. All-or-none responses were observed for HRG and EGF (biphasic statistics) at around 10-20 min. The emergence of time-dependent collective behaviors of expression occurred through bifurcation of a coherent expression state (CES). In the ensemble of mRNA expression, the self-organized CESs reveals distinct characteristic expression domains for biphasic statistics, which exhibits notably the presence of criticality in the expression profile as a route for genomic transition. In time-dependent changes in the expression domains, the dynamics of CES reveals that the temporal development of the characteristic domains is characterized as autonomous bistable switch, which exhibits dynamic criticality (the temporal development of criticality) in the genome-wide coherent expression dynamics. It is expected that elucidation of the biophysical origin for such critical behavior sheds light on the underlying mechanism of the control of whole genome.

Topics: Algorithms; Computational Biology; Epidermal Growth Factor; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genome-Wide Association Study; Genomics; Humans; MCF-7 Cells; Models, Genetic; Models, Statistical; Neoplasms; Neuregulin-1; Protein Structure, Tertiary; RNA, Messenger; Time Factors

Balanced activity of protein tyrosine kinases and phosphatases (PTPs) controls tyrosine phosphorylation levels and, consequently, is needed to prevent pathologies like cancer. Phosphatase activity is tightly regulated in space and time. Thus, in order to understand how phospho-tyrosine signalling is regulated, the intracellular dynamics of PTPs should be investigated. Here, we have studied the intracellular dynamics of PTPD1, a FERM (four-point-one, ezrin, radixin, moesin) domain-containing PTP that is over expressed in cancer cells and potentiates EGFR signalling. Whereas PTPD1 was excluded from E-cadherin rich cell-cell adhesions in epithelial cell monolayers, it diffused from the cytoplasm to those membranes in contact with the extracellular medium. Localisation of PTPD1 at the plasma membrane was mediated by its FERM domain and enabled the formation of EGFR/PTPD1-containing signalling complexes that pre-existed at the plasma membrane before EGF stimulation. PTPD1 and EGFR transiently co-localised at EGF stimulation sites until the formation of macropinosomes containing active species of EGFR. Interference of PTPD1 expression caused a decrease in EGFR phosphorylated species at the periphery of the cell. Presented data suggest that the transient formation of dynamic PTPD1/EGFR signalling complexes strengthens EGF signalling by promoting the spatial propagation of EGFR phosphorylated species.

Topics: Cell Adhesion; Cell Membrane; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Gene Expression Regulation; Humans; MCF-7 Cells; Neoplasms; Phosphorylation; Protein Structure, Tertiary; Protein Tyrosine Phosphatases, Non-Receptor; RNA, Small Interfering; Signal Transduction

Activation of nuclear factor κB (NFκB) is a central event in the responses of normal cells to inflammatory signals, and the abnormal constitutive activation of NFκB is important for the survival of most cancer cells. In nonmalignant human cells, EGF stimulates robust activation of NFκB. The kinase activity of the EGF receptor (EGFR) is required, because the potent and specific inhibitor erlotinib blocks the response. Down-regulating EGFR expression or inhibiting EGFR with erlotinib impairs constitutive NFκB activation in several different types of cancer cells and, conversely, increased activation of NFκB leads to erlotinib resistance in these cells. We conclude that EGF is an important mediator of NFκB activation in cancer cells. To explore the mechanism, we selected an erlotinib-resistant cell line in which the guanine nucleotide exchange factor Son of Sevenless 1 (SOS1), well known to be important for EGF-dependent signaling to MAP kinases, is overexpressed. Increased expression of SOS1 increases NFκB activation in several different types of cancer cells, and ablation of SOS1 inhibits EGF-induced NFκB activation in these cells, indicating that SOS1 is a functional component of the pathway connecting EGFR to NFκB activation. Importantly, the guanine nucleotide exchange activity of SOS1 is not required for NFκB activation.

Topics: Breast; Cell Line, Tumor; Cells, Cultured; Drug Resistance, Neoplasm; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Erlotinib Hydrochloride; Female; Gene Knockdown Techniques; Humans; Neoplasms; NF-kappa B; Protein Kinase Inhibitors; Quinazolines; Signal Transduction; SOS1 Protein; Up-Regulation

Cancer metastasis is considered a major challenge in cancer therapy. Recently, epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR) signaling has been shown to induce epithelial-mesenchymal transition (EMT) and thereby to promote cancer metastasis. However, the underlying mechanism has not been fully elucidated. We demonstrate that EGF can induce EMT in human prostate and lung cancer cells and thus promote invasion and migration. EGF-induced EMT has been characterized by the cells acquiring mesenchymal spindle-like morphology and increasing their expression of N-cadherin and fibronectin, with a concomitant decrease of E-cadherin. Both protein and mRNA expression of transcription factor Snail rapidly increases after EGF treatment. The knockdown of Snail significantly attenuates EGF-induced EMT, suggesting that Snail is crucial for this process. To determine the way that Snail is accumulated, we demonstrate (1) that EGF promotes the stability of Snail via inhibiting the activity of glycogen synthase kinase 3 beta (GSK-3β), (2) that protein kinase C (PKC) rather than the phosphatidylinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is responsible for GSK-3β inhibition and (3) that GSK-3β inhibition promotes the transcription of Snail. Taken together, these results reveal that the PKC/GSK-3β signaling pathway controls both the stability and transcription of Snail, which is crucial for EMT induced by EGF in PC-3 and A549 cells. Our study suggests a novel signaling pathway for Snail regulation and provides a better understanding of growth-factor-induced tumor EMT and metastasis.

Topics: Cell Line, Tumor; Cell Movement; Cell Nucleus; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Neoplasm Invasiveness; Neoplasms; Protein Kinase C; Protein Stability; Protein Transport; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transcription, Genetic; Up-Regulation

In the three decades since the discovery of the Wnt1 proto-oncogene in virus-induced mouse mammary tumours, our understanding of the signalling pathways that are regulated by the Wnt proteins has progressively expanded. Wnts are involved in an complex signalling network that governs multiple biological processes and cross-talk with multiple additional signalling cascades, including the Notch, FGF (fibroblast growth factor), SHH (Sonic hedgehog), EGF (epidermal growth factor) and Hippo pathways. The Wnt signalling pathway also illustrates the link between abnormal regulation of the developmental processes and disease manifestation. Here we provide an overview of Wnt-regulated signalling cascades and highlight recent advances. We focus on new findings regarding the dedicated Wnt production and secretion pathway with potential therapeutic targets that might be beneficial for patients with Wnt-related diseases.

Topics: Animals; Epidermal Growth Factor; Fibroblast Growth Factors; Hedgehog Proteins; Hippo Signaling Pathway; Humans; Mice; Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Mas; Receptors, Notch; Wnt Signaling Pathway; Wnt1 Protein

K-Ras is frequently mutated in human cancers. Mutant (mt) K-Ras can stimulate both oncogenic transformation and apoptosis through activation of extracellular signal-regulated kinase (ERK) and AKT pathways and the MST2 pathway, respectively. The biological outcome is determined by the balance and cross talk between these pathways. In colorectal cancer (CRC), a K-Ras mutation is negatively correlated with MST2 expression, as mt K-Ras can induce apoptosis by activating the MST2 pathway. However, wild-type (wt) K-Ras can prevent the activation of the MST2 pathway upon growth factor stimulation and enable transformation by mt K-Ras in CRC cells that express MST2. Here we have investigated the mechanism by which wt and mt K-Ras differentially regulate the MST2 pathway and MST2-dependent apoptosis. The ability of K-Ras to activate MST2 and MST2-dependent apoptosis is determined by the differential activation kinetics of mt K-Ras and wt K-Ras. Chronic activation of K-Ras by mutation or overexpression of Ras exchange factors results in the activation of MST2 and LATS1, increased MST2-LATS1 complex formation, and apoptosis. In contrast, transient K-Ras activation upon epidermal growth factor (EGF) stimulation prevents the formation of the MST2-LATS1 complex in an AKT-dependent manner. Our data suggest that the close relationship between Ras prosurvival and proapoptotic signaling is coordinated via the differential regulation of the MST2-LATS1 interaction by transient and chronic stimuli.

Topics: Apoptosis; Cell Line, Tumor; Enzyme Activation; Epidermal Growth Factor; Genes, ras; HeLa Cells; Humans; Mutation; Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; ras Proteins; Serine-Threonine Kinase 3; Signal Transduction; Tumor Suppressor Proteins

The Functional Assessment of Cancer Therapy-Epidermal Growth Factor Receptor Inhibitor 18 (FACT-EGFRI-18) is a patient-reported outcomes questionnaire developed to assess the effect of EGFRI on patients. The FACT-EGFR-18 was translated into Dutch and evaluated in order to document that the translation adequately captures the concepts of the original English-language version of the questionnaire and is readily understood by subjects in the target population.. Translation of the FACT-EGFRI-18 from English to Dutch was accomplished by employing the Functional Assessment of Chronic Illness Therapy (FACIT) multilingual translation methodology. Ten native-speaking residents of the target country who reported EGFRI associated dermatological adverse events (dAEs) were asked to review the translation of the harmonized FACT-EGFRI-18.. Participants generally found the Dutch FACT-EGFRI-18 easy to understand and complete. In addition, the translation retained the original meaning of the FACT-EGFRI-18 items and instructions. Based on the results of the cognitive debriefing interviews, no changes to improve clarity and comprehension of translations were identified.. The Dutch FACT-EGFRI-18 demonstrates content validity and linguistic validity, and was found conceptually equivalent to its English source, thus confirming linguistic validation. The results suggest that the Dutch FACT-EGFRI-18 can be applied to measure dAE related health related quality of life in Dutch-speaking patients undergoing EGFRI therapy. Formal validation of the Dutch FACT-EGFRI-18 is ongoing.

Topics: Aged; Comprehension; Drug Eruptions; Drug-Related Side Effects and Adverse Reactions; England; Epidermal Growth Factor; Female; Humans; Linguistics; Male; Middle Aged; Neoplasms; Netherlands; Quality of Life; Risk Assessment; Self Report; Surveys and Questionnaires; Translating; Treatment Outcome

Soluble epidermal growth factor receptor (sEGFR) is a circulating serum biomarker in cancer patients. Recent studies suggest that baseline serum sEGFR concentrations may predict responsiveness to EGFR-targeted therapy. Here, we demonstrate that sEGFR is generated through proteolytic cleavage of a cell surface precursor of an alternately spliced EGF receptor isoform and that sEGFR binds to EGF with high affinity. Proteolytic cleavage is stimulated by an anti-α5/β1 integrin antibody and 4-aminophenylmercuric acetate, and inhibited by fibronectin. Two FDA-approved therapeutic anti-EGFR antibodies also inhibit shedding of sEGFR, thus implicating the cell surface precursor of sEGFR as a competing target for anti-EGFR antibodies in human tissues. These observations parallel trastuzumab regulation of HER2 shedding and have implications for patient stratification in future clinical trials of EGFR-targeted antibodies.

Topics: Alternative Splicing; Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal, Humanized; Biomarkers, Tumor; Cetuximab; CHO Cells; Clinical Trials as Topic; Cricetinae; Epidermal Growth Factor; ErbB Receptors; Humans; Integrins; Neoplasms; Neoplastic Cells, Circulating; Protein Isoforms

Non-steroidal anti-inflammatory drugs (NSAIDs) were shown to reduce the risk of colorectal cancer recurrence and are widely used to modulate inflammatory responses. Indomethacin is an NSAID. Herein, we reported that indomethacin can suppress cancer cell migration through its influence on the focal complexes formation. Furthermore, endothelial growth factor (EGF)-mediated Ca2+ influx was attenuated by indomethacin in a dose dependent manner. Our results identified a new mechanism of action for indomethacin: inhibition of calcium influx that is a key determinant of cancer cell migration.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Calcium; Calcium Signaling; Cell Line, Tumor; Cell Movement; Cyclooxygenase 2; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Indomethacin; Neoplasms; Phosphorylation

Aberrant localization of proteins is increasingly being suggested as a causal player in epithelial cancers. Despite this, few studies have investigated how mislocalization of a protein can alter individual biological processes that contribute to cancer progression. Using Ras as a model of transformation, we investigate how localization of the polarity protein Scribble contributes to its tumor-suppressive properties. Wild-type Scribble has been shown to modulate Ras-mitogen-activated protein kinase (MAPK) transformation both in vitro and in vivo. By utilizing a construct that carries a mutation in the LRR domain of Scribble (Scribble P305L) resulting in a cytosolic rather than the usual membrane-bound localization, we report that discrete tumor suppressive properties of Scribble are differentially sensitive to the localization of Scribble. We find that although the Scribble P305L mislocalization mutant can no longer suppress Ras-MAPK-induced invasion or epithelial to mesenchymal transition phenotypes, mislocalized Scribble can still suppress anchorage-independent cell growth. This study illustrates that the manner in which protein mislocalization contributes to cancer is likely complex and highlights the need for careful interrogation as to how cell polarity protein mislocalization, its secondary consequences, and the mutations that give rise to their mislocalization may contribute to specific aspects of cancer progression.

Topics: Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Humans; MAP Kinase Signaling System; Membrane Proteins; Mutation; Neoplasms; ras Proteins; Transforming Growth Factor beta; Tumor Suppressor Proteins

Receptor-targeted imaging is emerging as a promising strategy for diagnosis of human cancer. Herein, we developed an epidermal growth factor-based nanoprobe (EGF-NP) for in vivo optical imaging of epidermal growth factor receptor (EGFR), an important target for cancer imaging. The self-quenched EGF-NP is fabricated by sequentially conjugating a near-infrared (NIR) fluorophore (Cy5.5) and a quencher (BHQ-3) to EGF, a low-molecular weight polypeptide (6.2 kDa), compared to EGFR antibody (150 kDa). The self-quenched EGF-NP presented great specificity to EGFR, and rapidly internalized into the cells, as monitored by time-lapse imaging. Importantly, the self-quenched EGF-NP boosted strong fluorescence signals upon EGFR-targeted uptake into EGFR-expressing cells, followed by lysosomal degradation, as confirmed by lysosomal marker cell imaging. Consistent with cellular results, intravenous injection of EGF-NP into tumor-bearing mice induced strong NIR fluorescence intensity in the target tumor tissue with high specificity against EGFR-expressing cancer cells. Signal accumulation of EGF-NP in tumor was much faster than that of EGFR monoclonal antibody (Cetuximab)-Cy5.5 conjugates due to the rapid clearance from the body and tissue permeability of low-molecular weight EGF. This self-quenched, EGF-based imaging probe can be applied for diagnosis of various cancers.

Topics: Animals; Cell Line, Tumor; Diagnostic Imaging; Epidermal Growth Factor; ErbB Receptors; Fluorescence; Humans; Intracellular Space; Mice; Nanoparticles; Neoplasms; Spectroscopy, Near-Infrared

FAK is a non-receptor tyrosine kinase contributing to migration and proliferation downstream of integrin and/or growth factor receptor signaling of normal and malignant cells. In addition to well-characterized tyrosine phosphorylations, FAK is phosphorylated on several serines, whose role is not yet clarified. We observed that phosphorylated FAK on serine 732 (P-FAKSer732) is present at variable levels in vitro, in several melanoma, ovarian and thyroid tumor cell lines and in vivo, in tumor cells present in fresh ovarian cancer ascites. In vitro P-FAKSer732 was barely detectable during interphase while its levels strongly increased in mitotic cells upon activation of the EGFR/MEK/ERK axis in an integrin-independent manner. P-FAKSer732 presence was crucial for the maintenance of the proliferation rate and its levels were inversely related to the levels of acetylated α-tubulin. P-FAKSer732 localized at the microtubules (MTs) of the spindle, biochemically associated with MTs and contributed to MT depolymerization. The lack of the phosphorylation on Ser732 as well as the inhibition of CDK5 activity by roscovitine impaired mitotic spindle assembly and correct chromosome alignment during mitosis. We also identified, for the first time, that the EGF-dependent EGFR activation led to increased P-FAKSer732 and polymerized MTs. Our data shed light on the multifunctional roles of FAK in neoplastic cells, being involved not only in integrin-dependent migratory signaling but also in integrin-independent MT dynamics and mitosis control. These findings provide a new potential target for inhibiting the growth of tumor cells in which the EGFR/MEK/ERK/CDK5 pathway is active.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin-Dependent Kinase 5; Epidermal Growth Factor; ErbB Receptors; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesions; Humans; Integrins; MAP Kinase Signaling System; Microtubules; Mitosis; Neoplasms; Phosphorylation; Phosphoserine; Polymerization; Spindle Apparatus

The clinical efficacy of anti-angiogenic therapies has been difficult to predict, and biomarkers that can predict responsiveness are sorely needed in this era of personalized medicine. CVX-060 is an angiopoietin-2 (Ang2) targeting therapeutic, consisting of two peptides that bind Ang2 with high affinity and specificity, covalently fused to a scaffold antibody. In order to optimize the use of this compound in the clinic the construction of a predictive model is described, based on the efficacy of CVX-060 in 13 cell line and 2 patient-derived xenograft models. Pretreatment size tumors from each of the models were profiled for the levels of 27 protein markers of angiogenesis, SNP haplotype in 5 angiogenesis genes, and somatic mutation status for 11 genes implicated in tumor growth and/or vascularization. CVX-060 efficacy was determined as tumor growth inhibition (TGI%) at termination of each study. A predictive statistical model was constructed based on the correlation of these efficacy data with the marker profiles, and the model was subsequently tested by prospective analysis in 11 additional models. The results reveal a range of CVX-060 efficacy in xenograft models of diverse tissue types (0-64% TGI, median = 27%) and define a subset of 3 proteins (Ang1, EGF, Emmprin), the levels of which may be predictive of TGI by Ang2 blockade. The direction of the associations is such that better efficacy correlates with high levels of target and low levels of compensatory/antagonizing molecules. This effort has revealed a set of candidate predictive markers for CVX-060 efficacy that will be further evaluated in ongoing clinical trials.

Topics: Angiogenesis Inhibitors; Angiopoietin-1; Angiopoietin-2; Animals; Basigin; Biomarkers, Pharmacological; Cell Line, Tumor; Disease Models, Animal; Epidermal Growth Factor; Female; Gene Expression; Gene Expression Profiling; Humans; Mice; Molecular Targeted Therapy; Neoplasms; Peptides; Polymorphism, Single Nucleotide; Predictive Value of Tests; Tumor Burden; Xenograft Model Antitumor Assays

Recent preclinical evidence substantially supports the successful combination of chemotherapies and active immunotherapy for cancer treatment. These data sustain the effect of sequential combination schemes (vaccine plus chemotherapy or vice versa), which could be difficult to implement in clinical practice. Since chemotherapy is the standard treatment for most cancers, ethical issues forbid its delay and make difficult the evaluation of other treatments such as using an immunotherapeutic agent. Besides, vaccines must be applied as soon as possible to advanced cancer patients, in order to give them time to develop an effective immune response. Thus, a clinically attractive scenario is the concomitant application of treatments. However, little is known about the specific effect of different chemotherapeutic agents when combined with a cancer vaccine in such concomitant treatment. In this work, we analyze the influence of high-dose carboplatin or paclitaxel in the generation of a specific immune response when administered concomitantly with an OVA vaccine. Interestingly, neither carboplatin nor paclitaxel affects the humoral and CTL in vivo response generated by the vaccine. Moreover, an enhancement of the overall anti-tumor effect was observed in animals treated with OVA/CF vaccine combined with cytotoxic drugs. Moreover, the effect of the concomitant treatment was tested using a tumor-related antigen, the epidermal growth factor (EGF). Animals administered with EGF-P64k/Montanide and cytotoxic agents showed an antibody response similar to that from control animals. Therefore, our study suggests that carboplatin and paclitaxel can be concomitantly combined with active immunotherapies in the clinical practice of advanced cancer patients.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cancer Vaccines; Carboplatin; Combined Modality Therapy; Epidermal Growth Factor; Female; Humans; Immunotherapy, Active; Lymphocytes; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Paclitaxel

Cancer is one of the most life-threatening diseases, which causes 7.6 million deaths and around 1 trillion dollars economic loss every year. Theranostic materials are expected to improve early detection and safe treatment through personalized medicine. Driven by the needs, we report the development of a theranostic plasmonic shell-magnetic core star shape nanomaterial based approach for the targeted isolation of rare tumor cells from the whole blood sample, followed by diagnosis and photothermal destruction. Experimental data with whole blood sample spiked with SK-BR-3 cancer cell shows that Cy3 attached S6 aptamer conjugated theranostic plasmonic/magnetic nanoparticles can be used for fluorescence imaging and magnetic separation even in 0.001% mixtures. A targeted photothermal experiment using 1064 nm near-IR light at 2-3 W/cm(2) for 10 min resulted in selective irreparable cellular damage to most of the SK-BR-3 cancer cells. We discuss the possible mechanism and operating principle for the targeted imaging, separation, and photothermal destruction using theranostic magnetic/plasmonic nanotechnology. After the optimization of different parameters, this theranostic nanotechnology-driven assay could have enormous potential for applications as contrast agent and therapeutic actuators for cancer.

Topics: Animals; Cell Line, Tumor; Epidermal Growth Factor; Humans; Magnetics; Nanoparticles; Nanotechnology; Neoplasms; Phototherapy; Rabbits

Lewis Y (LeY) antigen is an oligosaccharide that is highly expressed at the cell surface in various human cancers. Increased LeY expression activates epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) and promotes cell proliferation in EGFR-overexpressing cells. However, the effect of downregulation of LeY expression on cell proliferation in HER2-overexpressing cells remains unknown. FUT1 encodes α1,2-fucosyltransferase, a key enzyme for LeY synthesis. We knocked down FUT1 by short interfering RNA (siRNA) in four HER2-overexpressing human cancer cell lines, including NCI-N87, MKN7, SKBr3 and BT474. We investigated whether downregulation of LeY and alteration in the glycosylation status of these cells affect cell proliferation and HER2 activation. Knocking down FUT1 expression markedly inhibited proliferation of NCI-N87, which highly expressed EGFR and was sensitive to EGFR deprivation. Furthermore, FUT1 siRNA downregulated the total amount of HER2 protein, phosphorylation of HER2 and EGFR, and phosphorylation of extracellular signal-regulated kinase (ERK) in this cell line. Moreover, the marked downregulation of phosphorylation of HER2 and ERK was observed following short-time EGF-stimulation. These effects were not observed in the other three cell lines. Our results suggest that knockdown of FUT1 downregulates HER2 signaling via EGFR downregulation. FUT1 may serve as a new molecular target for HER2-overexpressing human cancers with activated EGFR signaling.

Topics: Apoptosis; Blotting, Western; Cell Cycle; Cell Proliferation; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Fucosyltransferases; Galactoside 2-alpha-L-fucosyltransferase; Humans; Lewis Blood Group Antigens; National Cancer Institute (U.S.); Neoplasms; Phosphorylation; Real-Time Polymerase Chain Reaction; Receptor, ErbB-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Tumor Cells, Cultured; United States

HER2, a member of the epidermal growth factor receptor (ErbB) family, is over-expressed in many cancers. Trastuzumab and Pertuzumab are two monoclonal antibodies targeting different extracellular domains of HER2 for cancer therapy. As Pertuzumab binds to the dimerization arm of HER2, it can block HER2 heterodimerization and in turn ErbB signaling. Whether Trastuzumab has the same function is unclear. In this work, we have applied living-cell single-molecule force spectroscopy (SMFS) by Atomic Force Microscopy (AFM) to investigate the effect of Trastuzumab, as well as Pertuzumab, on HER2-modulated EGF-EGFR interaction. The results demonstrated that EGF bound to EGFR more stably in the cells co-expressing EGFR and HER2, and the binding enhancement in the presence of HER2 was inhibited by either Trastuzumab or Pertuzumab. Trastuzumab is expected to exert a similar inhibition effect on HER2/EGFR dimerization as Pertuzumab, although it does not bind directly to the dimerization arm of HER2.. Living-cell single-molecule force spectroscopy (SMFS) combined by Atomic Force Microscopy (AFM) was used by this team of scientists to investigate the effect of two monoclonal antibodies used in cancer therapy, Trastuzumab and Pertuzumab, on HER2-modulated EGF-EGFR interaction, demonstrating the utility of this technique in characterizing the effects of protein-based therapeutics on membrane receptors.

Topics: Antibodies, Monoclonal, Humanized; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Ligands; Microscopy, Atomic Force; Neoplasms; Protein Binding; Receptor, ErbB-2; Trastuzumab

Raf kinases are essential for normal Ras-Raf-MEK-ERK pathway signaling, and activating mutations in components of this pathway are associated with a variety of human cancers, as well as the related developmental disorders Noonan, LEOPARD, and cardiofaciocutaneous syndromes. Although the Raf kinases are known to dimerize during normal and disease-associated Raf signaling, the functional significance of Raf dimerization has not been fully elucidated. Here, using mutational analysis and a peptide inhibitor, we show that dimerization is required for normal Ras-dependent Raf activation and for the biological function of disease-associated Raf mutants with moderate, low, or impaired kinase activity. However, dimerization is not needed for the function of B-Raf mutants with high catalytic activity, such as V600E-B-Raf. Importantly, we find that a dimer interface peptide can effectively block Raf dimerization and inhibit Raf signaling when dimerization is required for Raf function, thus identifying the Raf dimer interface as a therapeutic target.

Topics: Amino Acid Substitution; Animals; Cell Line; Enzyme Activation; Epidermal Growth Factor; Humans; MAP Kinase Signaling System; Mice; Mutagenesis, Site-Directed; Mutation, Missense; Neoplasms; Peptide Fragments; Platelet-Derived Growth Factor; Protein Interaction Domains and Motifs; Protein Kinase Inhibitors; Protein Multimerization; raf Kinases; ras Proteins

Regenerating islet-derived family member, 4 (Reg IV) is a secreted protein and member of the C-type lectin superfamily. Expression analyses have characterized Reg IV as a prognostic marker for certain cancers; however, the functional role of Reg IV in cancer, including downstream signaling, has only begun to be elucidated. To investigate the biological role of Reg IV in cancer, phosphorylation events were studied in cancer cell lines in the context of either Reg IV stimulation (HCT116 cells) or knockdown of endogenous Reg IV (PC3 and KM12 cells). In addition to the previously observed impact on epidermal growth factor receptor and Akt phosphorylation, we observed modulation in the phosphorylation of multiple additional receptor tyrosine kinases (RTKs), including insulin receptor, insulin-like growth factor receptor as well as their downstream effectors, mitogen-activated protein kinase and phosphatidylinositol-3-kinase pathways. Furthermore, knockdown of Reg IV impacted the ability of insulin and EGF to stimulate downstream tyrosine phosphorylation. Knockdown of Reg IV in cancer cell lines inhibited anchorage-dependent and anchorage-independent (both soft-agar and spheroid assays) cell growth and induced cell cycle arrest. This was accompanied by upregulation of p21 and p27. Transiently silencing Reg IV in cancer cells induced apoptosis and downregulated Bcl-2. Conversely, stimulation of HCT116 cells with recombinant Reg IV induced Bcl-2. Hsp27, a molecule implicated in drug resistance, was similarly modulated by Reg IV. Consistent with our observations with Reg IV siRNA-mediated knockdown, monoclonal antibodies directed against Reg IV inhibited PC3 and KM12 cell growth. Collectively, Reg IV plays an important role in cancer by modulation of key signaling molecules including Hsp27, Bcl-2 and multiple RTKs.

Topics: Apoptosis; Cell Cycle; Cell Cycle Checkpoints; Cell Growth Processes; Cell Line, Tumor; Drug Resistance, Neoplasm; Epidermal Growth Factor; Gene Knockdown Techniques; HCT116 Cells; Heat-Shock Proteins; HEK293 Cells; HSP27 Heat-Shock Proteins; Humans; Insulin; Lectins, C-Type; Mitogen-Activated Protein Kinases; Molecular Chaperones; Neoplasms; Pancreatitis-Associated Proteins; Phosphatidylinositol 3-Kinase; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Receptor Protein-Tyrosine Kinases; Signal Transduction; Tyrosine; Up-Regulation

Previously, it was shown that a novel 4-(N)-stearoyl gemcitabine nanoparticle formulation was more effective than gemcitabine hydrochloride in controlling the growth of model mouse or human tumors pre-established in mice. In the present study, the feasibility of targeting the stearoyl gemcitabine nanoparticles (GemC18-NPs) into tumor cells that over-express epidermal growth factor receptor (EGFR) to more effectively control tumor growth was evaluated. EGFR is over-expressed in a variety of tumor cells, and EGF is a known natural ligand of EGFR. Recombinant murine EGF was conjugated onto the GemC18-NPs. The ability of the EGF to target the GemC18-NPs to human breast adenocarcinoma cells that expressed different levels of EGFR was evaluated in vitro and in vivo. In culture, the extent to which the EGF-conjugated GemC18-NPs were taken up by tumor cells was correlated to the EGFR density on the tumor cells, whereas the uptake of untargeted GemC18-NPs exhibited no difference among those same cell lines. The relative cytotoxicity of the EGF-conjugated GemC18-NPs to tumor cells in culture was correlated to EGFR expression as well. In vivo, EGFR-over-expressing MDA-MB-468 tumors in mice treated with the EGF-conjugated GemC18-NPs grew significantly slower than in mice treated with untargeted GemC18-NPs, likely due to that the EGF-GemC18-NPs were more anti-proliferative, anti-angiogenic, and pro-apoptotic. Fluorescence intensity data from ex vivo imaging showed that the EGF on the nanoparticles helped increase the accumulation of the GemC18-NPs into MDA-MB-468 tumors pre-established in mice by more than 2-fold as compared to the un-targeted GemC18-NPs. In conclusion, active targeting of the GemC18-NPs into EGFR-over-expressed tumors can further enhance their anti-tumor activity.

Topics: Animals; Antimetabolites, Antineoplastic; Cell Line, Tumor; Deoxycytidine; Epidermal Growth Factor; ErbB Receptors; Gemcitabine; Humans; Mice; Mice, Nude; Molecular Targeted Therapy; Nanoparticles; Neoplasms; Ovalbumin; Tumor Burden

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a membrane-anchored protein, known as proHB-EGF. ProHB-EGF is cleaved by metalloproteases through a process referred to as 'ectodomain shedding', resulting in the formation of soluble HB-EGF. Both proHB-EGF and soluble HB-EGF are biologically active; the former acts on neighbouring cells through juxtacrine signalling, whereas the latter can move to distant locations. Elevated HB-EGF expression has been observed in ovarian and some other cancers. CRM197, a diphtheria toxin (DT) mutant, binds directly to the epidermal growth factor (EGF)-like domain and represses the mitogenic activity of HB-EGF. Recently, monoclonal antibodies (mAbs) specific for human HB-EGF were generated by immunizing HB-EGF-deficient mice with human HB-EGF (Hamaoka et al. (2010) J. Biochem. 148, 55-69). Most of the mAbs can bind to the EGF-like domain of HB-EGF, but fail to inhibit the mitogenic activity of soluble HB-EGF. However, some mAbs prevented the ectodomain shedding of proHB-EGF and inhibited the proliferation of EGF receptor-expressing cells stimulated by proHB-EGF-expressing cells. Hamaoka et al. showed that CRM197 prevents the ectodomain shedding of proHB-EGF. Thus, these mAbs function as specific inhibitors for the ectodomain shedding of HB-EGF and may be useful for treating cancers exhibiting elevated levels of HB-EGF.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Bacterial Proteins; Binding Sites; Cell Proliferation; Epidermal Growth Factor; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Metalloproteases; Mice; Mice, Knockout; Mutation; Neoplasms; Protein Binding; Protein Precursors

Analysis of cellular signaling networks typically involves targeted measurements of phosphorylated protein intermediates. However, phosphoproteomic analyses usually require affinity enrichment of phosphopeptides and can be complicated by artifactual changes in phosphorylation caused by uncontrolled preanalytical variables, particularly in the analysis of tissue specimens. We asked whether changes in protein expression, which are more stable and easily analyzed, could reflect network stimulation and inhibition. We employed this approach to analyze stimulation and inhibition of the epidermal growth factor receptor (EGFR) by EGF and selective EGFR inhibitors. Shotgun analysis of proteomes from proliferating A431 cells, EGF-stimulated cells, and cells co-treated with the EGFR inhibitors cetuximab or gefitinib identified groups of differentially expressed proteins. Comparisons of these protein groups identified 13 proteins whose EGF-induced expression changes were reversed by both EGFR inhibitors. Targeted multiple reaction monitoring analysis verified differential expression of 12 of these proteins, which comprise a candidate EGFR inhibition signature. We then tested these 12 proteins by multiple reaction monitoring analysis in three other models: 1) a comparison of DiFi (EGFR inhibitor-sensitive) and HCT116 (EGFR-insensitive) cell lines, 2) in formalin-fixed, paraffin-embedded mouse xenograft DiFi and HCT116 tumors, and 3) in tissue biopsies from a patient with the gastric hyperproliferative disorder Ménétrier's disease who was treated with cetuximab. Of the proteins in the candidate signature, a core group, including c-Jun, Jagged-1, and Claudin 4, were decreased by EGFR inhibitors in all three models. Although the goal of these studies was not to validate a clinically useful EGFR inhibition signature, the results confirm the hypothesis that clinically used EGFR inhibitors generate characteristic protein expression changes. This work further outlines a prototypical approach to derive and test protein expression signatures for drug action on signaling networks.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cetuximab; Chromatography, Liquid; Colorectal Neoplasms; Epidermal Growth Factor; ErbB Receptors; Gastritis, Hypertrophic; Gefitinib; Humans; Mice; Neoplasms; Neoplasms, Glandular and Epithelial; Phosphorylation; Prospective Studies; Proteomics; Quinazolines; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transplantation, Heterologous

Surface plasmon resonance (SPR) biosensor detects intracellular signaling events as a change of the angle of resonance (AR). We previously reported that the activation of epidermal growth factor receptor (EGFR) on keratinocytes causes a unique triphasic change of AR, whereas the activation of other receptors, such as IgE receptor and adenosine A3 receptor on mast cells, causes a transient monophasic increase of AR. To study the mechanism of AR changes induced by EGFR activation, we introduced wild and mutated EGFR cDNAs into Chinese hamster ovary (CHO) cells and analyzed changes of AR in response to EGF. CHO cells expressing wild-type EGFR showed a triphasic change of AR, whereas cells expressing kinase-dead EGFR (K721M) showed minimum change of AR. A phosphatidylinositol 3-kinase inhibitor, wortmannin, attenuated the third phase of AR change in CHO cells expressing wild-type EGFR. The pattern of AR change was independent on the concentration of EGF. We also analyzed changes of AR with a nontumorigenic keratinocyte cell line, HaCaT, and several cell lines of carcinoma to explore the feasibility of SPR biosensor as a tool for clinical diagnosis. The activation of HaCaT cells and one out of six carcinoma cell lines showed a full triphasic change of AR. In contrast, five out of the six cell lines showed mono- or bi-phasic change of AR. These results suggest that EGF induces the SPR signals via the phosphorylation of EGFR, and provide a possibility that the SPR biosensor could be applied to the real-time detection and diagnosis of malignant tumors.

Topics: Animals; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Phosphorylation; Surface Plasmon Resonance

Topics: Epidermal Growth Factor; Genetic Predisposition to Disease; Humans; Neoplasms; Polymorphism, Genetic

Cancer is known to be a complex disease and its therapy is difficult. Much information is available on molecules and pathways involved in cancer onset and progression and this data provides a valuable resource for the development of predictive computer models that can help to identify new potential drug targets or to improve therapies. Modeling cancer treatment has to take into account many cellular pathways usually leading to the construction of large mathematical models. The development of such models is complicated by the fact that relevant parameters are either completely unknown, or can at best be measured under highly artificial conditions. Here we propose an approach for constructing predictive models of such complex biological networks in the absence of accurate knowledge on parameter values, and apply this strategy to predict the effects of perturbations induced by anti-cancer drug target inhibitions on an epidermal growth factor (EGF) signaling network. The strategy is based on a Monte Carlo approach, in which the kinetic parameters are repeatedly sampled from specific probability distributions and used for multiple parallel simulations. Simulation results from different forms of the model (e.g., a model that expresses a certain mutation or mutation pattern or the treatment by a certain drug or drug combination) can be compared with the unperturbed control model and used for the prediction of the perturbation effects. This framework opens the way to experiment with complex biological networks in the computer, likely to save costs in drug development and to improve patient therapy.

Topics: Computer Simulation; Epidermal Growth Factor; Humans; Monte Carlo Method; Neoplasms; Protein Kinase Inhibitors; Signal Transduction; Systems Biology

Phage display has identified the dodecapeptide YHWYGYTPQNVI (GE11) as a ligand that binds to the epidermal growth factor receptor (EGFR) but does not activate the receptor. Here, we compare the EGFR binding affinities of GE11, EGF, and their polyethyleneimine-polyethyleneglycol (PEI-PEG) conjugates. We found that although GE11 by itself does not exhibit measurable affinity to the EGFR, tethering it to PEI-PEG increases its affinity markedly, and complex formation with polyinosine/cytosine (polyIC) further enhances the affinity to the submicromolar range. PolyIC/PPGE11 has a similar strong antitumor effect against EGFR overexpressing tumors in vitro and in vivo, as polyIC/polyethyleneimine-polyetheleneglycol-EGF (polyIC/PP-EGF). Absence of EGFR activation, as previously shown by us and easier production of GE11 and GE11 conjugates, confer polyIC/PPGE11 a significant advantage over similar EGF-based polyplexes as a potential therapy of EGFR overexpressing tumors.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Drug Delivery Systems; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Ligands; Mice; Mice, Nude; Neoplasms; Peptides; Poly I-C; Polyethylene Glycols; Polyethyleneimine; Xenograft Model Antitumor Assays

Aberrant expression and activation of EGF receptor (EGFR) has been implicated in the development and progression of many human cancers. As such, targeted therapeutic inhibition of EGFR, for example by antibodies, is a promising anticancer strategy. The overall efficacy of antibody therapies results from the complex interplay between affinity, valence, tumor penetration and retention, and signaling inhibition. To gain better insight into this relationship, we studied a panel of EGFR single-chain Fv (scFv) antibodies that recognize an identical epitope on EGFR but bind with intrinsic monovalent affinities varying by 280-fold. The scFv were converted to Fab and IgG formats, and investigated for their ability to bind EGFR, compete with EGF binding, and inhibit EGF-mediated downstream signaling and proliferation. We observed that the apparent EGFR-binding affinity for bivalent IgG plateaus at intermediate values of intrinsic affinity of the cognate Fab, leading to a biphasic curve describing the ratio of IgG to Fab affinity. Mathematical modeling of antibody-receptor binding indicated that the biphasic effect results from nonequilibrium assay limitations. This was confirmed by further observation that the potency of EGF competition for antibody binding to EGFR improved with both intrinsic affinity and antibody valence. Similarly, both higher intrinsic affinity and bivalent binding improved the potency of antibodies in blocking cellular signaling and proliferation. Overall, our work indicates that higher intrinsic affinity combined with bivalent binding can achieve avidity that leads to greater in vitro potency of antibodies, which may translate into greater therapeutic efficacy.

Topics: Antibodies, Monoclonal; Antibody Affinity; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Humans; Neoplasms; Phosphorylation; Protein Binding

Cationic liposome-DNA complexes (lipoplexes) are used for the delivery of plasmid DNA to cultured cells and various tissues in vivo. In this chapter, we describe the preparation and evaluation of plain and targeted lipoplexes, using targeting ligands, including epidermal growth factor and transferrin. Ligand-associated lipoplexes may be used to target DNA or other nucleic acid drugs to specific cells, particularly cancer cells that overexpress the receptors for the ligands. We provide examples of the enhancement of gene expression mediated by epidermal growth factor in murine and human oral squamous cell carcinoma cells, and human hepatoblastoma and rat colon adenocarcinoma cells. We also summarize the studies on the use of transferrin-lipoplexes for enhancing gene delivery to cervical carcinoma, murine colon carcinoma, and African green monkey kidney cells. We outline two animal models in which transferrin-lipoplexes have been used for antitumor therapy by delivering either the gene encoding interleukin-12 or a suicide gene: a CT26 murine colon carcinoma, and a syngeneic, orthotopic murine oral squamous cell carcinoma.

Topics: Animals; Cell Line, Tumor; Cell Survival; Chlorocebus aethiops; Epidermal Growth Factor; Female; Genes, Reporter; Genes, Transgenic, Suicide; Genetic Therapy; Green Fluorescent Proteins; Humans; Interleukin-12; Liposomes; Luciferases; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Nanocapsules; Nanomedicine; Neoplasm Transplantation; Neoplasms; Plasmids; Rats; Transfection; Transferrin

The cytoplasmic domains of EGF-like ligands, including EGF cytoplasmic domain (EGFcyt), have important biological functions. Using specific constructs and peptides of human EGF cytoplasmic domain, we demonstrate that EGFcyt facilitates lysosomal and proteasomal protein degradation, and this coincided with growth inhibition of human thyroid and glioma carcinoma cells. EGFcyt and exon 22-23-encoded peptide (EGF22.23) enhanced procathepsin B (procathB) expression and procathB-mediated lysosomal degradation of EGFR/ErbB1 as determined by inhibitors for procathB and the lysosomal ATPase inhibitor BafA1. Presence of mbEGFctF, EGFcyt, EGF22.23, and exon 23-encoded peptides suppressed the expression of the deubiqitinating enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1). This coincided with hyperubiquitination of total cellular proteins and ErbB1/2 and reduced proteasome activity. Upon small interfering RNA-mediated silencing of endogenously expressed UCH-L1, a similar hyperubiquitinylation phenotype, reduced ErbB1/2 content, and attenuated growth was observed. The exon 23-encoded peptide region of EGFcyt was important for these biologic actions. Structural homology modeling of human EGFcyt showed that this molecular region formed an exposed surface loop. Peptides derived from this EGFcyt loop structure may aid in the design of novel peptide therapeutics aimed at inhibiting growth of cancer cells.

Topics: Cathepsin B; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Exons; Humans; Lysosomes; Models, Molecular; Neoplasms; Peptides; Proteasome Endopeptidase Complex; Protein Interaction Domains and Motifs; Proteolysis; Receptor, ErbB-2; RNA Interference; Signal Transduction; Ubiquitin; Ubiquitin Thiolesterase; Ubiquitination

In this work, we have evaluated the ability of targeted lipoplexes to enhance transgene expression in EGF receptor (EGFR) overexpressing tumor cells by using lipoplexes.. We prepared DOTAP/cholesterol liposomes modified with EGF at 0.5/1, 1/1, 2/1 and 5/1 lipid/DNA (+/-) charge ratio by sequentially mixing the liposomes with the ligand and adding the reporter or the therapeutic plasmid gene, pCMVLuc (pVR1216) or pCMVIL12, respectively. HepG2, DHDK12proB and SW620 cells were used for in vitro experiments, which were performed in the presence of 60% serum.. The characterization of EGF-lipoplexes indicated a size close to 300 nm and a variable net surface charge as a function of the amount of EGF associated to the cationic liposomes. EGF-lipoplexes, which showed an increased transfection activity, were positively charged, noncytotoxic and highly effective in protecting DNA from DNase I attack. Transfection activity in vitro resulted in an enhancement in the luciferase and IL-12 expression by EGF-lipoplexes compared with those without ligand (plain-lipoplexes) and to naked DNA. The results observed in SW620 cells, which are deficient in EGFR, confirmed that DNA uptake was predominantly via EGFR-mediated endocytosis. In vivo transfection activity was confirmed by luciferase imaging in living mice. Bioluminiscence could be detected mainly in the lung with a maximum signal 24 h after application. The resulting EGF-lipoplexes significantly increased the level of gene expression in mice compared with control or naked DNA.. These findings indicate that these nanovectors may be an adequate alternative to viral vectors for gene therapy.

Topics: Animals; Cell Line, Tumor; Deoxyribonucleases; DNA; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Genetic Therapy; Genetic Vectors; Humans; Interleukin-12; Liposomes; Mice; Mice, Inbred BALB C; Neoplasms; Transfection; Up-Regulation

Rapid growth of tumor cells coupled with inadequate vascularization leads to shortage of oxygen and nutrients. The unfolded protein response (UPR), a defense cellular mechanism activated during such stress conditions, is a complex process that includes upregulation of the endoplasmic reticulum chaperones, such as glucose-regulated protein 78 (GRP78). Due to its central role in UPR, GRP78 is overexpressed in many cancers; it is implicated in cancer cell survival through supporting of drug- and radioresistance as well as metastatic dissemination, and is generally associated with poor outcome. This is the reason why selective destruction of GRP78 could become a novel anticancer strategy. GRP78 is the only known substrate of the proteolytic A subunit (SubA) of a bacterial AB(5) toxin, and the selective SubA-induced cleavage of GRP78 leads to massive cell death. Targeted delivery of SubA into cancer cells via specific receptor-mediated endocytosis could be a suitable strategy for assaulting tumor cells. We fused SubA to epidermal growth factor (EGF), whose receptor (EGFR) is frequently overexpressed in tumor cells, and demonstrated that the resulting EGF-SubA immunotoxin is an effective killer of EGFR-positive tumor cells. Furthermore, because of its unique mechanism of action, EGF-SubA synergizes with UPR-inducing drugs, which opens a possibility for the development of mechanism-based combination regimens for effective anticancer therapy. In this chapter, we provide experimental protocols for the assessment of the effects of EGF-SubA on EGFR-positive cancer cells, either alone or in combination with UPR-inducing drugs.

Topics: Animals; Antineoplastic Agents; Catechin; Cell Line; Drug Screening Assays, Antitumor; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Heat-Shock Proteins; Humans; Neoplasms; Thapsigargin; Unfolded Protein Response

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Benzimidazoles; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Hedgehog Proteins; Humans; Neoplasm Proteins; Neoplasms; Papillomaviridae; Side-Population Cells; Signal Transduction

Topics: Apoptosis; Cell Differentiation; Cell Proliferation; Cell Survival; Epidermal Growth Factor; Humans; Neoplasm Invasiveness; Neoplasms; Prognosis; Signal Transduction; Transforming Growth Factor beta

In cancer research, it is important to evaluate the performance of a biomarker (e.g., molecular, genetic, or imaging) that correlates patients' prognosis or predicts patients' response to treatment in a large prospective study. Due to overall budget constraint and high cost associated with bioassays, investigators often have to select a subset from all registered patients for biomarker assessment. To detect a potentially moderate association between the biomarker and the outcome, investigators need to decide how to select the subset of a fixed size such that the study efficiency can be enhanced. We show that, instead of drawing a simple random sample from the study cohort, greater efficiency can be achieved by allowing the selection probability to depend on the outcome and an auxiliary variable; we refer to such a sampling scheme as outcome and auxiliary-dependent subsampling (OADS). This article is motivated by the need to analyze data from a lung cancer biomarker study that adopts the OADS design to assess epidermal growth factor receptor (EGFR) mutations as a predictive biomarker for whether a subject responds to a greater extent to EGFR inhibitor drugs. We propose an estimated maximum-likelihood method that accommodates the OADS design and utilizes all observed information, especially those contained in the likelihood score of EGFR mutations (an auxiliary variable of EGFR mutations) that is available to all patients. We derive the asymptotic properties of the proposed estimator and evaluate its finite sample properties via simulation. We illustrate the proposed method with a data example.

Topics: Biomarkers, Tumor; Computer Simulation; Epidermal Growth Factor; Humans; Lung Neoplasms; Methods; Mutation; Neoplasms; Patient Selection; Precision Medicine; Predictive Value of Tests; Prognosis

Doxorubicin nano-aggregate was prepared for the purpose of epidermal growth factor receptor targeted anti-cancer therapy. An epidermal growth factor fragment composed of 11 amino acids was conjugated to an amino terminal of bi-functional poly(ethylene glycol) and doxorubicin was subsequently conjugated to the other carboxyl terminal of the conjugate. A mixture of the conjugate, free doxorubicin, and triethylamine spontaneously formed nano-sized aggregates in aqueous phase, which was confirmed by transmission electron microscopy and dynamic light scattering. A549 cells incubated with doxorubicin nano-aggregates with the epidermal growth factor fragment showed increased endocytic uptakes of the aggregates compared to unmodified aggregates. Pre-blocking of the epidermal growth factor receptor on the cell significantly decreased a degree of the cellular uptakes. Cytotoxicity of the nano-aggregates was significantly increased when epidermal growth factor fragments were decorated on the surface of doxorubicin nano-aggregates, which was confirmed by a live/dead cell assay and a MTT-based cytotoxicity assay. When doxorubicin nano-aggregates were administered to model animals bearing human lung carcinoma, the epidermal growth factor fragment significantly strengthened in vivo anti-cancer effects of doxorubicin nano-aggregates compared to native doxorubicin or unmodified nano-aggregates. Thus, doxorubicin nano-aggregates decorated with an epidermal growth factor fragment is expected to be a potent anti-cancer agent aiming to tumor tissue over-expressing epidermal growth factor receptors.

Topics: Animals; Antibiotics, Antineoplastic; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Doxorubicin; Drug Delivery Systems; Epidermal Growth Factor; Female; Humans; Mice; Mice, Inbred ICR; Mice, Nude; Nanoparticles; Neoplasms; Peptide Fragments

The epidermal growth factor receptor (EGFR) overexpressed in many epithelial tumors is an attractive target for tumor therapy since numerous blocking agents of EGFR signaling have proven their anti-tumor activity. Here we report a novel monoclonal antibody (mAb), A13, which was generated from mice immunized with human cervical carcinoma A431 cells. In addition to binding to soluble EGFR with affinity of K(D) approximately 5.8nM, mAb A13 specifically bound to a variety of tumor cells and human placenta tissues expressing EGFR. A13 efficiently inhibited both EGF-dependant EGFR tyrosine phosphorylation in cervical and breast tumor cells and also in vitro colony formation of EGFR-overexpressing lung tumors. Competition and sandwich ELISAs, competitive surface plasmon resonance, and domain-level epitope mapping analyses demonstrated that mAb A13 competitively bound to the domain III (amino acids 302-503) of EGFR with EGF, but recognized distinct epitopes from those of cetuximab (Erbitux). Our results demonstrated that anti-EGFR mAb A13 interfered with EGFR proliferation signaling by blocking EGF binding to EGFR with different epitopes from those of cetuximab, suggesting that combination therapies of mAb A13 with cetuximab may prove beneficial for anti-tumor therapy.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Base Sequence; Binding Sites, Antibody; Cell Line, Tumor; Cell Proliferation; Cetuximab; Epidermal Growth Factor; Epitope Mapping; Epitopes; ErbB Receptors; Female; Humans; Immunohistochemistry; Male; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Neoplasms; Phosphorylation; Uterine Cervical Neoplasms

Src kinases are key regulators of cellular proliferation, survival, motility, and invasiveness. They play important roles in the regulation of inflammation and cancer. Overexpression or hyperactivity of c-Src has been implicated in the development of various types of cancer, including lung cancer. Src inhibition is currently being investigated as a potential therapy for non-small cell lung cancer in Phase I and II clinical trials. The mechanisms of Src implication in cancer and inflammation are linked to the ability of activated Src to phosphorylate multiple downstream targets that mediate its cellular effector functions. In this study, we reveal that inducible nitric-oxide synthase (iNOS), an enzyme also implicated in cancer and inflammation, is a downstream mediator of activated Src. We elucidate the molecular mechanisms of the association between Src and iNOS in models of inflammation induced by lipopolysaccharide and/or cytokines and in cancer cells and tissues. We identify human iNOS residue Tyr(1055) as a target for Src-mediated phosphorylation. These results are shown in normal cells and cancer cells as well as in vivo in mice. Importantly, such posttranslational modification serves to stabilize iNOS half-life. The data also demonstrate interactions and co-localization of iNOS and activated Src under inflammatory conditions and in cancer cells. This study demonstrates that phosphorylation of iNOS by Src plays an important role in the regulation of iNOS and nitric oxide production and hence could account for some Src-related roles in inflammation and cancer.

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; Enzyme Activation; Enzyme Stability; Epidermal Growth Factor; Epithelium; Half-Life; Humans; Lung; Mice; Mice, Inbred C57BL; Models, Biological; Neoplasms; Nitric Oxide Synthase Type II; Phosphorylation; Phosphoserine; Pneumonia; Protein Transport; src-Family Kinases

Targeted delivery of intracellularly active diagnostics and therapeutics in vivo is a major challenge in cancer nanomedicine. A nanocarrier should possess long circulation time yet be small and stable enough to freely navigate through interstitial space to deliver its cargo to targeted cells. Herein, it is shown that by adding targeting ligands to nanoparticles that mimic high-density lipoprotein (HDL), tumor-targeted sub-30-nm peptide-lipid nanocarriers are created with controllable size, cargo loading, and shielding properties. The size of the nanocarrier is tunable between 10 and 30 nm, which correlates with a payload of 15-100 molecules of fluorescent dye. Ligand-directed nanocarriers targeting epidermal growth factor receptor (EGFR) are confirmed both in vitro and in vivo. The nanocarriers show favorable circulation time, tumor accumulation, and biodistribution with or without the targeting ligand. The EGFR targeting ligand is proved to be essential for the EGFR-mediated tumor cell uptake of the nanocarriers, a prerequisite of intracellular delivery. The results demonstrate that targeted HDL-mimetic nanocarriers are useful delivery vehicles that could open new avenues for the development of clinically viable targeted nanomedicine.

Topics: Animals; Cell Line, Tumor; Drug Carriers; Drug Delivery Systems; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Fluorescence; Green Fluorescent Proteins; Humans; Lipoproteins, HDL; Mice; Mice, Nude; Microscopy, Confocal; Molecular Mimicry; Nanoparticles; Neoplasms; Peptides; Reproducibility of Results; Xenograft Model Antitumor Assays

Epidermal growth factor (EGF) stimulates apoptosis in tumor cells depending on the expression levels of EGF receptor and Her2. We examined the protein levels in 22 different cancer cell lines and assessed the responses to EGF. EGF-induced cytotoxicity was not correlated with the levels of either EGFR or Her2. Fourteen cell lines exhibited decreased cell proliferation, whereas 293T cells did not display any noticeable changes and degraded transiently expressed EGFR following EGF treatment. EGF treatment resulted in significant tumor growth inhibition in some xenografts. Our data indicate that exogenous EGF treatment leads to growth inhibition rather than inducing tumor cell proliferation.

Topics: Animals; Caspases; Cell Death; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Mice; Mice, Inbred BALB C; Neoplasms; Receptor, ErbB-2; Xenograft Model Antitumor Assays

The adaptor protein p140Cap/SNIP is a novel Src-binding protein that regulates Src activation through C-terminal Src kinase (Csk). Here, by gain and loss of function approaches in breast and colon cancer cells, we report that p140Cap immobilizes E-cadherin at the cell membrane and inhibits EGFR and Erk1/2 signalling, blocking scatter and proliferation of cancer cells. p140Cap-dependent regulation of E-cadherin/EGFR cross-talk and cell motility is due to the inhibition of Src kinase. However, rescue of Src activity is not sufficient to restore Erk1/2 phosphorylation and proliferation. Indeed, p140Cap also impairs Erk1/2 phosphorylation by affecting Ras activity, downstream to the EGFR. In conclusion, p140Cap stabilizes adherens junctions and inhibits EGFR and Ras signalling through the dual control of both Src and Ras activities, thus affecting crucial cancer properties such as invasion and growth. Interestingly, p140Cap expression is lost in more aggressive human breast cancers, showing an inverse correlation with EGFR expression. Therefore, p140Cap mechanistically behaves as a tumour suppressor that inhibits signalling pathways leading to aggressive phenotypes.

Topics: Adaptor Proteins, Vesicular Transport; Animals; Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Membrane; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; Neoplasms; Protein Stability; ras Proteins; Receptor Cross-Talk; Signal Transduction; src-Family Kinases

Epithelial-mesenchymal transition (EMT) is a key event in the generation of invasive tumor cells. A hallmark of EMT is the repression of E-cadherin expression, which is regulated by various signal transduction pathways including extracellular signal-regulated kinase (ERK) and Wnt. These pathways are highly interconnected via multiple coupled feedback loops (CFL). As the function of such coupled feedback regulations is difficult to analyze experimentally, we used a systems biology approach where computational models were designed to predict biological effects that result from the complex interplay of CFLs. Using epidermal growth factor (EGF) and Wnt as input and E-cadherin transcriptional regulation as output, we established an ordinary differential equation model of the ERK and Wnt signaling network containing six feedback links and used extensive computer simulations to analyze the effects of these feedback links in isolation and different combinations. The results show that the feedbacks can generate a rich dynamic behavior leading to various dose-response patterns and have a decisive role in determining network responses to EGF and Wnt. In particular, we made two important findings: first, that coupled positive feedback loops composed of phosphorylation of Raf kinase inhibitor RKIP by ERK and transcriptional repression of RKIP by Snail have an essential role in causing a switch-like behavior of E-cadherin expression; and second, that RKIP expression inhibits EMT progression by preventing E-cadherin suppression. Taken together, our findings provide us with a system-level understanding of how RKIP can regulate EMT progression and may explain why RKIP is downregulated in so many metastatic cancer cells.

Topics: Animals; Cadherins; Epidermal Growth Factor; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Feedback, Physiological; Humans; MAP Kinase Signaling System; Mesoderm; Models, Biological; Neoplasms; Phosphatidylethanolamine Binding Protein; Snail Family Transcription Factors; Transcription Factors; Transcription, Genetic; Wnt Proteins

Topics: Animals; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Signal Transduction

Tumor cell motility and invasion is governed by dynamic regulation of the cortical actin cytoskeleton. The actin-binding protein cortactin is commonly upregulated in multiple cancer types and is associated with increased cell migration. Cortactin regulates actin nucleation through the actin related protein (Arp)2/3 complex and stabilizes the cortical actin cytoskeleton. Cortactin is regulated by multiple phosphorylation events, including phosphorylation of S405 and S418 by extracellular regulated kinases (ERK)1/2. ERK1/2 phosphorylation of cortactin has emerged as an important positive regulatory modification, enabling cortactin to bind and activate the Arp2/3 regulator neuronal Wiskott-Aldrich syndrome protein (N-WASp), promoting actin polymerization and enhancing tumor cell movement.. In this report we have developed phosphorylation-specific antibodies against phosphorylated cortactin S405 and S418 to analyze the subcellular localization of this cortactin form in tumor cells and patient samples by microscopy. We evaluated the interplay between cortactin S405 and S418 phosphorylation with cortactin tyrosine phosphorylation in regulating cortactin conformational forms by Western blotting. Cortactin is simultaneously phosphorylated at S405/418 and Y421 in tumor cells, and through the use of point mutant constructs we determined that serine and tyrosine phosphorylation events lack any co-dependency. Expression of S405/418 phosphorylation-null constructs impaired carcinoma motility and adhesion, and also inhibited lamellipodia persistence monitored by live cell imaging.. Cortactin phosphorylated at S405/418 is localized to sites of dynamic actin assembly in tumor cells. Concurrent phosphorylation of cortactin by ERK1/2 and tyrosine kinases enables cells with the ability to regulate actin dynamics through N-WASp and other effector proteins by synchronizing upstream regulatory pathways, confirming cortactin as an important integration point in actin-based signal transduction. Reduced lamellipodia persistence in cells with S405/418A expression identifies an essential motility-based process reliant on ERK1/2 signaling, providing additional understanding as to how this pathway impacts tumor cell migration.

Topics: Actins; Blotting, Western; Cell Line, Tumor; Cell Movement; Cortactin; Epidermal Growth Factor; Green Fluorescent Proteins; Humans; Microscopy, Confocal; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Molecular Dynamics Simulation; Neoplasms; Phosphorylation; Protein Conformation; Pseudopodia; RNA Interference; Serine; Tyrosine; Wiskott-Aldrich Syndrome Protein

The understanding of cellular signaling pathways in malignant tumors is an important aspect of cancer research and modern targeted therapy strategies. Growth factors and their receptors in particular are critical to modern cancer therapy research, because these factors control all phases of tumor development and metastasis. Most importantly, growth factors are responsible for cell survival under cytotoxic drugs and radiotherapy. These growth factor signaling pathways are composed of complex networks that have adapted to efficiently respond to certain disturbances, such as a single agent that targets one aspect of the pathway. Meanwhile, multiple insults to the pathway, such as combination therapy regimens, are known to be effective in shutting down these pathways and, consequently, killing the tumor cell. Research is currently under way to find new ways to exploit fragile aspects of oncogenic networks, such as uncommon, multiple perturbations that target essential hubs through immunotherapy, combinations of antibodies, heat shock protein inhibitors, or novel drug combinations. Complex growth factor signaling networks and novel methods to shut down these networks are described within a framework of engineering and mathematical concepts.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Survival; Combined Modality Therapy; Epidermal Growth Factor; Heat-Shock Proteins; Humans; Immunotherapy; Models, Theoretical; Molecular Targeted Therapy; Neoplasms; Signal Transduction; Translational Research, Biomedical

This study was conducted to explore the relationship between different treatment-modulated EGFR expression and gefitinib sensitivity.. Gefitinib-sensitive (A431) and -resistant (A375, MALME-3M, and SK-MEL 5) tumour cell lines were treated with epidermal growth factor (EGF), gefitinib or radiation in vitro, and EGFR expression levels were measured by using ELISA.. EGF, and gefitinib treatment resulted in significantly higher levels of total and/or phosphorylated EGFR in sensitive than in resistant tumours and this was associated with gefitinib IC(50). In contrast, radiation-modulated EGFR expression, both total and phosphorylated, did not correlate with the efficacy of gefitinib. Stimulation of proliferation by EGF was significantly stronger in A431 than in the other three lines, indicating sensitive tumours were more EGFR-dependent than resistant tumours for cell proliferation.. These findings imply a potential role of EGF- and gefitinib-modulated EGFR expression in predicting gefitinib sensitivity.

Topics: Antineoplastic Agents; Cell Growth Processes; Cell Line, Tumor; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Melanoma; Neoplasms; Protein Kinase Inhibitors; Quinazolines

ACK1 (activated Cdc42-associated kinase 1) is a cytoplasmic tyrosine kinase implicated in trafficking through binding to epidermal growth factor (EGF) receptor and clathrin. Here, we have identified a new ACK1-binding partner, the E3 ubiquitin ligase Nedd4-2, which binds ACK1 via a conserved PPXY-containing region. We show that this motif also binds Nedd4-related proteins and several other WW domain-containing proteins, including the tumor suppressor oxidoreductase Wwox. In HeLa cells ACK1 colocalizes with Nedd4-2 in clathrin-rich vesicles, requiring this PPXY motif. Nedd4-2 strongly down-regulates ACK1 levels when coexpressed, and this process can be blocked by proteasome inhibitor MG132. ACK1 degradation via Nedd4 requires their mutual interaction and a functional E3 ligase; it is also driven by ACK1 activity. ACK1 is polyubiquitinated in vivo, and dominant inhibitory Nedd4 blocks endogenous ACK1 turnover in response to acute EGF treatment. Because EGF stimulation activates ACK1 ( Galisteo, M., Y., Y., Urena, J., and Schlessinger, J. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 9796-9801 ), our result suggest that EGF receptor-mediated ACK1 activation allows Nedd4-2 to drive kinase degradation. Thus the interplay between Nedd4-2-related E3 ligases that regulate ACK1 levels and Cbl that modifies EGF receptor impinges on cell receptor dynamics. These processes are particularly pertinent given the report of genomic amplification of the ACK1 locus in metastatic tumors.

Topics: Amino Acid Motifs; Animals; Chlorocebus aethiops; Clathrin; Clathrin-Coated Vesicles; COS Cells; Cysteine Proteinase Inhibitors; Down-Regulation; Endosomal Sorting Complexes Required for Transport; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Enzymologic; HeLa Cells; Humans; Leupeptins; Nedd4 Ubiquitin Protein Ligases; Neoplasm Metastasis; Neoplasms; Oxidoreductases; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; Protein Structure, Tertiary; Protein Transport; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-cbl; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Ubiquitination; WW Domain-Containing Oxidoreductase

It has been previously shown that gefitinib reverses breast cancer resistance protein (BCRP)-mediated drug resistance. Here, the impact of BCRP on gefitinib-mediated inhibition in epidermal growth factor receptor (EGFR) signaling is evaluated.. Sensitivity to gefitinib was determined by growth inhibition assay, and intracellular gefitinib levels were measured with HPLC. Western blotting was performed to detect EGFR signaling molecules.. BCRP reduced intracellular gefitinib levels and attenuated inhibitory activities of gefitinib to EGF-dependent EGFR signalings including downstream MAPK and Akt pathways in gefitinib-sensitive PC-9 cells. However, gefitinib did not inhibit MAPK and Akt signalings in KB-3-1 and HCT-116 cells, and BCRP-mediated gefitinib-resistance shown in PC-9 cells was not observed in gefitinib-insensitive KB-3-1 and HCT-116 cells.. BCRP transports gefitinib and suppresses its inhibitory effects on EGFR phosphorylation. However, effects of BCRP on gefitinib activity in the EGFR signaling and on gefitinib-resistance were limited in the gefitinib-sensitive cells only.

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Blotting, Western; Chromatography, High Pressure Liquid; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Neoplasm Proteins; Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinazolines; Signal Transduction; Tumor Cells, Cultured

Our previous findings indicated that RSK2 plays a critical role in proliferation and cell transformation induced by tumor promoters, such as epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate, and that kaempferol, a natural compound found in edible plants, selectively inhibits RSK2 activity. However, the molecular mechanism for RSK2 activation is unclear. Herein, we provide evidence showing that NH(2)-terminal kinase domain (NTD) activation of RSK2 is required for the activation of the extracellular signal-regulated kinase-mediated COOH-terminal kinase domain (CTD). We also found that the NTD plays a key role in substrate phosphorylation and that kaempferol binds with the NTD but not the CTD in both the active and inactive forms. Homology modeling of the RSK2 NH(2)-terminal domain and small-molecule docking, validated by mutagenesis experiments, clearly showed that Val(82) and Lys(100) are critical amino acids for kaempferol binding and RSK2 activity. Furthermore, immunohistofluorescence and Western blot results indicated that the RSK2 protein level is markedly higher in cancer cell lines as well as cancer tissues compared with nonmalignant cell lines or normal tissues. In addition, kaempferol inhibited proliferation of malignant human cancer cell lines, including A431, SK-MEL-5 and SK-MEL-28, and HCT-116. These results indicate that targeting RSK2 with natural compounds, such as kaempferol, might be a good strategy for chemopreventive or chemotherapeutic application.

Topics: Binding Sites; Cell Division; Cell Line, Tumor; Cell Transformation, Neoplastic; Epidermal Growth Factor; Humans; Kaempferols; Kinetics; Lysine; Neoplasms; Phosphorylation; Protein Kinases; Recombinant Proteins; Reference Values; Ribosomal Protein S6 Kinases, 90-kDa; Skin; Tetradecanoylphorbol Acetate; Tissue Array Analysis; Valine

Rapid progress in the elucidation of candidate genes and proteins that play a role in disease processes such as cancer has been possible with widespread use of genomics and proteomics in the last ten years. It is becoming important to adapt the knowledge gained from mass analytical techniques to visual techniques that enable spatial-temporal discernment of molecular events. This is significant, particularly for the study of pathways that regulate dynamic processes such as cell migration or early events associated with differentiation such as Ca(2+) signaling. This paper describes the use of techniques that create sharp growth factor gradients suitable for local activation of cell surface receptors. The methods involve retardation of the direct flow to create steep gradients at the surface plane where cells are grown. These methods are shown to be suitable for rapid biological assays such as Ca(2+) transients that occur within 1 min of receptor activation, demonstrating that the speed and level of Ca(2+) transients are related to gradient strength. A microfabricated chamber is also determined to be suitable for longer-term analyses such as cancer cell chemotaxis.

Topics: Animals; Calcium; Chemotaxis; Epidermal Growth Factor; Neoplasms; PC12 Cells; Rats; Receptors, CXCR4

Epidermal growth factor receptor (EGFR) inhibitors are increasingly used in combination with radiotherapy in the treatment of various EGFR-overexpressing cancers. However, little is known about the effects of cell cycle status on EGFR inhibitor-mediated radiosensitization. Using EGFR-overexpressing A431 and UMSCC-1 cells in culture, we found that radiation activated the EGFR and extracellular signal-regulated kinase pathways in quiescent cells, leading to progression of cells from G(1) to S, but this activation and progression did not occur in proliferating cells. Inhibition of this activation blocked S-phase progression and protected quiescent cells from radiation-induced death. To determine if these effects were caused by EGFR expression, we transfected Chinese hamster ovary (CHO) cells, which lack EGFR expression, with EGFR expression vector. EGFR expressed in CHO cells also became activated in quiescent cells but not in proliferating cells after irradiation. Moreover, quiescent cells expressing EGFR underwent increased radiation-induced clonogenic death compared with both proliferating CHO cells expressing EGFR and quiescent wild-type CHO cells. Our data show that radiation-induced enhancement of cell death in quiescent cells involves activation of the EGFR and extracellular signal-regulated kinase pathways. Furthermore, they suggest that EGFR inhibitors may protect quiescent tumor cells, whereas radiosensitization of proliferating cells may be caused by downstream effects such as cell cycle redistribution. These findings emphasize the need for careful scheduling of treatment with the combination of EGFR inhibitors and radiation and suggest that EGFR inhibitors might best be given after radiation in order to optimize clinical outcome.

Topics: Animals; Cell Cycle; Cell Line, Tumor; Cell Proliferation; CHO Cells; Cricetinae; Cricetulus; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Humans; Neoplasms; Radiation-Sensitizing Agents

Targeting dysregulated signaling pathways in tumors has led to the development of a novel class of signal transduction inhibitors, including inhibitors of the epidermal growth factor (EGF) receptor (EGFR). To dissect oncogenic pathways, identify key pathway determinants, and evaluate the efficacy of targeted agents, it is vital to develop technologies that allow the detection of temporal signaling events under physiological conditions. Here we report the application of a label-free optical biosensor to reveal the rapid response of cancer cells to EGF, expressed as a dynamic mass redistribution (DMR) signal. In response to EGF, squamous cell carcinoma of the head and neck cells exhibited a rapid rise in DMR signal, whereas lung adenocarcinoma cells showed a biphasic DMR profile, suggesting a cell type-dependent DMR response. Pharmacological studies suggested the importance of EGFR and the phosphatidylinositol-3 kinase pathway in mediating the EGF-induced DMR response. The defined DMR signatures offer a simple yet sensitive tool for evaluating EGFR-targeted agents, as shown with gefitinib and erlotinib. The assay can also be used for cell-based high-throughput screening of EGF pathway inhibitors, as demonstrated by its robust performance in a 384-well plate format (Z' > 0.5). This technology is applicable to other oncogenic pathways for the discovery of novel therapeutic agents for the treatment of various cancers.

Topics: Antineoplastic Agents; Biosensing Techniques; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Gefitinib; Humans; Neoplasms; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Quinazolines; Signal Transduction

The epidermal growth factor receptor (EGFR) is frequently overexpressed in various tumours of epidermal origin and is held responsible for tumourigenicity and tumour persistence. Increased nuclear factor (NF)-kappaB activity has been suggested to be involved in the malignant behaviour of EGFR-overexpressing cells. However, the mechanisms that regulate EGF-induced NF-kappaB activation are still largely unknown. Here we show that EGF can induce NF-kappaB-dependent gene expression independently from IkappaBalpha degradation or p100 processing in EGFR-overexpressing HEK293T cells. Moreover, EGF-induced NF-kappaB activation could be inhibited by overexpression of ABINs, which were previously identified as intracellular inhibitors of tumour necrosis factor, interleukin-1 and lipopolysaccharide-induced NF-kappaB activation. Knockdown of ABIN-1 by RNA interference boosted the NF-kappaB response upon EGF stimulation. The C-terminal ubiquitin-binding domain containing region of ABINs was crucial and sufficient for NF-kappaB inhibition. Adenoviral gene transfer of ABINs reduced constitutive NF-kappaB activity as well as the proliferation of EGFR-overexpressing A431 and DU145 human carcinoma cells. Altogether, these results demonstrate an important role for an ABIN-sensitive non-classical NF-kappaB signalling pathway in the proliferation of EGFR-overexpressing tumour cells, and indicate a potential use for ABIN gene therapy in the treatment of cancer.

Topics: Adaptor Proteins, Signal Transducing; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA-Binding Proteins; Epidermal Growth Factor; ErbB Receptors; Genetic Therapy; Humans; I-kappa B Proteins; Neoplasms; NF-kappa B; NF-kappa B p52 Subunit; NF-KappaB Inhibitor alpha; Phosphorylation; Protein Structure, Tertiary; RNA Interference; Signal Transduction

Cell motility is involved in physiological and pathological processes such as the invasion and migration of cells. c-Jun N-terminal kinase (JNK) cascades are involved in the invasion and metastasis of cancer cells. However, little is known about the downstream signaling of JNK. In the present study, we used small interfering RNA (siRNA) directed against JNK1 to reduce its expression. We used microarray techniques to compare the gene expression profiles of epidermal growth factor (EGF)-stimulated HeLa cells with and without JNK1 siRNA treatment. We identified a JNK1 target gene, myosin phosphatase-Rho interacting protein (M-RIP). RNA interference-mediated inhibition of JNK1 strongly inhibited M-RIP mRNA expression induced by EGF, as well as the invasion of HeLa cells. In addition, M-RIP siRNA-treated cells showed significantly reduced invasive activity. Thus, a functional analysis of JNK1 and M-RIP with RNA interference reveals a critical role for this cascade in the invasive behavior of cancer cells.

Topics: Adaptor Proteins, Signal Transducing; Cell Movement; Epidermal Growth Factor; Eukaryotic Initiation Factor-4E; Focal Adhesion Kinase 1; Gene Expression Profiling; HeLa Cells; Humans; Mitogen-Activated Protein Kinase 8; Neoplasm Invasiveness; Neoplasms; RNA, Small Interfering; Signal Transduction

Hepatoma-derived growth factor (HDGF) is highly expressed in human cancer and its expression is correlated with poor prognosis of cancer. The growth factor is known to stimulate cell growth while the underlying mechanism is however not clear. Transfection with HDGF cDNA stimulated while its specific antisense oligonucleotides repressed the growth of human hepatocellular carcinoma HepG2 cells. Furthermore, knock-down of HDGF by antisense oligos also induced apoptosis in HepG2 cells and in other human cancer cells, e.g. human squamous carcinoma A431 cells. HDGF knock-down was found to induce the expression of the pro-apoptotic protein Bad and also inactivate ERK and Akt, which in turn led to dephosphorylation of Bad at Ser-112, Ser-136, and activation of the intrinsic apoptotic pathway, i.e. depolarization of the mitochondrial membrane, release of mitochondrial cytochrome c, increase in the processing of caspase 9 and 3. As HDGF knock-down not only suppresses the growth but also induces apoptosis in human cancer cells, HDGF may therefore serve as a survival factor for human cancer cells and a potential target for cancer therapy.

Topics: Apoptosis; bcl-Associated Death Protein; Cell Line, Tumor; Cell Proliferation; Colforsin; Down-Regulation; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; Intercellular Signaling Peptides and Proteins; Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-akt

Protein phosphorylation mediates many critical cellular responses and is essential for many biological functions during development. About one-third of cellular proteins are phosphorylated, representing the phosphor-proteome, and phosphorylation can alter a protein's function, activity, localization and stability. Tyrosine phosphorylation events mediated by aberrant activation of Receptor Tyrosine Kinase (RTK) pathways have been proven to be involved in the development of several diseases including cancer. To understand the systems biology of RTK activation, we have developed a phosphor-proteome focused on tyrosine phosphorylation events under insulin and EGF signaling pathways using the PhosphoScan technique coupled with high-throughput mass spectrometry analysis. Comparative proteomic analyses of all these tyrosine phosphorylation events revealed that around 70% of these pY events are conserved in human orthologs and paralogs. A careful analysis of published in vivo tyrosine phosphorylation events from literature and patents revealed that around 38% of pY events from Drosophila proteins conserved on 185 human proteins are confirmed in vivo tyrosine phosphorylation events. Hence the data are validated partially based on available reports, and the credibility of the remaining 62% of novel conserved sites that are unpublished so far is very high but requires further follow-up studies. The novel pY events found in this study that are conserved on human proteins could potentially lead to the discovery of drug targets and biomarkers for the detection of various cancers and neurodegenerative diseases.

Topics: Animals; Conserved Sequence; Drosophila; Drosophila Proteins; Epidermal Growth Factor; ErbB Receptors; Humans; Insulin; Neoplasms; Neurodegenerative Diseases; Phosphopeptides; Phosphoproteins; Protein-Tyrosine Kinases; Proteome; Receptor Protein-Tyrosine Kinases; Signal Transduction; Species Specificity

Soluble isoforms of the epidermal growth factor receptor (sEGFR) previously have been identified in the conditioned culture media (CCM) of the vulvar adenocarcinoma cell line, A431 and within exosomes of the keratinocyte cell line HaCaT. Here, we report that the extracellular domain (ECD) of EGFR is shed from the cell surface of human carcinoma cell lines that express 7x10(5) receptors/cell or more. We purified this proteolytic isoform of EGFR (PI-sEGFR) from the CCM of MDA-MB-468 breast cancer cells. The amino acid sequence of PI-sEGFR was determined by reverse-phase HPLC nano-electrospray tandem mass spectrometry of peptides generated by trypsin, chymotrypsin or GluC digestion. The PI-sEGFR protein is identical in amino acid sequence to the EGFR ECD. The release of PI-sEGFR from MDA-MB-468 cells is enhanced by phorbol 12-myristate 13-acetate, heat-inactivated fetal bovine serum, pervanadate, and EGFR ligands (i.e., EGF and TGF-alpha). In addition, 4-aminophenylmercuric acetate, an activator of metalloproteases, increased PI-sEGFR levels in the CCM of MDA-MB-468 cells. Inhibitors of metalloproteases decreased the constitutive shedding of EGFR while the PMA-induced shedding was inhibited by metalloprotease inhibitors, by the two serine protease inhibitors leupeptin and 3,4-dichloroisocoumarin (DCI), and by the aspartyl inhibitor pepstatin. These results suggest that PI-sEGFR arises by proteolytic cleavage of EGFR via a mechanism that is regulated by both PKC- and phosphorylation-dependent pathways. Our results further suggest that when proteolytic shedding of EGFR does occur, it is correlated with a highly malignant phenotype.

Topics: Amino Acid Sequence; Animals; Cattle; Cell Line, Tumor; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; Molecular Sequence Data; Neoplasms; Peptides; Phenylmercuric Acetate; Protease Inhibitors; Protein Isoforms; Protein Kinase C; Protein Structure, Tertiary; Sequence Alignment; Spectrometry, Mass, Electrospray Ionization; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha; Vanadates

Previous studies demonstrated the EGF-targeted phagemid particles carrying siRNA against Akt could be expressed efficiently in the presence of hydroxycamptothecin (HCPT). However, no significant cell growth inhibition was obtained. This study was to further investigate whether the EGF-targeted phagemid particles carrying siRNA would be a promising tool for anti-cancer siRNA delivery.. We found that pSi4.1-siFAK phagemid particles could significantly inhibit the expression of focal adhesion kinase in the HCPT-treated cells. Moreover, we also observed that the particles could potently suppress cell growth and cell invasion.. These results indicated that EGF-targeted phagemid particles might be a promising tool for anti-cancer siRNA delivery in the presence of HCPT.

Topics: Antineoplastic Agents, Phytogenic; Bacteriophages; Base Sequence; Camptothecin; Cell Growth Processes; Cell Line, Tumor; Cell Movement; Combined Modality Therapy; Epidermal Growth Factor; Focal Adhesion Protein-Tyrosine Kinases; Genetic Vectors; Humans; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasms; RNA Interference; RNA, Small Interfering

Cripto is a cell surface protein highly expressed in certain solid tumors, and overexpression of Cripto protein is oncogenic. Cripto-1 protein is encoded by CRIPTO1 gene. CRIPTO3, a presumed pseudogene, has an open reading frame with six amino acid differences from Cripto-1. We show that CRIPTO3 mRNA is the CRIPTO message expressed in many cancer samples. A CRIPTO3 SAGE tag was found in several cancer SAGE libraries, while the CRIPTO1 tag was found in ES cell libraries. In vitro experiments indicate both Cripto-1 and Cripto-3 proteins are functional in the Nodal-dependent signal pathway. Our data indicate that CRIPTO3 is an expressed gene, particularly in certain cancers, and suggest a potentially novel mechanism of oncogenesis through activation of a retrogene.

Topics: Amino Acid Sequence; Cell Line, Tumor; Cell Transformation, Neoplastic; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Molecular Sequence Data; Neoplasm Proteins; Neoplasms; Pseudogenes; Transcription, Genetic

We have previously shown that treatment of human epidermoid carcinoma A431 cells with grape seed proanthocyanidins (GSPs) induces apoptosis of A431 cells. Here, we report that treatment of A431 cells with GSPs inhibits constitutive as well as EGF-induced higher levels of phosphorylated proteins of MAPK family in a dose-dependent manner. This effect is associated with the reactivation of MAP kinase phosphatases. Western blot analysis reveals that GSPs decrease: (i) the levels of phosphatidylinositol 3-kinase (PI3K) and the phosphorylation of Akt at ser473, and (ii) the constitutive activation of NF-kappaB/p65. As NF-kappaB-targeted genes play crucial roles in tumor cell proliferation and differentiation, we assessed the effect of GSPs on proteins encoded by these genes. Treatment with GSPs results in inhibition of the expression of COX-2, iNOS, PCNA, cyclin D1 and MMP-9 in A431 cells compared with non-GSPs-treated controls. Treatment of athymic nude mice with GSPs by oral gavage (50 or 100 mg/kg body weight/mouse) reduces the growth of A431-xenografts in mice, which is associated with the inhibition of tumor cell proliferation in xenografts as indicated by the inhibition of mRNA expression of PCNA and cyclin D1, and of NF-kappaB activity. Together, the data suggest that GSPs might be effective in the treatment of skin cancers.

Topics: Animals; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms; NF-kappa B; Proanthocyanidins; Signal Transduction

Lewis Y (LeY) antigen is highly expressed in a variety of human carcinomas of epithelial cell origin. Recent studies suggest functional blockade of LeY may provide a novel therapeutic approach for the treatment of cancers. However, suppressing LeY expression by genetic manipulation and its impact on neoplastic cell proliferation has not been investigated. We report here that different fucosyltransferases (FUTs) were expressed with the greatest expression of fucosyltransferase I or IV (FUT1/4), the two key enzymes for the synthesis of LeY in human epidermoid carcinoma A431 cells. Knocking down FUT1/4 expression by short interfering RNA technique dramatically reduced the expression of FUT1/4 and LeY and inhibited cell proliferation through decreasing epidermal growth factor receptor (EGFR) signaling pathway. Treatment of A431 cells that were inoculated into the nude mice with FUT1 siRNA or FUT4 siRNA greatly impeded tumor growth. Suppressing FUT1/4 expression also blocked EGF-induced tyrosine phosphorylation of EGFR and mitogen-activated protein kinases. In conclusion, suppressing the expression of FUT1/4 by RNAi technology reduces the synthesis of LeY and inhibits cancer growth. It may serve as a potential methodology for the treatment of cancers that express LeY glycoconjugates.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Fucosyltransferases; Galactoside 2-alpha-L-fucosyltransferase; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Lewis Blood Group Antigens; Lewis X Antigen; MAP Kinase Signaling System; Mice; Mice, Nude; Mitogen-Activated Protein Kinases; Neoplasms; Phosphotyrosine; RNA, Small Interfering; Transfection; Xenograft Model Antitumor Assays

Class IA PI3Ks (phosphoinositide 3-kinases) generate the secondary messenger PtdIns(3,4,5)P(3), which plays an important role in many cellular responses. The accumulation of PtdIns(3,4,5)P(3) in cell membranes is routinely measured using GFP (green fluorescent protein)-labelled PH (pleckstrin homology) domains. However, the kinetics of membrane PtdIns(3,4,5)P(3) synthesis and turnover as detected by PH domains have not been validated using an independent method. In the present study, we measured EGF (epidermal growth factor)-stimulated membrane PtdIns(3,4,5)P(3) production using a specific monoclonal anti-PtdIns(3,4,5)P(3) antibody, and compared the results with those obtained using PH-domain-dependent methods. Anti-PtdIns(3,4,5)P(3) staining rapidly accumulated at the leading edge of EGF-stimulated carcinoma cells. PtdIns(3,4,5)P(3) levels were maximal at 1 min, and returned to basal levels by 5 min. In contrast, membrane PtdIns(3,4,5)P(3) production, measured by the membrane translocation of an epitope-tagged (BTK)PH (PH domain of Bruton's tyrosine kinase), remained approx. 2-fold above basal level throughout 4-5 min of EGF stimulation. To determine the reason for this disparity, we measured the rate of PtdIns(3,4,5)P(3) hydrolysis by measuring the decay of the PtdIns(3,4,5)P(3) signal after LY294002 treatment of EGF-stimulated cells. LY294002 abolished anti-PtdIns(3,4,5)P(3) membrane staining within 10 s of treatment, suggesting that PtdIns(3,4,5)P(3) turnover occurs within seconds of synthesis. In contrast, (BTK)PH membrane recruitment, once initiated by EGF, was relatively insensitive to LY294002. These data suggest that sequestration of PtdIns(3,4,5)P(3) by PH domains may affect the apparent kinetics of PtdIns(3,4,5)P(3) accumulation and turnover; consistent with this hypothesis, we found that GRP-1 (general receptor for phosphoinositides 1) PH domains [which, like BTK, are specific for PtdIns(3,4,5)P(3)] inhibit PTEN (phosphatase and tensin homologue deleted on chromosome 10) dephosphorylation of PtdIns(3,4,5)P(3) in vitro. These data suggest that anti-PtdIns(3,4,5)P(3) antibodies are a useful tool to detect localized PtdIns(3,4,5)P(3), and illustrate the importance of using multiple approaches for the estimation of membrane phosphoinositides.

Topics: Amino Acid Motifs; Animals; Cell Line, Tumor; Epidermal Growth Factor; Epitopes; Gastrointestinal Hormones; Gene Expression Regulation; Immunoassay; Kinetics; Neoplasms; Phosphatidylinositol Phosphates; PTEN Phosphohydrolase; Rats; Sensitivity and Specificity

Angiogenesis, the growth of new blood vessels from the pre-existing vasculature is associated with physiological (for example wound healing) and pathological conditions (tumour development). Vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) are the major angiogenic regulators. We have identified a natural product (cheiradone) isolated from a Euphorbia species which inhibited in vivo and in vitro VEGF- stimulated angiogenesis but had no effect on FGF-2 or EGF activity. Two primary cultures, bovine aortic and human dermal endothelial cells were used in in vitro (proliferation, wound healing, invasion in Matrigel and tube formation) and in vivo (the chick chorioallantoic membrane) models of angiogenesis in the presence of growth factors and cheiradone. In all cases, the concentration of cheiradone which caused 50% inhibition (IC50) was determined. The effect of cheiradone on the binding of growth factors to their receptors was also investigated.. Cheiradone inhibited all stages of VEGF-induced angiogenesis with IC50 values in the range 5.20-7.50 microM but did not inhibit FGF-2 or EGF-induced angiogenesis. It also inhibited VEGF binding to VEGF receptor-1 and 2 with IC50 values of 2.9 and 0.61 microM respectively.. Cheiradone inhibited VEGF-induced angiogenesis by binding to VEGF receptors -1 and -2 and may be a useful investigative tool to study the specific contribution of VEGF to angiogenesis and may have therapeutic potential.

Topics: Angiogenesis Inhibitors; Animals; Cattle; Cell Differentiation; Chick Embryo; Chorioallantoic Membrane; Collagen; Diterpenes; Drug Combinations; Endothelial Cells; Epidermal Growth Factor; Euphorbia; Fibroblast Growth Factor 2; Humans; Laminin; Neoplasm Invasiveness; Neoplasms; Neovascularization, Pathologic; Neovascularization, Physiologic; Phytotherapy; Plant Preparations; Proteoglycans; Receptors, Vascular Endothelial Growth Factor; Wound Healing

We examined the role of the actin nucleation promoters neural Wiskott-Aldrich syndrome protein (N-WASP) and WAVE2 in cell protrusion in response to epidermal growth factor (EGF), a key regulator in carcinoma cell invasion. We found that WAVE2 knockdown (KD) suppresses lamellipod formation and increases filopod formation, whereas N-WASP KD has no effect. However, simultaneous KD of both proteins results in the formation of large jagged protrusions with lamellar properties and increased filopod formation. This suggests that another actin nucleation activity is at work in carcinoma cells in response to EGF. A mammalian Diaphanous-related formin, mDia1, localizes at the jagged protrusions in double KD cells. Constitutively active mDia1 recapitulated the phenotype, whereas inhibition of mDia1 blocked the formation of these protrusions. Increased RhoA activity, which stimulates mDia1 nucleation, was observed in the N-WASP/WAVE2 KD cells and was shown to be required for the N-WASP/WAVE2 KD phenotype. These data show that coordinate regulation between the WASP family and mDia proteins controls the balance between lamellar and lamellipodial protrusion activity.

Topics: Actin Cytoskeleton; Animals; Carcinoma; Carrier Proteins; Cell Line, Tumor; Cell Movement; Cell Surface Extensions; Cytochrome-B(5) Reductase; Down-Regulation; Epidermal Growth Factor; Formins; Neoplasm Invasiveness; Neoplasms; Pseudopodia; Rats; rhoA GTP-Binding Protein; Wiskott-Aldrich Syndrome Protein Family; Wiskott-Aldrich Syndrome Protein, Neuronal

Galectin-3 (Gal-3) is a pleiotropic beta-galactoside-binding protein expressed at relatively high levels in human neoplasms. Its carbohydrate recognition domain (CRD) contains a hydrophobic pocket that can accommodate the farnesyl moiety of K-Ras. Binding of K-Ras to Gal-3 stabilizes K-Ras in its active (GTP-bound) state. Gal-3, which does not interact with N-Ras, was nevertheless shown to reduce N-Ras-GTP in BT-549 cells by an unknown mechanism that we explored here. First, comparative analysis of various cancer cell lines (glioblastomas, breast cancer cells and ovarian carcinomas) showed a positive correlation between low N-Ras-GTP/high K-Ras-GTP phenotype and Gal-3 expression levels. Next we found that epidermal growth factor-stimulated GTP loading of N-Ras, but not of K-Ras, is blocked in cells expressing high levels of Gal-3. Activation of Ras guanine nucleotide releasing proteins (RasGRPs) by phorbol 12-myristate 13-acetate (PMA) or downregulation of Gal-3 by Gal-3 shRNA increased the levels of N-Ras-GTP in Gal-3 expressing cells. We further show that the N-terminal domain of Gal-3 interacts with and inhibits RasGRP4-mediated GTP loading on N-Ras and H-Ras proteins. Growth of BT-549 cells stably expressing the Gal-3 N-terminal domain was strongly attenuated. Overall, these experiments demonstrate a new control mechanism of Ras activation in cancer cells whereby the Gal-3 N-terminal domain inhibits activation of N-Ras and H-Ras proteins.

Topics: Animals; Blotting, Western; Cricetinae; Epidermal Growth Factor; Galectin 3; Genes, ras; Guanosine Triphosphate; Humans; Immunoprecipitation; Neoplasms; ras Guanine Nucleotide Exchange Factors; ras Proteins; Rats; RNA, Small Interfering; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The events occurring during tumor formation and progression display similarities to some of the steps in embryonic morphogenesis. The family of AP-2 proteins consists of five different transcription factors (alpha, beta, gamma, delta, and epsilon) that play relevant roles in embryonic development, as demonstrated by the phenotypes of the corresponding knockout mice. Here, we show that AP-2alpha and AP-2gamma proteins play an essential role in tumorigenesis. Down-modulation of AP-2 expression in tumor cells by RNA interference (RNAi) led to enhanced tumor growth and reduced chemotherapy-induced cell death, as well as migration and invasion. Most of these biological modulations were rescued by AP-2 overexpression. We observed that increased xenotransplant growth was mostly due to highly enhanced proliferation of the tumor cells together with reduced innate immune cell recruitment. Moreover, we showed that migration impairment was mediated, at least in part, by secreted factors. To identify the genetic programs involved in tumorigenesis, we performed whole genome microarray analysis of AP-2alpha knockdown cells and observed that AP-2alpha regulates specific genes involved in cell cycle, cell death, adhesion, and migration. In particular, we showed that ESDN, EREG, and CXCL2 play a major role in AP-2 controlled migration, as ablation of any of these genes severely altered migration.

Topics: Animals; Base Sequence; Breast Neoplasms; Cell Death; Cell Division; Cell Line, Tumor; Cell Movement; Chemokine CXCL2; Epidermal Growth Factor; Epiregulin; Female; HeLa Cells; Humans; Membrane Proteins; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Neoplasms; Oligonucleotide Array Sequence Analysis; RNA Interference; RNA, Small Interfering; Transcription Factor AP-2; Transplantation, Heterologous

Cancer cells in their respective microenvironments must endure various growth-constraining stresses. Under these conditions, the cancer cell-derived factors are thought to modulate the signaling pathways between cell growth and dormancy. Here, we describe a cancer cell-derived regulatory system that modulates the phosphatidylinositol 3'-kinase (PI3K)-Akt pathway under serum deprivation stress. Through the use of biochemical purification, we reveal that cancer cell-secreted insulin-like growth factor 1 (IGF-1) and clusterin, an extracellular stress protein, constitute this regulatory system. We show that secreted clusterin associates with IGF-1 and inhibits its binding to the IGF-1 receptor and hence negatively regulates the PI3K-Akt pathway during serum deprivation. This inhibitory function of clusterin appears to prefer IGF-1, as it fails to exert any effects on epidermal growth factor signaling. We demonstrate furthermore that the constitutive activation of oncogenic signaling downstream of IGF-1 confers insensitivity to the inhibitory effects of clusterin. Thus, the interplay between cancer cell-derived clusterin and IGF-1 may dictate the outcome of cell growth and dormancy during tumorigenic progression.

Topics: Amino Acid Sequence; Animals; Cell Line; Cell Proliferation; Clusterin; Culture Media, Serum-Free; Enzyme Activation; Epidermal Growth Factor; Humans; Insulin-Like Growth Factor I; Mice; Molecular Sequence Data; Neoplasms; Phosphatidylinositol 3-Kinases; Protein Binding; Proto-Oncogene Proteins c-akt; Receptor, IGF Type 1; Signal Transduction; Zebrafish

We have shown that one of the principle mechanisms of chemotherapy resistance involves the activation of nuclear factor kappa-B (NF-kappaB). In an effort to identify NF-kappaB-regulated chemotherapy response genes, we performed a microarray assay and observed that heparin-binding EGF-like growth factor (HB-EGF) was significantly upregulated by SN38 (a strong inducer of NF-kappaB activity) in colon cancer cells. Further studies revealed that HB-EGF was rapidly induced following a variety of chemotherapy treatments. Using RNA interference, we demonstrated that the chemotherapy-induced HB-EGF was largely dependent on activator protein-1 (AP-1) and NF-kappaB activation. Constitutive HB-EGF expression rescued AP-1/NF-kappaB small interfering RNA (siRNA) cells from chemotherapy-induced apoptosis. Meanwhile, we found that the enzymatic shedding of HB-EGF was also regulated by chemotherapy treatment, resulting in the elevated release of soluble HB-EGF from the cellular membrane. Induction of HB-EGF expression and ectodomain shedding synergistically led to robust epidermal growth factor receptor (EGFR) phosphorylation, whereas inhibition of HB-EGF expression by use of the HB-EGF inhibitor (CRM197) or siRNA resulted in the suppression of chemotherapy-induced EGFR phosphorylation. These results suggest that the chemotherapy-induced EGFR activation is regulated by HB-EGF. Finally, we demonstrated that overexpression of HB-EGF led to apoptotic resistance to chemotherapy, whereas suppression of HB-EGF expression by siRNA resulted in a dramatic increase in cell death. In summary, our study suggests that chemotherapy-induced HB-EGF activation represents a critical mechanism of inducible chemotherapy resistance. Therefore, therapeutic intervention aimed at inhibiting HB-EGF activity may be useful in cancer prevention and treatments.

Topics: Apoptosis; Bacterial Proteins; Cell Line, Tumor; Drug Resistance, Neoplasm; Epidermal Growth Factor; Gene Expression; Gene Expression Regulation, Neoplastic; Genes, fos; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Neoplasms; NF-kappa B; RNA, Small Interfering; Transcription Factor AP-1; Transcription Factor RelA

All ligands of the epidermal growth factor receptor (EGFR), which has important roles in development and disease, are made as transmembrane precursors. Proteolytic processing by ADAMs (a disintegrin and metalloprotease) regulates the bioavailability of several EGFR-ligands, yet little is known about the enzyme responsible for processing the recently identified EGFR ligand, epigen. Here we show that ectodomain shedding of epigen requires ADAM17, which can be stimulated by phorbol esters, phosphatase inhibitors and calcium influx. These results suggest that ADAM17 might be a good target to block the release of bioactive epigen, a highly mitogenic ligand of the EGFR which has been implicated in wound healing and cancer.

Topics: ADAM Proteins; ADAM17 Protein; Animals; Cells, Cultured; Epidermal Growth Factor; Epigen; ErbB Receptors; Fibroblasts; Growth Substances; Humans; Ligands; Membrane Proteins; Mice; Neoplasms; Protease Inhibitors; Protein Precursors; Protein Processing, Post-Translational; Protein Structure, Tertiary; Wound Healing

Adenovirus (Ad)-based vectors are useful gene delivery vehicles for a variety of applications. Despite their attractive properties, many in vivo applications require modulation of the viral tropism. Targeting approaches applied to adenoviral vectors included genetic modification of the viral capsid, controlled expression of the transgene and combinatorial approaches that combine two or more targeting elements in single vectors. Most of these studies confirmed successful retargeting in cell cultures, however, in vivo gains of targeted adenoviral vectors have not been widely demonstrated. We have developed a combinatorial retargeting approach utilizing metabolically biotinylated Ad, where the biotin acceptor peptide was incorporated in one of the fibers in a dual fiber viral particle resulting in metabolically biotinylated fiber-mosaic Ad (mBfMAd). We have utilized this vector in complex with epidermal growth factor (EGF)-Streptavidin to retarget fiber-mosaic virus to EGF receptor (EGFR) expressing cells in vitro and confirmed an increased infectivity of the retargeting complex. Most importantly, the utility of this strategy was demonstrated in vivo in two distinct animal models. In both models tested, retargeted mBfMAd demonstrated an increased ratio of gene expression in target tissues compared to the liver expression profile. Thus, metabolically biotinylated fiber-mosaic virus in combination with appropriate adapters can be successfully exploited for adenoviral retargeting strategies.

Topics: Adenoviridae; Animals; Biotinylation; Cell Line, Tumor; Coxsackie and Adenovirus Receptor-Like Membrane Protein; Epidermal Growth Factor; ErbB Receptors; Female; Gene Targeting; Genetic Therapy; Genetic Vectors; Humans; Luciferases; Lung; Mice; Mice, SCID; Mice, Transgenic; Models, Animal; Neoplasms; Receptors, Virus; Recombinant Fusion Proteins; Streptavidin; Transduction, Genetic; Transgenes

EphA2 overexpression has been reported in many cancers and is believed to play an important role in tumor metastasis and angiogenesis. We show that the activated epidermal growth factor receptor (EGFR) and the cancer-specific constitutively active EGFR type III deletion mutant (EGFRvIII) induce the expression of EphA2 in mammalian cell lines, including the human cancer cell lines A431 and HN5. The regulation is partially dependent on downstream activation of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase and is a direct effect on the EphA2 promoter. Furthermore, EGFR and EphA2 both localize to the plasma membrane and EphA2 coimmunoprecipitates with activated EGFR and EGFRvIII. Ligand activation of EphA2 and EphA2 knockdown by small interfering RNA inhibit EGF-induced cell motility of EGFR-overexpressing human cancer cells, indicating a functional role of EphA2 in EGFR-expressing cancer cells.

Topics: Cell Line, Tumor; Cell Membrane; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Humans; MAP Kinase Kinase Kinases; Neoplasms; Promoter Regions, Genetic; Protein Biosynthesis; Protein Kinase Inhibitors; Receptor, EphA2; RNA, Small Interfering; Transcriptional Activation

Systems wide modeling and analysis of signaling networks is essential for understanding complex cellular behaviors, such as the biphasic responses to different combinations of cytokines and growth factors. For example, tumor necrosis factor (TNF) can act as a proapoptotic or prosurvival factor depending on its concentration, the current state of signaling network and the presence of other cytokines. To understand combinatorial regulation in such systems, new computational approaches are required that can take into account non-linear interactions in signaling networks and provide tools for clustering, visualization and predictive modeling.. Here we extended and applied an unsupervised non-linear dimensionality reduction approach, Isomap, to find clusters of similar treatment conditions in two cell signaling networks: (I) apoptosis signaling network in human epithelial cancer cells treated with different combinations of TNF, epidermal growth factor (EGF) and insulin and (II) combination of signal transduction pathways stimulated by 21 different ligands based on AfCS double ligand screen data. For the analysis of the apoptosis signaling network we used the Cytokine compendium dataset where activity and concentration of 19 intracellular signaling molecules were measured to characterise apoptotic response to TNF, EGF and insulin. By projecting the original 19-dimensional space of intracellular signals into a low-dimensional space, Isomap was able to reconstruct clusters corresponding to different cytokine treatments that were identified with graph-based clustering. In comparison, Principal Component Analysis (PCA) and Partial Least Squares - Discriminant analysis (PLS-DA) were unable to find biologically meaningful clusters. We also showed that by using Isomap components for supervised classification with k-nearest neighbor (k-NN) and quadratic discriminant analysis (QDA), apoptosis intensity can be predicted for different combinations of TNF, EGF and insulin. Prediction accuracy was highest when early activation time points in the apoptosis signaling network were used to predict apoptosis rates at later time points. Extended Isomap also outperformed PCA on the AfCS double ligand screen data. Isomap identified more functionally coherent clusters than PCA and captured more information in the first two-components. The Isomap projection performs slightly worse when more signaling networks are analyzed; suggesting that the mapping function between cues and responses becomes increasingly non-linear when large signaling pathways are considered.. We developed and applied extended Isomap approach for the analysis of cell signaling networks. Potential biological applications of this method include characterization, visualization and clustering of different treatment conditions (i.e. low and high doses of TNF) in terms of changes in intracellular signaling they induce.

Topics: Algorithms; Apoptosis; Cluster Analysis; Cytokines; Discriminant Analysis; Epidermal Growth Factor; Humans; Insulin; Least-Squares Analysis; Metabolic Networks and Pathways; Models, Biological; Neoplasms; Neural Networks, Computer; Principal Component Analysis; Signal Transduction; Tumor Necrosis Factor-alpha

With the completion of the human genome project, analysis of enriched phosphotyrosyl proteins from epidermal growth factor (EGF)-induced phosphotyrosine proteome permits the identification of novel downstream substrates of the EGF receptor (EGFR). Using cICAT-based LC-MS/MS method, we identified and relatively quantified the tyrosine phosphorylation levels of 21 proteins between control and EGF-treated A431 human cervical cancer cells. Of these, Endofin, DCBLD2, and KIAA0582 were validated to be novel tyrosine-phosphorylation targets of EGF signaling and Iressa, a highly selective inhibitor of EGFR. In addition, EGFR activity was shown to be necessary for EGF-induced localization of Endofin, an FYVE domain-containing protein regulated by phosphoinositol lipid and engaged in endosome-mediated receptor modulation. Although several groups have conducted phosphoproteomics of EGF signaling in recent years, our study is the first to identify and validate Endofin, DCBLD2, and KIAA0582 as part of a complex EGF phosphotyrosine signaling network. These novel data will provide new insights into the complex EGF signaling and may have implications on target-directed cancer therapeutics.

Topics: Amino Acid Sequence; Cell Line, Tumor; Epidermal Growth Factor; Gefitinib; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Microtubule-Associated Proteins; Molecular Sequence Data; Neoplasms; Phosphoproteins; Phosphotyrosine; Proteomics; Quinazolines; Serine Endopeptidases; Signal Transduction; Tandem Mass Spectrometry

The marine natural chamigrane-type sesquiterpenoid, dactylone, is closely related to secondary metabolites of some edible species of red algae. In the present study, the effect of dactylone was tested on the mouse skin epidermal JB6 P+ Cl41 cell line and its stable transfectants as well as on several human tumor cell lines, including lung (H460), colon (HCT-116), and skin melanomas (SK-MEL-5 and SK-MEL-28). This natural product was effective at nontoxic doses as a cancer-preventive agent, which exerted its actions, at least in part, through the inhibition of cyclin D3 and Cdk4 expression and retinoblastoma tumor suppressor protein (Rb) phosphorylation. The inhibition of these cell cycle components was followed by cell cycle arrest at the G1-S transition with subsequent p53-independent apoptosis. Therefore, these data showed that application of dactylone and related compounds may lead to decreased malignant cell transformation and/or decreased tumor cell proliferation.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Cycle; Cell Line, Tumor; Cell Transformation, Neoplastic; Epidermal Growth Factor; Flow Cytometry; G1 Phase; Humans; Mice; Neoplasms; Phenotype; S Phase; Sesquiterpenes; Transfection

To determine the importance of single-chain Fv (scFv) affinity on binding, uptake, and cytotoxicity of tumor-targeting nanoparticles, the affinity of the epidermal growth factor receptor (EGFR) scFv antibody C10 was increased using molecular evolution and yeast display. A library containing scFv mutants was created by error-prone PCR, displayed on the surface of yeast, and higher affinity clones selected by fluorescence activated cell sorting. Ten mutant scFv were identified that had a 3-18-fold improvement in affinity (KD=15-88 nM) for EGFR-expressing A431 tumor cells compared to C10 scFv (KD=264 nM). By combining mutations, higher affinity scFv were generated with KD ranging from 0.9 nM to 10 nM. The highest affinity scFv had a 280-fold higher affinity compared to that of the parental C10 scFv. Immunoliposome nanoparticles (ILs) were prepared using EGFR scFv with a 280-fold range of affinities, and their binding and uptake into EGFR-expressing tumor cells was quantified. At scFv densities greater than 148 scFv/IL, there was no effect of scFv affinity on IL binding and uptake into tumor cells, or on cytotoxicity. At lower scFv densities, there was less uptake and binding for ILs constructed from the very low affinity C10 scFv. The results show the importance of antibody fragment density on nanoparticle uptake, and suggest that engineering ultrahigh affinity scFv may be unnecessary for optimal nanoparticle targeting.

Topics: Amino Acid Sequence; Antibodies; Antibody Affinity; Cell Line, Tumor; Cell Survival; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Humans; Liposomes; Molecular Sequence Data; Mutation; Nanoparticles; Neoplasms; Sequence Alignment; Solubility; Topotecan

Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein that is frequently overexpressed in a variety of carcinomas. This pan-carcinoma antigen has served as the target for a plethora of immunotherapies. Innovative therapeutic approaches include the use of trifunctional antibodies (trAbs) that recruit and activate different types of immune effector cells at the tumour site. The trAb catumaxomab has dual specificity for EpCAM and CD3. In patients with malignant ascites, catumaxomab significantly increased the paracentesis-free interval, corroborating the high efficacy of this therapeutic antibody. Here, we characterised the monoclonal antibody (mAb) HO-3, that is, the EpCAM-binding arm of catumaxomab. Peptide mapping indicated that HO-3 recognises a discontinuous epitope, having three binding sites in the extracellular region of EpCAM. Studies with glycosylation-deficient mutants showed that mAb HO-3 recognised EpCAM independently of its glycosylation status. High-affinity binding was not only detected for mAb HO-3, but also for the monovalent EpCAM-binding arm of catumaxomab with an excellent K(D) of 5.6 x 10(-10) M. Furthermore, trAb catumaxomab was at least a 1000-fold more effective in eliciting the eradication of tumour cells by effector peripheral blood mononuclear cells compared with mAb HO-3. These findings suggest the great therapeutic potential of trAbs and clearly speak in favour of EpCAM-directed cancer immunotherapies.

Topics: Amino Acid Sequence; Antibodies, Bispecific; Antigens, Neoplasm; Base Sequence; Cell Adhesion Molecules; Cell Line, Tumor; DNA Primers; Epidermal Growth Factor; Epithelial Cell Adhesion Molecule; Epitopes; Glycosylation; Humans; Immunotherapy; Molecular Sequence Data; Neoplasms

Daidzein and genistein are isoflavones found in soybean. Genistein is known to exhibit anticarcinogenic activities and inhibit tyrosine kinase activity. However, the underlying molecular mechanisms of the chemopreventive activities of daidzein and its metabolite, equol, are not understood. Here we report that equol inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ mouse epidermal cells by targeting the MEK/ERK/p90RSK/activator protein-1 signaling pathway. TPA-induced neoplastic cell transformation was inhibited by equol, but not daidzein, at noncytotoxic concentrations in a dose-dependent manner. Equol dose-dependently attenuated TPA-induced activation of activator protein-1 and c-fos, whereas daidzein did not exert any effect when tested at the same concentrations. The TPA-induced phosphorylation of ERK1/2, p90RSK, and Elk, but not MEK or c-Jun N-terminal kinase, was inhibited by equol but not by daidzein. In vitro kinase assays revealed that equol greatly inhibited MEK1, but not Raf1, kinase activity, and an ex vivo kinase assay also demonstrated that equol suppressed TPA-induced MEK1 kinase activity in JB6 P+ cell lysates. Equol dose-dependently inhibited neoplastic transformation of JB6 P+ cells induced by epidermal growth factor or H-Ras. Both in vitro and ex vivo pull-down assays revealed that equol directly bound with glutathione S-transferase-MEK1 to inhibit MEK1 activity without competing with ATP. These results suggested that the antitumor-promoting effect of equol is due to the inhibition of cell transformation mainly by targeting a MEK signaling pathway. These findings are the first to reveal a molecular basis for the anticancer action of equol and may partially account for the reported chemopreventive effects of soybean.

Topics: Animals; Binding Sites; Cell Line; Epidermal Growth Factor; Equol; Extracellular Signal-Regulated MAP Kinases; Glycine max; Isoflavones; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mice; Models, Molecular; Molecular Structure; Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-fos; ras Proteins; Replication Protein C; Ribosomal Protein S6 Kinases, 90-kDa; Tetradecanoylphorbol Acetate

Death-associated protein kinase (DAPK) is a calmodulin-regulated serine/threonine kinase and elicits tumor suppression function through inhibiting cell adhesion/migration and promoting apoptosis. Despite these biological functions, the signaling mechanisms through which DAPK is regulated remain largely elusive. Here, we show that the leukocyte common antigen-related (LAR) tyrosine phosphatase dephosphorylates DAPK at pY491/492 to stimulate the catalytic, proapoptotic, and antiadhesion/antimigration activities of DAPK. Conversely, Src phosphorylates DAPK at Y491/492, which induces DAPK intra-/intermolecular interaction and inactivation. Upon EGF stimulation, a rapid Src activation leads to subsequent LAR downregulation, and these two events act in synergism to inactivate DAPK, thereby facilitating tumor cell migration and invasion toward EGF. Finally, DAPK Y491/492 hyperphosphorylation is found in human cancers in which Src activity is aberrantly elevated. These results identify LAR and Src as a DAPK regulator through their reciprocal modification of DAPK Y491/492 residues and establish a functional link of this DAPK-regulatory circuit to tumor progression.

Topics: Apoptosis Regulatory Proteins; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line, Tumor; Death-Associated Protein Kinases; Epidermal Growth Factor; Humans; Neoplasms; Nerve Tissue Proteins; Phosphorylation; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins pp60(c-src); Receptor-Like Protein Tyrosine Phosphatases, Class 2; Receptors, Cell Surface; Signal Transduction; Tumor Suppressor Proteins

Macromolecular complexes exhibit reduced diffusion in biological membranes; however, the physiological consequences of this characteristic of plasma membrane domain organization remain elusive. We report that competition between the galectin lattice and oligomerized caveolin-1 microdomains for epidermal growth factor (EGF) receptor (EGFR) recruitment regulates EGFR signaling in tumor cells. In mammary tumor cells deficient for Golgi beta1,6N-acetylglucosaminyltransferase V (Mgat5), a reduction in EGFR binding to the galectin lattice allows an increased association with stable caveolin-1 cell surface microdomains that suppresses EGFR signaling. Depletion of caveolin-1 enhances EGFR diffusion, responsiveness to EGF, and relieves Mgat5 deficiency-imposed restrictions on tumor cell growth. In Mgat5(+/+) tumor cells, EGFR association with the galectin lattice reduces first-order EGFR diffusion rates and promotes receptor interaction with the actin cytoskeleton. Importantly, EGFR association with the lattice opposes sequestration by caveolin-1, overriding its negative regulation of EGFR diffusion and signaling. Therefore, caveolin-1 is a conditional tumor suppressor whose loss is advantageous when beta1,6GlcNAc-branched N-glycans are below a threshold for optimal galectin lattice formation.

Topics: Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Caveolin 1; Cell Membrane; Cytoskeleton; Diffusion; Embryonic Stem Cells; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Fluorescence Recovery After Photobleaching; Galectins; Mice; Models, Biological; Neoplasms; Nerve Tissue Proteins; Phosphorylation; Protein Structure, Tertiary; Protein Transport; Recombinant Fusion Proteins; Signal Transduction; Thiazolidines

Topics: Amino Acid Sequence; Animals; Cell Cycle Proteins; Epidermal Growth Factor; Guanine Nucleotide Exchange Factors; Humans; Mass Spectrometry; Mice; Molecular Sequence Data; Neoplasms; p21-Activated Kinases; Phosphorylation; Protein Binding; Rho Guanine Nucleotide Exchange Factors

Gamma-catenin (plakoglobin) exists in cells either as a component of adherens junctions, along with beta-catenin and alpha-catenin, or in association with desmoplakin in desmosomes, which are in turn coupled to the cytoskeleton linking to the plasma membrane. Although gamma-catenin overexpression is observed in many cancers, the molecular basis of its contribution to tumor progression remains unclear. In this study, we examined gamma-catenin overexpression-mediated effects leading to altered regulation of effector genes such as PTTG and c-Myc, as well as differential activation of signaling pathways. We found that overexpression of gamma-catenin caused: (1) a reduction in E-cadherin and corresponding increase in vimentin levels concomitant with increased cell mobility and migration; (2) enhancement in the levels of phosphorylated Akt and Erk in the presence of EGF and (3) an increase in PTTG and c-Myc protein levels, which are likely to accelerate chromosomal instability and uncontrolled proliferation, respectively, in the affected cells. These effects resulting from overexpression of gamma-catenin were further validated in converse experiments with the aid of siRNA knockdown of the endogenous gamma-catenin gene. In conclusion, our studies provide a molecular basis for the promotion of genomic instability and the oncogenic effects due to overexpression of gamma-catenin in human cancer.

Topics: Aneuploidy; Cell Movement; Epidermal Growth Factor; gamma Catenin; Gene Expression Regulation, Neoplastic; Genomic Instability; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Proteins; Neoplasms; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Securin; TCF Transcription Factors; Transcription Factor 7-Like 2 Protein; Transcription, Genetic; Up-Regulation

Factors predicting sensitivity to epidermal growth factor receptor (EGFR) blockade are largely unknown and new strategies are being sought to individualize cancer therapy. This study evaluated the variation in the expression of the early response gene c-fos as a distal effect of EGFR inhibition and its relationship to antitumor effects. The growth-inhibitory and c-fos-modulating effects of gefitinib and erlotinib in human cancer cell lines (A431, CAL27, HN11, HuCCT1, and Hep2) were determined. Next, these cell lines were xenografted in mice and treated for 14 days with gefitinib (A431 and HuCCT1) or erlotinib (CAL27, HN11, and Hep2). Fine needle aspiration biopsy of tumors was done at baseline and after 14 days of therapy for c-fos assessment. In addition, we tested the feasibility of analyzing this marker in five paired tumor samples from a clinical trial of gefitinib in patients with solid tumors. In culture, gefitinib and erlotinib decreased c-fos mRNA levels in the susceptible cell lines A431, CAL27, and HN11; however, both drugs failed to achieve c-fos inhibition in resistant cells. Gefitinib or erlotinib abrogated the increase in c-fos expression in vivo in EGFR-sensitive A431, CAL27, and HN11 tumors but not in resistant strains. Ex vivo evaluation was feasible and predicted in vivo effects. The feasibility study in paired human tumor biopsies showed that this biomarker can be reliably measured in clinical materials. In summary, variations in c-fos expression reflect the pharmacologic actions of EGFR inhibitors in in vitro and in vivo models.

Topics: Animals; Biomarkers, Tumor; Cell Growth Processes; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Female; Gefitinib; Genes, fos; Humans; Mice; Mice, Nude; Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-fos; Quinazolines; RNA, Messenger; Xenograft Model Antitumor Assays

Adenovirus early region 4 open reading frame 4 (E4orf4) protein is a novel cell death factor that selectively induces p53-independent apoptosis in cancer cells, but not in normal human cells. This study presents an approach for inhibiting p53-deficient tumor cell growth by using protein-based E4orf4 that had been genetically fused to epidermal growth factor (EGF) to ensure selective targeting of EGF receptor-overexpressing tumor cells. EGF-E4orf4 enables binding onto the cell surface and is then internalized into Saos-2 cells. The success of the process had been demonstrated by immunofluorescence assay and confocal laser microscopy. After prolonged exposure, E4orf4 remained mostly in the nuclei. EGF-E4orf4 treatment of Saos-2 cells showed dose-dependent cytotoxicity. Nearly 50% of the Saos-2 cells were killed at a concentration of 250 nmol/l. In contrast, EGF-E4orf4 showed no significant inhibitory effect iresn primary cells of human umbilical vein endothelial cells. To confirm the ability of EGF-E4orf4 to induce apoptosis, DNA fragmentation was detected using BrdUTP end-labeling. Flow cytometric analysis revealed a significant increase of apoptotic cells in Saos-2 cells treated with EGF-E4orf4, but not in the case of cells cultured in plain medium (t=0.028, P<0.05). In conclusion, these preliminary results indicate that EGF-E4orf4 could show promise as a new reagent that is more efficient and less toxic in anti-cancer therapy.

Topics: Cell Death; Cell Line, Tumor; Cell Nucleus; Dose-Response Relationship, Drug; Epidermal Growth Factor; Flow Cytometry; Humans; Microscopy, Confocal; Neoplasms; Protein Binding; Recombinant Fusion Proteins; Tumor Suppressor Protein p53; Viral Proteins

In contrast with the early view of metalloproteases as simple extracellular matrix-degrading entities, recent findings show that they are highly specific modulators of different signaling pathways involved, positively or negatively, in tumor development. Thus, before considering a given metalloprotease a therapeutic target, it seems advisable to characterize its function by identifying its repertoire of substrates. Here, we present a proteomic approach to identify ADAM17 substrates by difference gel electrophoresis. We found that the shedding of the extracellular domain of the transferrin receptor and those of two cell-cell adhesion molecules, activated leukocyte cell adhesion molecule (ALCAM) and desmoglein 2 (Dsg-2), is increased in cells overexpressing ADAM17. Genetic evidence shows that while ADAM17 is responsible for the shedding of ALCAM, both ADAM17 and ADAM10 can act on Dsg-2. Activation of the epidermal growth factor receptor leads to the upregulation of the shedding of Dsg-2 and to the concomitant upregulation of ADAM17, but not ADAM10, supporting the ability of overexpressed ADAM17 to shed Dsg-2. These results unveil a role of ADAM10 and ADAM17 in the shedding of cell-cell adhesion molecules. Since loss of cell adhesion is an early event in tumor development, these results suggest that ADAM17 is a useful target in anticancer therapy.

Topics: Activated-Leukocyte Cell Adhesion Molecule; ADAM Proteins; ADAM17 Protein; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Desmoglein 2; Electrophoresis, Gel, Two-Dimensional; Endopeptidases; Epidermal Growth Factor; Humans; Mice; Mutation; Neoplasms; Protein Structure, Tertiary; Proteomics; Substrate Specificity; Up-Regulation

It has been demonstrated that the actin-severing activity of cofilin can be downregulated by LIM kinase (LIMK)-dependent phosphorylation at residue Ser3. Chemotactic stimulation in various cell types induces cofilin dephosphorylation, suggesting that cofilin activation in these cells occurs by a dephosphorylation mechanism. However, resting metastatic carcinoma cells have the majority of their cofilin in a dephosphorylated but largely inactive state. Stimulation with epidermal growth factor (EGF) induces an increase in cofilin activity after 60 seconds together with an increase in phosphorylated cofilin (p-cofilin), indicating that cofilin dephosphorylation is not coupled to cofilin activation in these cells. Suppression of LIMK function by inhibiting Rho-associated protein kinase (ROCK) or LIMK siRNA inhibited the EGF-induced cofilin phosphorylation but had no effect on cofilin activity or cofilin-dependent lamellipod protrusion induced by EGF. Correlation analysis revealed that cofilin, p-cofilin and LIMK are not colocalized, and changes in the location of these proteins upon stimulation with EGF indicate that they are not functionally coupled. Phospholipase C, which has been implicated in cofilin activation following stimulation with EGF, does not regulate p-cofilin levels following stimulation with EGF. Therefore, our results do not support a model for the initial activation of cofilin by dephosphorylation in response to chemoattractant stimulation in metastatic carcinoma cells.

Topics: Actin Depolymerizing Factors; Animals; Cytoskeleton; Enzyme Activation; Epidermal Growth Factor; Intracellular Signaling Peptides and Proteins; Lim Kinases; Neoplasms; Octoxynol; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Protein Transport; Pseudopodia; Rats; rho-Associated Kinases; RNA, Small Interfering; Tumor Cells, Cultured; Type C Phospholipases

To improve the targeting of adenovirus vector for gene therapy, a fusion gene sCAR-EGF, in which epidermal growth factor gene was fused to the 3' end of extracellular Coxsackie virus-adenovirus receptor gene, was constructed and cloned into shuttle plasmid pDC315 to obtain a recombinant plasmid pDC315-sCAR-EGF. With the AdMax system, AD-293 cells were co-transfected with pDC315-sCAR-EGF and adenovirus genomic plasmid pBHGloxdeltaE13cre. Through high efficiency site specific recombination, a replication-defective adenovirus Ad5-CMV-sCAR-EGF was constructed. The recombinant adenovirus was analyzed by PCR and Western blotting, the results indicated that Ad5-CMV-sCAR-EGF contained the fusion gene sCAR-EGF, and the adenovirus infected cells was induced to produce and secrete the fusion protein into the supernatant. We have demonstrated that the fusion protein sCAR-EGF is helpful for elevating the infection efficiency of Ad5-CMV-luc with the reporter gene in vitro, which providing a new approach to the gene therapy for tumors overexpressing EGFR.

Topics: Adenoviridae; Cell Line; Coxsackie and Adenovirus Receptor-Like Membrane Protein; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Genetic Therapy; Humans; Neoplasms; Polymerase Chain Reaction; Receptors, Virus; Recombinant Fusion Proteins

The presence of scattered tumor cells at the invading front of several carcinomas has clinical significance. These cells differ in their protein expression from cells in central tumor regions as recently shown for the EGF-TM7 receptor CD97. To understand the impact of such heterogeneity on tumor invasion, we investigated tumor cells with modified CD97 expression in vitro and in vivo. Applying an individual cell-based computer model approach, we linked specific cell properties of these cells to tumor invasion characteristics. CD97 overexpression promoted tumor growth in scid mice, stimulated single cell motility, increased proteolytic activity of matrix metalloproteinases, and secretion of chemokines in vitro in an isoform-specific manner. We demonstrated by computer simulation studies that these effects of CD97 can increase the invasion capacity of tumors. Furthermore, they can cause the appearance of scattered tumor cells at the invasion front. We identified local tumor environment interactions as triggers of these multiple capabilities. Experimentally, our simulation results are supported by the finding that CD97 expression in tumor cells is regulated by their environment. Our combined experimental-theoretical analysis provides novel insight to how variations of individual cell properties can be linked to individual patterns of tumor cell invasion.

Topics: Animals; Antigens, CD; Cell Movement; Cell Survival; Chemokines; Clone Cells; Computer Simulation; Contact Inhibition; Epidermal Growth Factor; Humans; Mice; Mice, Inbred BALB C; Mice, SCID; Models, Biological; Models, Molecular; Neoplasm Invasiveness; Neoplasms; Protein Processing, Post-Translational; Protein Structure, Tertiary

To analyze involvement of human beta-defensin-2 (hBD-2) in intracellular signaling in vitro.. A431cells were cultured in the presence of 1 microg/ml of recombinant hBD-2 and/or 10 ng/ml EGF. For evaluation of expression of mRNAs for p70S6 kinase, isoforms alpha and beta, RT-PCR analysis was applied. Expression and activity of p70S6K, phosphorylation of PDK1, ERK, JNK, p38 kinases and EGF receptor (EGFR) was evaluated using Western blot analysis.. 30 min incubation of A431 cells with 1 mug/ml of hBD-2 didn't influence autophosphorylation level of EGFR, but resulted in activation of p70S6K, 12 h treatment - in prominently increased level of mRNA for alpha and beta-isoforms of p70S6 kinase, whilst 24 h treatment - in elevation of p70S6K synthesis on protein level. Up-stream kinase phosphorylating p70S6K, PDK1, is also phosporylated upon influence of exogenous hBD-2 in vitro.. Our data point on the involvement of PDK1-p70S6K pathway in mediation of action of hBD-2 in A431 cells.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; beta-Defensins; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Phosphorylation; Protein Serine-Threonine Kinases; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Tumor Cells, Cultured

The tumor suppressor gene FHIT is inactivated by genetic and epigenetic changes in the majority of common human cancers. The human Fhit protein undergoes phosphorylation on tyrosine residue 114 by Src and related kinases both in vitro and in vivo. Src is a key cytoplasmic tyrosine kinase downstream to several growth factor receptors, including those of the EGF receptor family, which are overexpressed and activated in about one-third of human breast and ovarian carcinomas. However, the biological significance of Fhit phosphorylation by Src has remained elusive. In the present study, we demonstrate that FHIT acts as a checkpoint in cell proliferation mediated by activated tyrosine kinase receptors that recruit Src. Activation of EGF receptor family members induced Fhit phosphorylation by Src and the subsequent proteasome degradation of the phosphorylated Fhit protein. Indeed, the use of the Fhit mutant Y114F, which carries a phenylalanine instead of a tyrosine at position 114, unable to be phosphorylated on tyrosine 114 by Src, prevents Fhit degradation. Moreover, Fhit protein reduction is transient and occurs in a specific temporal window. During the signaling pathway of activated tyrosine kinase receptors, the phosphorylation of Fhit induces its degradation and the subsequent reduction in Fhit protein levels allows the transmission of the mitogenic signal; immediately thereafter, Fhit protein levels are restored. Such a scenario would suggest a key role for Fhit in the balance of proliferation/survival/apoptosis signals.

Topics: Acid Anhydride Hydrolases; Cell Line; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; Mitogens; Mitosis; Models, Biological; Neoplasm Proteins; Neoplasms; Phosphorylation; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins pp60(c-src); Signal Transduction

CD97, a membrane protein expressed at high levels on inflammatory cells and some carcinomas, is a member of the adhesion G protein-coupled receptor family, whose members have bipartite structures consisting of an extracellular peptide containing adhesion motifs noncovalently coupled to a class B 7-transmembrane domain. CD97alpha, the extracellular domain of CD97, contains 3 to 5 fibrillin class 1 epidermal growth factor (EGF)-like repeats, an Arg-Gly-Asp (RGD) tripeptide, and a mucin stalk. We show here that CD97alpha promotes angiogenesis in vivo as demonstrated with purified protein in a directed in vivo angiogenesis assay (DIVAA) and by enhanced vascularization of developing tumors expressing CD97. These data suggest that CD97 can contribute to angiogenesis associated with inflammation and tumor progression. Strong integrin alpha5beta1 interactions with CD97 have been identified, but alpha v beta3 also contributes to cell attachment. Furthermore, soluble CD97 acts as a potent chemoattractant for migration and invasion of human umbilical vein endothelial cells (HUVECs), and this function is integrin dependent. CD97 EGF-like repeat 4 is known to bind chondroitin sulfate. It was found that coengagement of alpha5beta1 and chondroitotin sulfate proteoglycan by CD97 synergistically initiates endothelial cell invasion. Integrin alpha5beta1 is the first high-affinity cellular counterreceptor that has been identified for a member within this family of adhesion receptors.

Topics: Animals; Antigens, CD; Cell Adhesion; Chondroitin Sulfates; Endothelium, Vascular; Epidermal Growth Factor; HT29 Cells; Humans; Integrin alpha5beta1; Integrin alphaVbeta3; Membrane Glycoproteins; Mice; Neoplasms; Neovascularization, Physiologic; NIH 3T3 Cells; Oligopeptides; Phenotype; Protein Structure, Tertiary; Rats; Receptors, G-Protein-Coupled; Umbilical Veins

EGF activates the ErbB1 receptor, but there appears only a limited correlation between its receptor binding affinity and mitogenic activity. This is indicated by our present observation that in cells with high ErbB1 expression, including SUM102 breast tumor cells, low affinity EGF/Notch chimeras have similarly high mitogenic activity as EGF, in spite of the fact that EGF is superior in inducing receptor tyrosine phosphorylation and p42/p44 MAP-kinase activity. However, as a result of receptor-mediated internalisation high-affinity ligands such as EGF are depleted much more rapidly from the extracellular medium than low-affinity EGF/Notch chimeras. As a consequence, the mitogenic activity of EGF on ErbB1 overexpressing cells is limited by substantial degradation of internalised ligand in the period before cells enter S-phase, a phenomenon that is not observed for low affinity mutant ligands. The mitogenic activity of EGF on ErbB1 overexpressing cells does therefore not only depend on the applied concentration but also on the total amount of ligand added, and is strongly underestimated when tested in a limited assay volume. No such dependence on the incubation volume was observed for EGF activity on cells with low ErbB1 expression levels and on cells for which EGF is growth inhibitory.

Topics: Animals; Cell Line, Tumor; Cell Transformation, Neoplastic; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Feedback, Physiological; Female; Growth Substances; Humans; Ligands; Mice; Mitosis; Mutation; Neoplasms; NIH 3T3 Cells; Protein Binding; Receptor Aggregation; Receptor, Notch1; Receptors, Cell Surface; Recombinant Fusion Proteins; S Phase; Transcription Factors

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Autocrine Communication; Cetuximab; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Exanthema; Humans; Ligands; Models, Biological; Neoplasm Proteins; Neoplasms; Paracrine Communication; Quinazolines; Skin; Transforming Growth Factor alpha; Treatment Outcome

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Clinical Trials as Topic; Epidermal Growth Factor; Female; Humans; Male; Neoplasms; Pain, Intractable; Piperazines; Pyridines

The breast tumor kinase (BRK) is a growth promoting non-receptor tyrosine kinase overexpressed in the majority of human breast tumors. BRK is known to potentiate the epidermal growth factor (EGF) response in these cells. Although BRK is known to phosphorylate the RNA-binding protein Sam68, the specific tyrosines phosphorylated and the exact role of this phosphorylation remains unknown. Herein, we have generated Sam68 phospho-specific antibodies against C-terminal phosphorylated tyrosine residues within the Sam68 nuclear localization signal. We show that BRK phosphorylates Sam68 on all three tyrosines in the nuclear localization signal. By indirect immunofluorescence we observed that BRK and EGF treatment not only phosphorylates Sam68 but also induces its relocalization. Tyrosine 440 was identified as a principal modulator of Sam68 localization and this site was phosphorylated in response to EGF treatment in human breast tumor cell lines. Moreover, this phosphorylation event was inhibited by BRK small interfering RNA treatment, consistent with Sam68 being a physiological substrate of BRK downstream of the EGF receptor in breast cancer cells. Finally, we observed that Sam68 suppressed BRK-induced cell proliferation, suggesting that Sam68 does indeed contain anti-proliferative properties that may be neutralized in breast cancer cells by phosphorylation.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Animals; Animals, Newborn; Astrocytes; Brain; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cells, Cultured; Disease Progression; DNA-Binding Proteins; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Fluorescent Antibody Technique, Indirect; Genetic Vectors; HeLa Cells; Humans; Immunoprecipitation; Microscopy, Fluorescence; Molecular Sequence Data; Neoplasm Proteins; Neoplasms; Peptides; Phosphoproteins; Phosphorylation; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Rats; RNA Interference; RNA-Binding Proteins; Sequence Homology, Amino Acid; Signal Transduction; Tyrosine

The transcription factor activator protein-1 (AP-1) has been implicated in a large variety of biological processes including oncogenic transformation. The tyrosine kinases of the epidermal growth factor receptor (EGFR) constitute the beginning of one signal transduction cascade leading to AP-1 activation and are known to control cell proliferation and differentiation. Drug discovery efforts targeting this receptor and other pathway components have centred on monoclonal antibodies and small molecule inhibitors. Resistance to such inhibitors has already been observed, guiding the prediction of their use in combination therapies with other targeted agents such as RNA interference (RNAi). This study examines the use of RNAi and kinase inhibitors for qualification of components involved in the EGFR/AP-1 pathway of ME180 cells, and their inhibitory effects when evaluated individually or in tandem against multiple components of this important disease-related pathway.. AP-1 activation was assessed using an ME180 cell line stably transfected with a beta-lactamase reporter gene under the control of AP-1 response element following epidermal growth factor (EGF) stimulation. Immunocytochemistry allowed for further quantification of small molecule inhibition on a cellular protein level. RNAi and RT-qPCR experiments were performed to assess the amount of knockdown on an mRNA level, and immunocytochemistry was used to reveal cellular protein levels for the targeted pathway components.. Increased potency of kinase inhibitors was shown by combining RNAi directed towards EGFR and small molecule inhibitors acting at proximal or distal points in the pathway. After cellular stimulation with EGF and analysis at the level of AP-1 activation using a beta-lactamase reporter gene, a 10-12 fold shift or 2.5-3 fold shift toward greater potency in the IC50 was observed for EGFR and MEK-1 inhibitors, respectively, in the presence of RNAi targeting EGFR.. EGFR pathway components were qualified as targets for inhibition of AP-1 activation using RNAi and small molecule inhibitors. The combination of these two targeted agents was shown to increase the efficacy of EGFR and MEK-1 kinase inhibitors, leading to possible implications for overcoming or preventing drug resistance, lowering effective drug doses, and providing new strategies for interrogating cellular signalling pathways.

Topics: Antibodies, Monoclonal; beta-Lactamases; Butadienes; Cell Differentiation; Cell Proliferation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Immunohistochemistry; Inhibitory Concentration 50; MAP Kinase Kinase 1; Neoplasms; Nitriles; Oligonucleotides, Antisense; Phosphorylation; Quinazolines; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; Signal Transduction; Transcription Factor AP-1; Transfection; Tyrphostins

Recent development in stem cell biology has indicated a new possible approach for the treatment of neurological diseases. However, in spite of tremendous hope generated, we are still on the way to understand if the use of stem cells to repair mature brain and spinal cord is a reliable possibility. In particular, we know very little on the in situ regulation of adult neural stem, and this also negatively impact on cell transplant possibilities. In this chapter we will discuss issues concerning the role and function of stem cells in neurological diseases, with regard to the impact of features of degenerating neurons and glial cells on in situ stem cells. Stem cell location and biology in the adult brain, brain host reaction to transplantation, neural stem cell reaction to experimental injuries and possibilities for exogenous regulation are the main topics discussed.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Bisbenzimidazole; Blotting, Western; Brain; Brain Diseases; Cell Differentiation; Cell Division; Cells, Cultured; Cerebral Ventricle Neoplasms; Encephalomyelitis, Autoimmune, Experimental; Epidermal Growth Factor; Fluorescent Antibody Technique; Glial Fibrillary Acidic Protein; In Vitro Techniques; Injections, Intraventricular; Intermediate Filament Proteins; Male; Microscopy, Confocal; Neoplasms; Nerve Degeneration; Nerve Growth Factor; Nerve Tissue Proteins; Nestin; Neural Cell Adhesion Molecule L1; Neuroglia; Neurons; Rats; Sialic Acids; Stem Cell Transplantation; Stem Cells; Wound Healing

The purpose of this study is to develop a high-throughput approach to detect protein expression from hundreds and thousands of samples and to apply this technology to profile circulating angiogenic factor protein levels in patients with gynecological tumors.. Analytes containing a mixture of protein are immobilized onto antibody-coated surface of support in array format. The presence of protein in analytes is detected with biotin-labeled antibody coupled with an enhanced chemiluminescence or fluorescence detection system. The exact amount of protein can be quantitatively measured. The expression levels of five angiogenic factors (angiogenin, interleukin 8, vascular endothelial growth factor, platelet-derived growth factor, and epidermal growth factor) from 157 samples were quantitatively measured using this novel protein array technology and were statistically analyzed. The expression patterns of angiogenic factors were analyzed using two-way hierarchical cluster analysis approach.. A novel protein array technology, which can simultaneously and quantitatively measure few protein levels from hundreds and thousands of samples was developed. Only minute amounts of sample are required for the assay. This approach also features high sensitivity and specificity. Using this novel protein array approach, we analyzed the plasma expression levels of five angiogenic factors in 137 patients diagnosed with a tumor and 20 controls. Statistical analysis reveals different expression levels of angiogenic factors between patients and controls. Cluster analysis suggests a possible classification of normal subjects from patients.. Enhanced protein profiling arrays provide a high-throughput and sensitive system to detect one or few protein from hundreds and thousands of samples. Such an approach should have broad application in biomedical discovery.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biotin; Cell Line, Tumor; Cluster Analysis; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Female; Genital Neoplasms, Female; Humans; Immunoglobulin G; Interleukin-8; Luminescent Measurements; Microscopy, Fluorescence; Middle Aged; Multigene Family; Neoplasms; Neovascularization, Pathologic; Oligonucleotide Array Sequence Analysis; Platelet-Derived Growth Factor; Protein Array Analysis; Ribonuclease, Pancreatic; Sensitivity and Specificity; Tissue Distribution; Vascular Endothelial Growth Factor A

Accumulation of beta-catenin, which leads to enhanced TCF/LEF-1 driven transcription and thereby contributes to tumor development, can result from mutation of beta-catenin itself, inactivation of the adenomatous polyposis coli (APC) protein, or Wnt pathway inhibition of the GSK-3beta kinase that together with APC promotes beta-catenin degradation. Nevertheless, emerging evidence shows that the activation of beta-catenin can occur independently of Wnt signaling to GSK-3beta. In response to EGF, tumor cells overexpressing EGF receptor display GSK-3beta-independent activation of beta-catenin, which may result from a combination of effects-EGF-stimulated, caveolin-1-dependent internalization of E-cadherin, resulting in release of beta-catenin from cell-cell contacts, and EGF-induced downregulation of caveolin-1, relieving the inhibition of signaling molecules sequestered by caveolin-1 at caveolae.

Topics: Adenomatous Polyposis Coli Protein; beta Catenin; Cadherins; Caveolin 1; Caveolins; Cytoskeletal Proteins; DNA-Binding Proteins; Epidermal Growth Factor; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Lymphoid Enhancer-Binding Factor 1; Neoplasms; Proto-Oncogene Proteins; Signal Transduction; Trans-Activators; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Wnt Proteins

Many years ago, diphtheria toxin (DT) showed antitumor activity in mice and in humans, but it was unclear whether this depended on the toxicity of the molecule only or on its strong inflammatory-immunological property as well. To deal with this open question, we planned to treat a group of cancer patients with cross-reacting material 197 (CRM197). CRM197 is a nontoxic mutant of DT that shares the immunological properties of the native molecule and its ability to bind to heparin-binding epidermal growth factor (HB-EGF), the specific cell-membrane receptor for DT that is often overexpressed in cancer.. 25 outpatients with various advanced tumors who were refractory to standard therapies (23 subjects) or had refused, in whole or in part, conventional therapies (2 subjects) were treated with CRM197 injected subcutaneously in the abdominal wall, on alternate days, for 6 days. Three different dosages (1.7, 2.6, or 3.5 mg/day) were used according to the patient's degree of immunological reactivity to DT/CRM197 (none, moderate, or high).. After the first administration of CRM197, a significant increase in the number of circulating neutrophils and in the serum level of TNF-alpha was detected. Toxicities were minimal. Only patients with delayed-type hypersensitivity to DT/CRM197 had irritating skin reactions in the injection sites and a flu-like syndrome with fever. Pharmacokinetics showed a mean peak concentration (12.7 ng/ml) 12 h after the first injection and a mean half-life of 18.1 h. There were two complete and one partial responses (metastatic breast carcinoma, neuroblastoma, and metastatic breast carcinoma) lasting 4, 45+, and 15 months, respectively. Six cases of stable disease, lasting from 1 to 15 months, were also recorded.. CRM197 injected subcutaneously elicited an inflammatory-immunological reaction, caused tolerable toxicities, was absorbed to a good extent into the circulatory system, and exerted some degree of biological antitumor activity. A possible role of neutrophils and TNF-alpha in the mode of action of the molecule is hypothesized.

Topics: Aged; Aged, 80 and over; Bacterial Proteins; Cell Line, Tumor; Clinical Trials as Topic; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Heparin; Humans; Inflammation; Male; Middle Aged; Neoplasms; Neutrophils; Time Factors; Treatment Outcome; Tumor Necrosis Factor-alpha

Topics: Antineoplastic Agents; Epidermal Growth Factor; ErbB Receptors; Genes, abl; Genes, erbB-2; Humans; Neoplasms; Proto-Oncogene Proteins c-kit; Tretinoin

We have previously reported the synthesis of SMA41, a unimolecular combination of an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) of the quinazoline class and a DNA-damaging monomethyltriazene termed "combimolecule". Hydrolysis of 1-[4-(m-tolylamino)-6-quinazolinyl]-3-methyltriazene (SMA41) gives rise to an intact TKI [6-amino-4-(3-methylanilino)quinazoline; SMA52] capable of inhibiting epidermal growth factor (EGF)-induced EGFR autophosphorylation and a DNA-targeting methyldiazonium species. Herein, we showed that SMA41 blocked EGF-induced EGFR autophosphorylation by an irreversible mechanism, suggesting that it may covalently damage the receptor in these cells. More importantly, this was associated with significant inhibition of mitogen-activated protein kinase activation in A431 cells. In cells treated with [14C]SMA41, radio-high-performance liquid chromatography detection of both N7- and O6-methylguanine revealed an almost complete repair of the O6-methylguanine lesions and a greater tolerance of the N7-methylguanine adducts 24 h post-treatment. In contrast to temozolomide (a cyclic triazene used in the clinic) and the reversible inhibitor SMA52, SMA41 induced significant cell cycle arrest in S, G2, and M phases 24 h after a 2-h drug exposure. Furthermore, in vivo studies demonstrated that SMA41 was well tolerated. At 200 mg/kg, it showed approximately 2-fold greater antiproliferative activity than SMA52 in A431 cells implanted in immunocompromised SCID mice. These results suggest that the binary targeting properties of SMA41 are associated with a binary cascade of events in the cells that seem to culminate into significant growth inhibition in vitro and in vivo.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Body Weight; Cell Cycle; Cell Line, Tumor; Chromatography, High Pressure Liquid; Comet Assay; DNA Damage; DNA, Neoplasm; Dose-Response Relationship, Drug; Drug Delivery Systems; Epidermal Growth Factor; Flow Cytometry; Humans; Mice; Mice, SCID; Mitogen-Activated Protein Kinases; Neoplasm Transplantation; Neoplasms; Quinazolines; Signal Transduction

Evidence suggests that CRIPTO-1 (CR-1) might be involved in the pathogenesis of human carcinoma. In the present study, we have screened the expression of CR-1 mRNA and protein in a wide panel of human cancer cell lines by using reverse transcriptase (RT)-PCR, real-time PCR and immunocytochemistry. Results of these experiments demonstrate that CR-1 is expressed in several, different carcinoma types. The anchorage-independent growth of colon, ovarian, lung and breast carcinoma cells was significantly inhibited by treatment with anti-CR-1 second generation antisense oligonucleotides. Similar results were obtained with anti-transforming growth factor alpha (TGF-alpha) and anti-amphiregulin (AR) antisense oligonucleotides. Treatment of carcinoma cells with CR-1 antisense oligonucleotides resulted in a significant reduction in the levels of expression of CR-1 mRNA and protein, and in the levels of activation of Akt. Finally, oral administration of either CR-1, AR or TGF-alpha antisense oligonucleotides produced a significant reduction in the growth of GEO colon carcinoma xenografts in nude mice that was associated with a reduction in the levels of expression of the target proteins. Taken together, these data strongly suggest that CR-1 might represent a novel target for therapeutic intervention in different carcinoma types.

Topics: Amphiregulin; Animals; Cell Line, Tumor; EGF Family of Proteins; Epidermal Growth Factor; Glycoproteins; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Mice; Neoplasm Proteins; Neoplasms; Oligonucleotides, Antisense; RNA, Messenger; Transforming Growth Factor alpha

Topics: Adenine Nucleotides; Antineoplastic Agents; Arabinonucleosides; Cancer Vaccines; Clofarabine; Cytokines; Epidermal Growth Factor; Glucocorticoids; Humans; Interleukin-2; Metalloporphyrins; Neoplasms; Proto-Oncogene Proteins c-bcl-2; Sirolimus

Increased expression of the epidermal growth factor receptor (EGFR) is common in cancer and correlates with neoplastic progression. Although the biology of this receptor has been the subject of intense investigation, surprisingly little is known about how increased expression of the wild-type EGFR affects downstream signal transduction in cells. We show that increasing the expression of the receptor results in dramatic shifts in signaling with attenuation of EGF-induced Ras, extracellular signal-related kinases (ERKs), and Akt activation, as well as amplification of STAT1 and STAT3 signaling. In this study, we focus on the mechanism of attenuated ERK signaling and present evidence suggesting that the mechanism of attenuated ERK signaling in EGFR-overexpressing cells is a sequestration of ERKs at the cell membrane in EGFR-containing complexes. Increased expression of the EGFR results in an aberrant localization of ERKs to the cell membrane. Furthermore, ERKs become associated with the EGFR in a physical complex in EGFR-overexpressing cells but not in control cells. The EGFR-ERK association is detected in unstimulated cells or on exposure to a low concentration of EGF; under these conditions, ERK activation is minimal. Exposure of these cells to saturating concentrations of EGF results in a decreased membrane localization of ERKs, a concomitant dissociation of ERKs from the EGFR, and restores ERK activation. A similar association can be detected between the EGFR and MEK1 in receptor-overexpressing cells, suggesting that multiple components of the ERK signaling pathway may become trapped in complexes with the EGFR. These findings can be demonstrated in cells transfected to express high levels of the EGFR as well as in cancer cells which naturally overexpress the EGFR and, thus, may be representative of altered EGFR signaling in human cancer.

Topics: DNA-Binding Proteins; Epidermal Growth Factor; ErbB Receptors; Humans; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Neoplasms; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; ras Proteins; STAT1 Transcription Factor; STAT3 Transcription Factor; Trans-Activators; Tumor Cells, Cultured; Tyrosine

Systemic application of surface-shielded transferrin-polyethylenimine/DNA complexes leads to predominant DNA uptake and gene expression in Neuro2a tumors in syngeneic A/J mice. Similarly, high expression levels were found in Huh-7 and HepG2 human tumor xenografts in SCID mice after systemic application of surface-shielded EGF-PEG-PEI/DNA complexes. Significant DNA uptake but low gene expression were found in the M-3 melanoma while no DNA uptake and no gene expression were found in KB, 518A2, A549, and SW480 xenograft tumor models. To elucidate the reasons for these differences, the tumors were analyzed for vascularization and infiltration of macrophages. Neuro2a, Huh-7, and HepG2 tumors are well vascularized, with a high density of partially immature blood vessels and low numbers of infiltrating macrophages. The M-3 melanoma is well vascularized correlating with significant DNA uptake, however, necrosis and intensive infiltration by macrophages lead to rapid degradation of DNA. In contrast, the KB, 518A2, A549, and SW480 tumors are poorly vascularized, correlating with undetectable DNA uptake and gene expression. Using two different vector systems the data indicate that gene delivery to tumors in vivo is affected by tissue-dependent factors. Uptake of DNA into the tumor depends on vascularization of the tumor, while necrosis and macrophage infiltration may facilitate degradation of the DNA.

Topics: Animals; DNA; Epidermal Growth Factor; Gene Expression; Genetic Therapy; Humans; Injections, Intravenous; Macrophages; Mice; Mice, Inbred DBA; Mice, Inbred Strains; Mice, SCID; Neoplasms; Neoplasms, Experimental; Neovascularization, Pathologic; Polyethyleneimine; Tissue Distribution; Transfection; Transferrin; Tumor Cells, Cultured

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents, Alkylating; Clinical Trials as Topic; Drug Resistance, Neoplasm; Epidermal Growth Factor; Humans; Neoplasms

Dual EGFR/erbB2 inhibition is an attractive therapeutic strategy for epithelial tumors, as ligand-induced erbB2/EGFR heterodimerization triggers potent proliferative and survival signals. Here we show that a small molecule, GW572016, potently inhibits both EGFR and erbB2 tyrosine kinases leading to growth arrest and/or apoptosis in EGFR and erbB2-dependent tumor cell lines. GW572016 markedly reduced tyrosine phosphorylation of EGFR and erbB2, and inhibited activation of Erk1/2 and AKT, downstream effectors of proliferation and cell survival, respectively. Complete inhibition of activated AKT in erbB2 overexpressing cells correlated with a 23-fold increase in apoptosis compared with vehicle controls. EGF, often elevated in cancer patients, did not reverse the inhibitory effects of GW572016. These observations were reproduced in vivo, where GW572016 treatment inhibited activation of EGFR, erbB2, Erk1/2 and AKT in human tumor xenografts. Erk1/2 and AKT represent potential biomarkers to assess the clinical activity of GW572016. Inhibition of activated AKT in EGFR or erbB2-dependent tumors by GW572016 may lead to tumor regressions when used as a monotherapy, or may enhance the anti-tumor activity of chemotherapeutics, since constitutive activation of AKT has been linked to chemo-resistance.

Topics: Antineoplastic Agents; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Lapatinib; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Quinazolines; Receptor, ErbB-2; Signal Transduction; Tumor Cells, Cultured

Cell migration requires precise control, which is altered or lost when tumor cells become invasive and metastatic. beta-catenin plays a dual role in this process: as a member of adherens junctions it is essential to link cadherins to the cytoskeleton thereby allowing tight intercellular adhesion, and as a member of the Wnt-signaling pathway, beta-catenin is translocated into the nucleus and serves together with the LEF1/TCF-transcription factors to drive gene expression necessary for the epithelial-to-mesenchymal transition (EMT). Activated beta-catenin signaling has been implicated in the genesis of a variety of tumors. Here we demonstrate a pivotal function for beta-catenin signaling in epithelial cell migration and tumorigenesis. Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) induce beta-catenin signaling under conditions where they stimulate cell motility. Ectopic expression of either stabilized beta-catenin or a regulatable form of activated beta-catenin induces cell migration in different cell types and cooperates with EGF and HGF in this process. Activation of beta-catenin signaling induces expression of the new target gene osteopontin during migration. Cells expressing stabilized beta-catenin also exhibit significantly increased capability to form tumors in a nude mouse xenograft model. The data suggest that a critical threshold of beta-catenin signaling, activated by cooperative mechanisms, may be important during the EMT and tumorigenesis.

Topics: Animals; Aphidicolin; beta Catenin; Carcinoma; Cell Line; Cell Movement; Cycloheximide; Cytoskeletal Proteins; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial Cells; Gene Targeting; Mice; Mice, Nude; Neoplasms; Osteopontin; Protein Synthesis Inhibitors; Rats; Sialoglycoproteins; Signal Transduction; Trans-Activators; Transcriptional Activation; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Topics: Advisory Committees; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Benzamides; Carcinoma, Non-Small-Cell Lung; Clinical Trials as Topic; Drug Approval; Drug Industry; Epidermal Growth Factor; Erlotinib Hydrochloride; Gefitinib; Humans; Imatinib Mesylate; Lung Neoplasms; Neoplasms; Patient Selection; Piperazines; Pyrimidines; Quinazolines; United States; United States Food and Drug Administration

The American Society of Clinical Oncology (ASCO) held its 38th annual meeting in Orlando, Florida on 17-22 May 2002. Leading oncologists in several subspecialties shared their expertise in nearly 200 educational sessions, and researchers reported the latest clinical cancer advances in more than 1800 abstract presentations. This review will focus on several new rationally designed agents and therapies with drugs directed at the epidermal growth factor (EGF) or its downstream pathway, farnesyltransferase and MAPK/ERK/kinase inhibition, and anti-angiogenesis drugs. Presented data at the ASCO-meeting are reviewed and placed into perspective.

Topics: Animals; Biomedical Research; Clinical Trials as Topic; Enzyme Inhibitors; Epidermal Growth Factor; Humans; Neoplasms; Neovascularization, Pathologic

Engineered phage-based vectors are an attractive alternative strategy for gene delivery because they possess no natural mammalian cell tropism and can be genetically modified for specific applications. Genotoxic treatments that increase the transduction efficiency of single-stranded adeno-associated virus were tested on cells transfected by single-stranded phage. Indeed, green fluorescent protein transgene expression by epidermal growth factor-targeted phagemid particles increased with heat shock, UV irradiation, and camptothecin (CPT) treatment. CPT resulted in transduction efficiencies of 30-45% in certain human carcinoma cell lines and reduced the minimal dose needed to detect green fluorescent protein-expressing cells to as low as 1-10 particles/cell. Targeted phage transduction was effective in many tumor cell lines and in prostate tumor xenografts with CPT treatment. Taken together, these data suggest the feasibility of using phage-based vectors for therapeutic gene delivery to cancer cells.

Topics: Animals; Antineoplastic Agents, Phytogenic; Bacteriophages; Camptothecin; Carcinoma; Combined Modality Therapy; Enzyme Inhibitors; Epidermal Growth Factor; Genetic Therapy; Genetic Vectors; Humans; Male; Mice; Mice, Nude; Neoplasms; Plasmids; Transduction, Genetic; Transfection; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Topics: Administration, Oral; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Lung Neoplasms; Neoplasms; Protein-Tyrosine Kinases; Quinazolines

Progression to an invasive, metastatic tumour requires the coordinated expression and function of a number of gene products, as well as their regulation in the context of invasion. The transcription factor AP-1 regulates expression of many of those genes necessary for implementation of the invasion programme. Two such gene products, CD44 and ezrin, are both upregulated in fibroblasts transformed by v-fos and are commonly implicated in cell motility and invasion. Here we report that CD44 and ezrin colocalise to membrane ruffles and microvilli of A431 cells after treatment with EGF. However, A431 cells expressing dominant-negative c-Jun (TAM67), and which as a consequence fail to invade in response to EGF, also fail to correctly localise CD44 and ezrin. CD44 and ezrin are both substrates for Protein Kinase C, and we show that their EGF-dependent colocalisation requires Protein Kinase C activity. Associated with TAM67 expression and disrupted CD44 and ezrin colocalisation is the increased expression and activation of the novel PKC theta isoform. Expression of PKC theta in A431 cells results in the inhibition of cell motility and disrupted localisation of CD44 and ezrin. We propose that AP-1 regulates the integrity of Protein Kinase C signalling and identifies PKC theta as a potential suppressor of the invasion programme.

Topics: Animals; Cell Compartmentation; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Cytoskeletal Proteins; Down-Regulation; Enzyme Inhibitors; Epidermal Growth Factor; Fluorescent Antibody Technique; Humans; Hyaluronan Receptors; Isoenzymes; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Phenotype; Phorbol Esters; Phosphoproteins; Protein Isoforms; Protein Kinase C; Protein Kinase C-theta; Protein Transport; Proto-Oncogene Proteins c-jun; Transcription Factor AP-1; Up-Regulation

An in vitro transformation system of carcinogen-treated Syrian hamster embryo (SHE) cell cultures represents multistep genetic and nongenetic changes that develop during the neoplastic progression of normal cells to tumor cells in vivo. During this neoplastic progression, SHE cells demonstrate an altered response to epidermal growth factor (EGF). In the present report, we examined the role of the adapter protein Gab1 (Grb2-associated binder-1) in the neoplastic progression of SHE cells. We used two asbestos-transformed SHE cell clones in different neoplastic stages: a 10W+8 clone, which is immortal and retains the ability to suppress the tumorigenicity of tumor cells in cell-cell hybrid experiments, and a 10W-1 clone, which has lost this tumor suppressor ability. 10W+8 cells expressed full-length 100-kDa Gab1 and associated 5.2-kb mRNA. Upon repeated cell passaging, 10W-1 cells showed increasing expression of a novel 87-kDa form of Gab1 as well as 4.6-kb mRNA with diminishing expression of the original 100-kDa Gab1. cDNA encoding the 87-kDa Gab1 predicts a form of Gab1 lacking the amino-terminal 103 amino acids (Gab1(Delta1-103)), which corresponds to loss of most of the pleckstrin homology (PH) domain. Gab1(Delta1-103) retains the ability to be phosphorylated in an EGF-dependent manner and to associate with the EGF receptor and SHP-2 upon EGF stimulation. The endogenous expression of Gab1(Delta1-103) in 10W-1 cells appeared closely related to EGF-dependent colony formation in soft agar. Moreover, transfection and expression of Gab1(Delta1-103), but not Gab1, in 10W+8 cells enhanced their EGF-dependent colony formation in soft agar. These results demonstrate that Gab1 is a target of carcinogen-induced transformation of SHE cells and that the expression of a Gab1 variant lacking most of the PH domain plays a specific role in the neoplastic progression of SHE cells.

Topics: Agar; Amino Acid Sequence; Animals; Asbestos; Base Sequence; Blood Proteins; Blotting, Northern; Blotting, Western; Carcinogens; Cell Division; Cell Line; Cells, Cultured; Cloning, Molecular; Cricetinae; Disease Progression; DNA, Complementary; Epidermal Growth Factor; Fluorescent Antibody Technique, Indirect; MAP Kinase Signaling System; Mesocricetus; Molecular Sequence Data; Neoplasms; Phenotype; Phosphoproteins; Phosphorylation; Plasmids; Precipitin Tests; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Time Factors; Transfection; Transformation, Genetic

TT-232 is a somatostatin analogue containing a five-residue ring structure. The present report describes TT-232-induced signalling events in A431 cells, where a 4-h preincubation with the peptide irreversibly induced a cell death program, which involves DNA-laddering and the appearance of shrunken nuclei, but is unrelated to somatostatin signalling. Early intracellular signals of TT-232 include a transient two-fold activation of the extracellular signal-regulated kinase (ERK2) and a strong and sustained activation of the stress-activated protein kinases c-Jun NH(2)-terminal kinase (JNK)/SAPK and p38MAPK. Blocking the signalling to ERK or p38MAPK activation had no effect on the TT-232-induced cell killing. At the commitment time for inducing cell death, TT-232 decreased EGFR-tyrosine phosphorylation and prevented epidermial growth factor (EGF)-induced events like cRaf-1 and ERK2 activation. Signalling to ERK activation by FCS, phorbol 12-myristate 13-acetate (PMA) and platelet-derived growth factor (PDGF) was similarly blocked. Our data suggest that TT-232 triggers an apoptotic type of cell death, concomitant with a strong activation of JNK and a blockade of cellular ERK2 activation pathways.

Topics: Antineoplastic Agents; Apoptosis; Drug Antagonism; Epidermal Growth Factor; Humans; Kinetics; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinases; Neoplasms; p38 Mitogen-Activated Protein Kinases; Peptides, Cyclic; Somatostatin; Tumor Cells, Cultured

We have previously described a rat monoclonal antibody, RS-11, which recognizes a tumor-associated antigen common to several species. In the present study, we have cloned and characterized the antigen recognized by RS-11. We screened a phage expression library prepared from HeLa cDNA and identified a clone that reacts with RS-11. DNA sequence analysis revealed that this clone contains sequences of keratin 18 (nucleotides 568-1196). We constructed several glutathione S-transferase fusion proteins and synthetic peptides based on this DNA sequence analysis and examined their reactivity with RS-11 to accurately map the RS-11 epitope. We determined that the epitope resides within a region of seven amino acids on the alpha-helix 2B domain of keratin 18 in which two amino acids (Leu(366) and Lys(370)) are completely conserved among intermediate filaments as well as other keratin members that are immunoreactive with RS-11. These two residues are sequentially discontinuous but spatially adjacent. The RS-11 epitope is constitutively present in human primary cultured hepatocytes; however, its immunoreactivity with RS-11 is up-regulated by malignant transformation or stimulation with either epidermal growth factor or transforming growth factor alpha.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Base Sequence; Blotting, Western; Cells, Cultured; DNA Primers; Epidermal Growth Factor; Epitope Mapping; Epitopes; HeLa Cells; Humans; Liver; Molecular Sequence Data; Neoplasms; Rats; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

A DNA fragment frequently hypermethylated in tumor cells was isolated using a novel screening strategy termed methylation-sensitive arbitrarily primed PCR. The isolated sequence corresponded to a CpG island at the 5' end of a previously unknown gene, TPEF (transmembrane protein containing epidermal growth factor and follistatin domains). Expression of TPEF was observed using Northern master blot analysis of a variety of normal tissues including colon, bladder, and prostate tissue. TPEF maps to human chromosome 2q33, where frequent loss of heterozygosity is seen in various human tumors, and TPEF was not expressed in most human colon and various other tumor cell lines examined by reverse transcription-PCR. Nine of 11 tumor cell lines were highly methylated in the 5' region and the first exon of the gene that demonstrated features characteristic of a CpG island. However the other two cell lines, which expressed TPEF, were hypomethylated in the 5' end of the gene. The region was also hypermethylated in 11 of 16 primary bladder tumors and in 3 of 4 primary colon tumors when compared with adjacent normal tissue. Our results suggest that potential tumor suppressor genes can be isolated from human tumors by virtue of their altered methylation patterns.

Topics: Amino Acid Sequence; Base Sequence; Chromosomes, Human, Pair 2; Colonic Neoplasms; CpG Islands; DNA Methylation; DNA, Neoplasm; Epidermal Growth Factor; Follistatin; Gene Expression Regulation, Neoplastic; Gene Silencing; Glycoproteins; Humans; Membrane Proteins; Molecular Sequence Data; Neoplasm Proteins; Neoplasms; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Tumor Cells, Cultured; Urinary Bladder Neoplasms

The aim of this study was to compare the profile of EGFr expression in transitional cell carcinoma of the bladder (TCC) and in oral squamous cell carcinoma (OSCC). In addition, to study the influence of EGF stimulation on the expression of major histocompatibility complex class I antigens, placental alkaline phosphatase (PLAP) as well as changes in tumour cell sensitivity to cisplatin using immunocytochemical staining, a colorimetric assay and SDS-gel electrophoresis. The results showed that: a) strong EGFr expression could be seen in 22/88 (27%) cases of TCCs. In oral tumours the values for non-invasive ameloblastoma and invasive OSCC were 4/25 (16%) and 30/41 (73%) respectively. b) EGFr expression in tumour cell lines paralleled that of tumour biopsies. The number of lines expressing high and low EGFr expression amongst TCCs were 4 and 4 and in OSCCs were 3 and 1 respectively. c) Exposure of tumour cell lines to EGF led to: i) an increase in EGFr expression (stimulatory indices SI, ranged from 1.06 to 2.58) for TCCs but a decrease in the case of OSCCs (SI ranged from 0.01 to 0.85). The corresponding SI values for class I antigens were 0.95-1.16 and 0.10-0.84. ii) A significant reduction in expression of PLAP by OSCC cell lines. iii) An increased susceptibility of OSCC cell lines to cisplatin by as much as 14% (p<0.001). These data demonstrated the overexpression of EGFr in a significant proportion of TCCs. As for oral tumours it depended on whether they were of an invasive or non-invasive type. In the invasive cases the majority overexpressed EGFr. The exposure of OSCC but not TCC tumour cells to EGF resulted in down regulation of EGFr and class I antigens. The expression of PLAP was also significantly reduced. Exposure of OSCC cells to EGF resulted in their increased susceptibility to cisplatin. The data supports the notion that the mitogenic activation of some tumour cells by EGF resulted in a reduction of their immune visibility, differentiation status and an increase in chemosensitivity.

Topics: Alkaline Phosphatase; Antineoplastic Agents; Biopsy; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Cisplatin; Dose-Response Relationship, Drug; Drug Interactions; Epidermal Growth Factor; ErbB Receptors; Histocompatibility Antigens Class I; Humans; Isoenzymes; Mouth Neoplasms; Neoplasms; Placenta; Tumor Cells, Cultured; Urinary Bladder; Urinary Bladder Neoplasms

Current adenoviral (Ad) vectors cannot be targeted to specific cell types due to the widespread distribution of the Ad receptor (CAR). Moreover, CAR and/or internalization receptors (alphav integrins) are absent or present at low levels on some cell types, rendering them resistant to Ad-mediated gene delivery. To address these problems, we have developed a novel vector targeting approach that takes advantage of the common cell signaling pathways initiated by ligation of alphav integrins and growth factor receptors. Recombinant growth factor/cytokines (TNF-alpha, IGF-1, EGF) which trigger phosphatidylinositol-3-OH kinase (PI3K) activation, a signaling molecule involved in adenovirus internalization, were fused to a monoclonal antibody specific for the viral penton base. Ad vectors complexed with these bifunctional mAbs increased gene delivery 10 to 50-fold to human melanoma cells lacking alphav integrins. The bifunctional mAbs also enhanced gene delivery by fiberless adenovirus particles which cannot bind to CAR. Improved gene delivery correlated with increased virus internalization and attachment as well as PI3K activity. The use of bifunctional mAbs to trigger specific cell signaling pathways offers a widely applicable method for bypassing the normal Ad receptors in gene delivery and potentially increasing the selectivity of gene transfer.

Topics: Adenoviridae; Androstadienes; Animals; Antigens, CD; beta-Galactosidase; Colorectal Neoplasms; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Gene Expression; Genetic Engineering; Genetic Therapy; Genetic Vectors; Humans; Insecta; Insulin-Like Growth Factor I; Integrin alphaV; Melanoma; Neoplasms; Phosphoinositide-3 Kinase Inhibitors; Receptors, Virus; Recombinant Fusion Proteins; Signal Transduction; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Wortmannin

The effects of chronic EGF exposure on expression of the alpha2beta1 collagen and alpha5beta1 fibronectin receptor in a pair of human carcinoma cell lines (A431 and A549) with differential responses to EGF in a short-term ECM-cell adhesion assay were investigated. Treatment with EGF at 10 ng/ml for 24 hr increased on both cell lines the expression of the alpha2- but not the beta1- or alpha5-integrin sub-units, and concomitantly cellular adhesion was increased on collagen IV but not on fibronectin. Increased collagen adhesion of A549 cells could be blocked by alpha2- and beta1-integrin-sub-unit antibodies down to control levels, while it was blocked by alpha2-integrin-sub-unit antibody only by 60% and completely by the beta1-integrin-sub-unit antibody on A431 cells. EGF induced disparate shifts in cell morphologies (dome-like structures, A431, vs. spindle-like fibroblastoid, A549) with concomitant opposite changes in the expression/localization of E-cadherin in cell-cell contacts. This could be taken as an indication for cell-type-specific differential changes in the ratio of cell-ECM vs. cell-cell contacts. The EGF-induced up-regulation of the alpha2beta1 integrin was instrumental in increasing collagen adhesion of A549 but only partly in the case of A431 cells, in which cells the alpha2beta1 integrin may have additional functions besides serving as cell-ECM receptor.

Topics: Adenocarcinoma; Antigens, CD; Carcinoma; Cell Adhesion; Cell Communication; Epidermal Growth Factor; ErbB Receptors; Humans; Integrin alpha2; Integrin alpha5; Neoplasm Proteins; Neoplasms; Tumor Cells, Cultured

Binding of epidermal growth factor (EGF) to the EGF receptor is known to trigger a number of biological responses in the target cells including EGF receptor phosphorylation and stimulation of DNA synthesis leading to cell proliferation. Agents that bind to the EGF receptors could have a significant role in the therapy of tumors that express increased numbers of receptors by blocking the stimulatory effect of EGF. Different monoclonal antibodies (MAbs) directed to the EGF receptor have been generated that inhibit EGF binding and do not induce activation of the receptor tyrosine kinase. When there is sufficient uptake these antibodies can be used for immunotherapy and, after labeling with an appropriate radionuclide, also for radioimmunotherapy. For evaluation of a ligand as a therapeutic agent, it is necessary to investigate its binding characteristics in tumor cells and experimental tumors in vivo. Because the effectiveness of the antitumor activity of the MAb is dependent upon the amount of receptors in the tissue and the penetration of the MAb into the tissue, the receptor density, biokinetics, and tumor distribution of the MAb or its fragments were evaluated in different tumor models. The results of the experimental studies with tumor cell spheroids and different xenotransplanted human tumors have shown that the uptake and distribution in the tumor tissue is dependent on the molecular weight of the ligand. The correlation between the uptake of the substances and the receptor density is an indication for a noninvasive scintigraphic characterization of human tumors using radiolabeled compounds with specific binding to the tumor receptor and for selection of an optimal therapeutic regimen or radionuclide targeting of the tumor.

Topics: Animals; Antibodies, Monoclonal; Cell Division; Epidermal Growth Factor; Epitopes, B-Lymphocyte; ErbB Receptors; Humans; Immunoglobulin Fab Fragments; Iodine Radioisotopes; Mice; Neoplasms; Radioimmunodetection; Spheroids, Cellular; Transplantation, Heterologous

8-Cl-cAMP, a cAMP analogue that antagonizes type I cAMP-dependent protein kinase, is a novel anti-tumor agent presently under investigation in clinical trials. Herein we report the effects of this agent on epidermal growth factor receptor expression and degradation in human KB cancer cells. Exposure to 10 microM 8-Cl-cAMP for 48 h induced a 65% increase in epidermal growth factor receptor surface expression while the receptor synthesis was 22-fold enhanced. Analysis of epidermal growth factor-dependent receptor internalization in 8-Cl-cAMP-treated cells showed a higher endocytosis rate as well as an accelerated degradation which occurred together with an increased receptor ubiquitination. The enhanced degradation of epidermal growth factor receptor correlated with the lack of epidermal growth factor-induced proliferation and mitogen-activated protein kinase stimulation. The disregulation of epidermal growth factor receptor internalization and ubiquitin-dependent degradation could underlay a new mechanism of the anti-tumor activity of 8-Cl-cAMP suggesting its combination with agents that disrupt epidermal growth factor receptor signalling.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Antineoplastic Agents; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; Cyclic AMP-Dependent Protein Kinases; Endocytosis; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Signal Transduction; Tumor Cells, Cultured; Ubiquitins

The epidermal growth factor (EGF) repeat superfamily of genes often encodes proteins that govern cellular proliferative responses. Using a high-throughput screening by hybridization approach, a novel human EGF repeat superfamily member that maps to human chromosome X was identified. Termed EGFL6, the gene encodes a predicted signal peptide, suggesting that it is secreted. Other predicted features include four and one-half EGF-like repeat domains, two N-linked glycosylation sites, an integrin association motif (RGD), and a tyrosine phosphorylation site. Importantly, its transcripts are expressed in brain and lung tumor and fetal tissues, but are generally absent from normal adult tissues. Implications with respect to cell cycle regulation and oncogenesis are discussed.

Topics: Adult; Amino Acid Sequence; Base Sequence; Calcium-Binding Proteins; Cell Adhesion Molecules; Cloning, Molecular; Epidermal Growth Factor; Female; Fetus; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Gene Library; Glycoproteins; Humans; Male; Membrane Glycoproteins; Middle Aged; Molecular Sequence Data; Multigene Family; Neoplasm Proteins; Neoplasms; Nucleic Acid Hybridization; Organ Specificity; Peptides; Repetitive Sequences, Amino Acid

We previously reported that retroviral vectors displaying epidermal growth factor (EGF) as part of a chimeric envelope glycoprotein are sequestered upon binding to EGF receptor (EGFR)-positive target cells, leading to loss of infectivity. In the current study, we have displayed stem cell factor (SCF) on beta-galactosidase-transducing ecotropic and amphotropic retroviral vector particles as a factor Xa protease-cleavable N-terminal extension of the envelope glycoprotein. Viral incorporation of the SCF chimeric envelopes was demonstrated by immunoblotting of pelleted virions and their specific attachment to Kit receptors was demonstrated by flow cytometry. Gene transfer studies showed that when SCF was displayed on an amphotropic envelope, the infectivity of the SCF-displaying vectors was selectively inhibited on Kit-expressing cells, but could be restored by adding soluble SCF to block the Kit receptors or by cleaving the displayed SCF domain from the vector particles with factor Xa protease. The host range properties of EGF-displaying and SCF-displaying vectors were then compared in cell mixing experiments. When EGFR-positive cancer cells and Kit-positive hematopoietic cells were mixed and exposed to the different engineered vector particles, the cancer cells were selectively transduced by the SCF-displaying vector and the hematopoietic cells were selectively transduced by the EGF-displaying vector. Retroviral display of polypeptide growth factors can therefore provide the basis for a novel inverse targeting strategy with potential use for selective transduction of hematopoietic or nonhematopoietic cells (eg, cancer cells) in a mixed cell population.

Topics: 3T3 Cells; Animals; Cell Line; Epidermal Growth Factor; ErbB Receptors; Factor Xa; Gene Targeting; Gene Transfer Techniques; Genetic Vectors; Hematopoietic Stem Cells; Humans; Mice; Neoplasms; Proto-Oncogene Proteins c-kit; Recombinant Fusion Proteins; Retroviridae; Stem Cell Factor

The development of vectors that are capable of efficient gene delivery is crucial to the success of gene therapy. We have developed both recombinant viral and nonviral vectors with the goal of correcting genetic abnormalities in cancer cells that are responsible for malignant transformation. Infection of cancer cells by recombinant adenovirus (Adv) indicates that the level of transduction is variable and dependent on the virus-to-cell ratio. Infection of cells with Adv/p53 resulted in levels of tumor suppressor p53 gene expression that could mediate tumor cell growth suppression and apoptosis, both in vitro and in vivo. The treatment of cancer cells with cisplatin prior to Adv transduction resulted in a higher level of therapeutic gene expression. Epidermal growth factor (EGF)/DNA complexes targeted to cancer cells overexpressing the EGF receptor resulted in efficient transduction of several lung cancer cell lines in vitro. As a result, these vectors provide improved methods with which to treat cancer in the clinical setting with gene therapy.

Topics: Adenoviridae; Analysis of Variance; Animals; Cell Line; Epidermal Growth Factor; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Mice; Mice, Nude; Neoplasms; Retroviridae; Tumor Cells, Cultured

The epidermal growth factor receptor (EGFR) is overexpressed in a significant percentage of carcinomas and contributes to the malignant phenotype. CP-358,774 is a directly acting inhibitor of human EGFR tyrosine kinase with an IC50 of 2 nM and reduces EGFR autophosphorylation in intact tumor cells with an IC50 of 20 nM. This inhibition is selective for EGFR tyrosine kinase relative to other tyrosine kinases we have examined, both in assays of isolated kinases and whole cells. At doses of 100 mg/kg, CP-358,774 completely prevents EGF-induced autophosphorylation of EGFR in human HN5 tumors growing as xenografts in athymic mice and of the hepatic EGFR of the treated mice. CP-358,774 inhibits the proliferation of DiFi human colon tumor cells at submicromolar concentrations in cell culture and blocks cell cycle progression at the G1 phase. This inhibitor produces a marked accumulation of retinoblastoma protein in its underphosphorylated form and accumulation of p27KIP1 in DiFi cells, which may contribute to the cell cycle block. Inhibition of the EGFR also triggers apoptosis in these cells as determined by formation of DNA fragments and other criteria. These results indicate that CP-358,774 has potential for the treatment of tumors that are dependent on the EGFR pathway for proliferation or survival.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Cell Cycle; Cell Division; DNA Fragmentation; DNA, Neoplasm; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Mice; Mice, Nude; Neoplasm Proteins; Neoplasms; Phosphorylation; Retinoblastoma Protein; Tumor Cells, Cultured

In order to study the potential of non-invasive scintigraphic evaluation of the epidermal growth factor (EGF) receptor status in vivo, the biokinetics and tumor binding of 125I-EGF and anti-(EGF receptor) mAb 425 were investigated in nude mice bearing human tumor xenografts with different EGF-receptor densities as determined by a radioreceptor assay. The results demonstrated a tumor uptake for both substances depending on the receptor level. The EGF receptor status, however, was reflected slightly better by the binding of EGF to tumor tissue compared to the mAb. The rapid blood clearance of EGF with a plasma half-life of less than 1 min led to a tumor-to-blood ratio of approximately 3 within 6 h after injection in tumors with a high receptor expression. A similar ratio for the mAb was not obtained before day 6 after injection. The absolute concentration of EGF, however, was low compared to the mAb. Therefore, it can be concluded that the EGF receptor status as a target for (radio)immunotherapy can be evaluated in vivo with EGF labeled with a short-life positron-emitting radionuclide or with monoclonal antibodies to the EGF receptor or their fragments.

Topics: Animals; Antibodies, Monoclonal; Carcinoma; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms; Tissue Distribution; Transplantation, Heterologous

We have previously shown that epidermal growth factor (EGF) enhances the in vitro and in vivo sensitivity of human ovarian carcinoma 2008 cells to cisplatin. EGF was found to enhance selectively the in vivo toxicity of cisplatin to 2008 cell xenografts without altering the toxicity of cisplatin to non-malignant target tissues such as the kidney or bone marrow. We now show that recombinant human EGF (rhEGF) enhances the cisplatin sensitivity of cell lines representative of many other types of malignancies in addition to ovarian carcinoma, including cancers of the head and neck, cervix, colon, pancreas and prostate, as well as non-small-cell carcinoma of the lung. In addition, rhEGF was found to sensitise cells to other platinum-containing drugs and several other classes of chemotherapeutic agents. rhEGF sensitised 2008 cells not only to cisplatin, but also to carboplatin and tetraplatin, as well as taxol, melphalan and 5-fluorouracil. We conclude that modulation of drug sensitivity by rhEGF is observed in cell lines representative of many human malignancies and for multiple classes of chemotherapeutic agents, indicating that it alters one or more components of the cellular damage response that are both common between cell lines and classes of drugs and fundamental to survival.

Topics: Antineoplastic Agents; Cell Division; Cisplatin; Drug Screening Assays, Antitumor; Drug Synergism; Epidermal Growth Factor; Female; Humans; Male; Neoplasms; Recombinant Proteins; Tumor Cells, Cultured

Topics: Adult; Amino Acid Sequence; Animals; Biglycan; Bone and Bones; Cartilage; Cattle; Cell Adhesion; Chimera; Collagen; Cytokines; Decorin; Embryo, Mammalian; Endothelial Growth Factors; Epidermal Growth Factor; Exostoses, Multiple Hereditary; Extracellular Matrix; Extracellular Matrix Proteins; Fetus; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Heparin; Heparin-binding EGF-like Growth Factor; Humans; Integrins; Intercellular Signaling Peptides and Proteins; Lymphokines; Marfan Syndrome; Membrane Glycoproteins; Mice; Molecular Sequence Data; Neoplasms; Osteogenesis; Proteoglycans; Proto-Oncogene Proteins; Rats; Syndecan-4; Tissue Extracts; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Topics: Epidermal Growth Factor; Fibroblast Growth Factor 2; Growth Substances; Humans; Neoplasms; Neovascularization, Pathologic; Transforming Growth Factor alpha

Gliomas, squamous carcinomas and different adenocarcinomas from breast, colon and prostate might have an increased number of epidermal growth factor (EGF) receptors. The receptors are, in these cases, candidates for binding of receptor specific toxic conjugates that might inactivate cellular proliferation. The purpose of this study was to evaluate whether it is reasonable to try ligand-dextran based conjugates for therapy.. EGF or TGF alpha were conjugated to dextran and binding, internalization, retention and degradation of eight types of such conjugates were analyzed in EGF-receptor amplified glioma cells. The conjugates were labelled with radioactive nuclides to allow detection and two of the conjugates were carrying boron in the form of carboranyl amino acids or aminoalkyl-carboranes. Comparative binding tests, applying 125I-EGF, were made with cultured breast, colon and prostate adenocarcinoma, glioma and squamous carcinoma cells. Some introductory tests to label with 76Br for positron emission tomography and with 131I for radionuclide therapy were also made.. The dextran part of the conjugates did not prevent receptor specific binding. The amount of receptor specific binding varied between the different types of conjugates and between the tested cell types. The dextran part improved intracellular retention and radioactive nuclides were retained for at least 20-24 h. The therapeutical effect improved when 131I was attached to EGF-dextran instead of native EGF.. The improved cellular retention of the ligand-dextran conjugates is an important property since it gives extended exposure time when radionuclides are applied and flexibility in the choice of time for application of neutrons in boron neutron capture therapy (BNCT). It is possible that ligand-dextran mediated BNCT might allow, if the applied neutron fields covers rather wide areas around the primary tumor, locally spread cells that otherwise would escape treatment to be inactivated.

Topics: Adenocarcinoma; Animals; Boron Compounds; Boron Neutron Capture Therapy; Carcinoma; Colonic Neoplasms; Dextrans; Drug Carriers; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Iodine Radioisotopes; Male; Mammary Neoplasms, Experimental; Models, Biological; Neoplasms; Neoplasms, Experimental; Prostatic Neoplasms; Rats; Rats, Sprague-Dawley; Transforming Growth Factor alpha; Tumor Cells, Cultured

Cluster 13 was defined by 2 independently derived murine monoclonal antibodies (MAbs), RS7 (IgG1) and MR54 (IgG2a), which were raised against human squamous-cell carcinoma of the lung and a human ovarian-carcinoma cell line, respectively. Immunologic and biochemical evidence demonstrated that RS7 and MR54, as well as 2 additional MAbs, MR6 (IgG2a) and MR23 (IgG1), generated in the same fusion as MR54, recognize the same antigen, a 46- to 48-kDa glycoprotein. Evaluation of the expression of antigen on the surface of tumor cell lines, Western blotting analyses, competitive binding studies, and double-determinant ELISA assays, support this conclusion. Two distinct epitopes are defined by these MAbs. In order to further characterize this antigen, amino-acid-sequence analyses were performed on peptides derived from antigen purified by affinity chromatography with MAb RS7. The sequence data obtained from 2 peptides, which were independently generated by CNBr cleavage and trypsin digestion respectively indicated identity to GA733-1. The GA733-1 genomic DNA sequence predicted a type-1 membrane protein of 35 kDa, with 4 potential N-linked glycosylation sites. The GA733-1 protein product has not been identified previously, and MAbs to this tumor-associated antigen were not previously known.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Blotting, Western; Carcinoma, Squamous Cell; Cell Adhesion Molecules; Cell Line; Epidermal Growth Factor; Epithelial Cell Adhesion Molecule; Female; Humans; Immunoglobulin G; Lung Neoplasms; Mice; Molecular Sequence Data; Multigene Family; Neoplasms; Ovarian Neoplasms; Sequence Homology, Amino Acid; Tumor Cells, Cultured

We developed a sensitive EGF receptor detection method for frozen tissue sections using biotinylated EGF as the primary reagent. The method was directly compared with an immunohistochemical technique based on an anti-EGF receptor monoclonal antibody (MAb EGFR1) in normal human and rat tissues and in human tumors. The method was more sensitive than a previously published biotinylated EGF-based technique. In normal human tissues and in 37 of the 50 tumors, the binding pattern mirrored that of positive staining with EGFR1. Five further tumors showed weak immunoreactivity, but in these no binding of biotinylated EGF was detected. The remaining eight tumors were negative by both techniques. The discordant cases may reflect a lower level of sensitivity of the ligand-binding technique or, alternatively, abnormal receptors may have been expressed in these tissues. EGF receptors could be detected in rat liver with biotinylated EGF but not with the antibody, indicating the usefulness of the ligand-based technique in cross-species studies.

Topics: Animals; Antibodies, Monoclonal; Biotin; Epidermal Growth Factor; ErbB Receptors; Female; Frozen Sections; Humans; Immunoenzyme Techniques; Ligands; Liver; Male; Mice; Microtomy; Neoplasms; Rats; Sensitivity and Specificity; Skin

The reduction of glucose supply induced the killing of tumor cells by tumor necrosis factor (TNF) in vivo and in vitro. In contrast, normal cell lines were resistant to TNF regardless of the presence or absence of glucose. Epidermal growth factor (EGF) did not exert a cytotoxic effect on tumor cells in the absence of glucose. Therefore, the killing mechanism of TNF under conditions of reduced glucose supply was investigated. Flow cytometry experiments and studies of kinetics revealed that the S-phase of the cell cycle was prolonged in the absence of glucose. After TNF treatment, the S-phase was found to be shortened and the rate of 3H-thymidine incorporation into DNA was increased, whereas EGF failed to exert such an effect. DNA synthesis and entry into mitosis are known to be regulated by cyclin A. In serum-starved tumor cells (HeLa) we have observed increased cyclin A synthesis within 10 hr, in parallel with enhancement of DNA synthesis and shortening of the S-phase after TNF treatment. We conclude that, under conditions of low glucose supply, TNF can assume the role of a growth factor in transformed cells.

Topics: Animals; Aphidicolin; Blotting, Northern; Blotting, Western; Cell Cycle; Cyclins; DNA, Neoplasm; Dose-Response Relationship, Drug; Epidermal Growth Factor; Glucose; Humans; Neoplasms; S Phase; Time Factors; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Previous studies have demonstrated quantitation of epidermal growth factor receptors (EGFR) to be of prognostic significance in breast, bladder, esophageal and other neoplasms. However, the relatively large quantity of unfixed tissue required for epidermal growth factor radioligand binding assays (RLBA) has precluded its application to cytologic specimens and small biopsy specimens. For this reason we evaluated reverse transcription intron differential polymerase chain reaction (RTIDPCR) as an assay of EGFR gene expression. Squamous cell carcinoma (A431 and SiHa), transitional cell carcinoma (HT1376, T24, RT4), mammary (MCF7) and endocervical (HeLa) adenocarcinoma, and leukemia (K562) cell lines were used to compare RTIDPCR and RLBA. RTIDPCR involved reverse transcription of RNA and amplification of cDNA using primers for beta-actin and EGFR. Good agreement was observed between the RLBA and RTIDPCR results. RNA extracted from fresh cells, Diff-Quik-stained smears and formalin-fixed, paraffin-embedded cell pellet sections yielded similar results. These data suggest that RTIDPCR may be useful in evaluating gene expression by cells processed as cytologic specimens.

Topics: Adenocarcinoma; Base Sequence; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Genes, Neoplasm; Humans; Introns; Leukemia, Myeloid; Molecular Sequence Data; Neoplasms; Polymerase Chain Reaction; RNA, Neoplasm; Staining and Labeling; Tissue Embedding; Tissue Preservation; Tumor Cells, Cultured

We report that cytosine arabinoside (Ara-C), a cytosine analogue that at low doses causes phenotypical changes on human leukemia cells in vitro and in vivo, induces growth inhibition of oropharyngeal cancer KB and lung adenocarcinoma A549 cell lines. An increase in the number of epidermal growth factor and transferrin receptors (EGFR, TrfR) is induced by Ara-C on these cells. Maximal EGFR up-regulation occurs 96 h after the beginning of Ara-C exposure while maximal TrfR up-regulation is detected 24 h later. These effects occur without changes in the affinity of EGFR and TrfR for their ligands. Two classes of EGF-binding sites with a Kd of 0.055 nM and 2.3 nM respectively, and one class of transferrin-binding sites with a Kd of about 4 nM are detected on both untreated and Ara-C-treated KB cells. [3H]Thymidine uptake is clearly stimulated on KB cells by nanomolar concentrations of EGF and transferrin, whereas in Ara-C-treated cells [3H]thymidine uptake is not increased by EGF and transferrin under conditions where maximal EGFR and TrfR up-regulation occurs. The enhanced EGF and transferrin binding is paralleled by a twofold increase of in vitro targeting of Ara-C-treated KB and A549 cells with anti-EGFR 108.1 mAb and anti-TrfR OKT9 mAb. We propose that Ara-C could provide a new approach for the improvement of the therapeutic index of anti-EGFR and anti-TrfR immunoconjugates.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Cell Division; Cytarabine; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Humans; Immunotherapy; Iodine Radioisotopes; KB Cells; Lung Neoplasms; Neoplasms; Radioimmunoassay; Receptors, Cell Surface; Receptors, Transferrin; Time Factors; Transferrin; Tumor Cells, Cultured; Up-Regulation

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous environmental contaminant that is produced inadvertently during the synthesis of some organochlorine compounds, such as the chlorinated phenoxy pesticides. It is biologically and ecologically persistent, with an estimated half-life of 7 years in humans. It possesses high acute toxicity in rodents and is a carcinogen, teratogen, and immunotoxin. In chronic bioassays for carcinogenicity, TCDD at a dose of 10 ng/kg/day increases the incidence of liver tumors in female rats, making it one of the most potent animal carcinogens ever tested. A recent study in humans has shown an increase in the incidence of respiratory tract tumors in workers in chlorinated phenoxy herbicide plants. Considerable controversy and uncertainty remain, however, concerning its carcinogenic potency in humans and the reliability of using animal data to predict human risks. It is generally accepted that most, if not all, of the effects of TCDD require its binding to the Ah receptor. In addition to its toxic effects, TCDD produces a number of biochemical effects, such as induction of CYP1A1, downregulation of binding activity of the estrogen and epidermal growth factor (EGF) receptors, and changes in cytokine pathways. These effects suggest that the Ah receptor plays an important role in regulating the cell cycle. A number of structural analogs of TCDD, such as the polychlorinated dibenzofurans, also interact with the Ah receptor, and they produce the same spectrum of responses as TCDD in animal and cell models. The potency of these compounds is strongly correlated with their binding affinity to the Ah receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Biomarkers, Tumor; Cricetinae; Cytochrome P-450 Enzyme System; Disease Susceptibility; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Humans; Isoenzymes; Liver; Lymphocytes; Mice; Neoplasms; Neoplasms, Experimental; Polychlorinated Dibenzodioxins; Rats; Rats, Sprague-Dawley; Receptors, Aryl Hydrocarbon; Receptors, Drug; Risk

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Endothelium, Vascular; Epidermal Growth Factor; Fibroblast Growth Factors; Humans; Neoplasms; Neovascularization, Pathologic; Signal Transduction; Transforming Growth Factor beta

Using a human keratinocyte model of tumor progression, we have examined the regulation of gene expression and secretion of a parathyroid hormone-like peptide (PLP) that has been implicated in the pathogenesis of hypercalcemia in cancer. A rapid and transient induction of PLP mRNA in response to serum stimulation was demonstrated in both established (HPK1A) and malignant (HPK1A-ras) cells; however the dose dependent increases were greater in HPK1A than in HPK1A-ras. Significant inhibition of this induction was noted with the addition of 1,25-dihydroxyvitamin D3 at a lower concentration in HPK1A than in HPK1A-ras. Amino-terminal PLP immunoreactivity and bioactivity correlated well (r = 0.98) when measured in conditioned medium. In the absence of mitogenic stimuli, malignant keratinocytes (HPK1A-ras) secreted significantly more PLP than established (HPK1A) keratinocytes. However, in response to increasing concentrations of epidermal growth factor and fetal bovine serum, PLP release was far greater from HPK1A (maximum 13 x basal) than from HPK1A-ras (maximum 3 x basal) cells. In addition, 1,25-dihydroxyvitamin D3 was more effective in inhibiting both basal and stimulated PLP secretion in HPK1A than in HPK1A-ras cultures. Reduction of extracellular Ca2+ from 2.0 mM to 0.5 mM appeared to be more effective at an early time point in reducing PLP secretion from the established cells compared with the malignant cells. These studies therefore demonstrate a progressive dysregulation of PLP expression and secretion in human keratinocytes in the transformation from established to malignant phenotype and may have important implications for understanding the pathogenetic mechanisms involved in vivo in the development of hypercalcemia in cancer.

Topics: Blotting, Northern; Calcitriol; Calcium; Cells, Cultured; Culture Media; Epidermal Growth Factor; Gene Expression; Humans; In Vitro Techniques; Keratinocytes; Neoplasm Proteins; Neoplasms; Parathyroid Hormone-Related Protein; Proteins; RNA, Messenger; Time Factors

Recent laboratory and clinical data suggest that some human neoplasms exhibit unusually high levels of cell-surface receptors for epidermal growth factor, and that this abnormality is associated with rapid cellular proliferation and poor prognosis. We propose that the existence of an abnormally high number of mitogen receptors is not merely correlated with rapid proliferation but is pathophysiologically responsible for such behavior. Cells with high levels of mitogen receptors may be rendered 'hypersensitive' to mitogenic stimuli, and hence may be stimulated to divide even when ambient mitogen concentrations are at a low 'background' level, insufficient to prompt the division of cells with a normal number of receptors. To investigate this hypothesis further, we have developed a mathematical model that describes proliferative behavior of cells as a function of mitogen concentration and receptor number. The model enables us to simulate the proliferative behavior of cells with various receptor levels at various mitogen concentrations and predicts a growth advantage associated with excess mitogen receptors. Computer simulations based on the model are consistent with previously published experimental data. This work provides support for the view that overexpression of genes encoding normal growth factor receptors can contribute to the inappropriate proliferation of neoplastic cells.

Topics: Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Models, Biological; Neoplasms; Tumor Cells, Cultured

Although under study to alleviate chemotherapy-induced bone marrow toxicity, cytokines can stimulate in vitro growth of solid human tumour cell lines. The effects of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and interleukin-3 (IL-3) on in vitro colony formation of primary human tumours was studied in a capillary soft-agar cloning system. Of 108 tumour specimens from 100 patients, 85 specimens were tested against all three factors at concentrations ranging from 0.1 to 1000 ng/ml. 44 of 100 tumours showed adequate growth in controls. 8 out of 43 (19%) specimens were significantly stimulated by GM-CSF, 6 of 40 (15%) by G-CSF and 10 of 44 (23%) by IL-3. Sensitivity to all three cytokines was observed in 4 of 44 (9%) specimens. By light microscopy the appearance of colonies from stimulated specimens was identical to that of controls. Sensitivity to cytokines was independent from sensitivity to epidermal growth factor, transferrin or insulin. Sensitivity to GM-CSF, G-CSF and IL-3 may be aberrantly expressed in a subgroup of solid human tumours.

Topics: Biopsy; Clone Cells; Dose-Response Relationship, Drug; Epidermal Growth Factor; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Insulin; Interleukin-3; Neoplasms; Recombinant Proteins; Stimulation, Chemical; Transferrin; Tumor Cells, Cultured; Tumor Stem Cell Assay

Topics: Antineoplastic Agents; Cell Division; Enzyme Activation; Epidermal Growth Factor; Gonadotropin-Releasing Hormone; Humans; Membrane Proteins; Neoplasms; Phosphoprotein Phosphatases; Phosphorylation; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Signal Transduction; Somatostatin

We examined the effects of epidermal growth factor (EGF) on the anchorage-dependent and -independent growth of four human squamous carcinoma cell lines that overexpress EGF receptors. While EGF inhibited anchorage-dependent growth, it stimulated anchorage-independent growth of all four cell lines tested. The results suggest that the proliferative responses to EGF are characterized by a preference for anchorage-independent, rather than -dependent growth, in cells overexpressing EGF receptors. Moreover, as EGF has been shown to stimulate the in vivo growth of squamous carcinoma cells overexpressing EGF receptors, it is also suggested that the in vitro EGF responsiveness of these cells in soft agar, but not in monolayer, better correlates with the in vivo EGF responsiveness.

Topics: Carcinoma, Squamous Cell; Cell Adhesion; Cell Count; Cell Division; DNA; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Tumor Cells, Cultured

Topics: Body Fluids; Endocrine System Diseases; Epidermal Growth Factor; Female; Humans; Male; Milk, Human; Neoplasms; Radioimmunoassay; Semen; Skin Diseases

Epidermal growth factor (EGF) was determined by radioimmunoassay in serum, plasma, and urine of 23 patients undergoing ablative therapy followed by bone marrow transplantation. The difference between the serum and plasma values reflected the amount of EGF released from the platelets at the time of blood coagulation. Platelet-derived EGF strongly correlated with platelet count (r + 0.850, P less than 0.0001), and the intercept of the regression line was very close to zero; one platelet contained approximately 2.5 x 10(-18) g EGF. Correspondingly, when the platelet count dropped after bone marrow ablation from 222 +/- 97 to 33 +/- 13 x 10(9)/l, the serum EGF decreased from 603 +/- 222 to 65 +/- 41 pg/ml (P less than 0.0001). Plasma EGF content did not correlate with the platelet count and did not change significantly after bone marrow ablation (before and after the ablation, correspondingly, 290 +/- 80 and 332 +/- 99 pg/ml, P = 0.194). High-performance liquid chromatographic fractionation of serum and plasma showed different molecular mass distribution of EGF-immunoreactive fractions. The main molecular mass components of the plasma EGF did not change after bone marrow ablation. Urine excretion remained unchanged (320 +/- 133 and 314 +/- 173 pmol EGF/mmol creatinine). We conclude that whereas platelets are the source of serum EGF, the origin of plasma EGF is different and the search of its origin is warranted.

Topics: Adult; Blood Platelets; Bone Marrow; Bone Marrow Transplantation; Epidermal Growth Factor; Female; Humans; Male; Middle Aged; Neoplasms; Plasma; Platelet Count; Radioimmunoassay

Human epithelial cells that had grown out from a maxillary carcinoma were examined for their responsiveness to putative growth-controlling factors in a serum-free medium. Among the factors examined, bovine brain acidic fibroblast growth factor (FGF) at 1 to 10 ng/ml significantly promoted DNA synthesis of the cells in the presence of 5 U/ml heparin, whereas type beta transforming growth factor inhibited it in a dose-dependent manner. Fetal bovine serum at 0.6% inhibited DNA synthesis of the cells by approximately 15%, but no significant influence was observed at higher concentrations up to 10%. Epidermal growth factor, bovine pituitary gland FGF and basic FGF exhibited no significant effect on DNA synthesis of the cells. The present result suggests that acidic FGF, a known mitogen for endothelial cells, is also mitogenic for human epithelial cells derived from maxillary carcinoma.

Topics: Carcinoma; DNA; Epidermal Growth Factor; Fibroblast Growth Factors; Humans; Neoplasms; Transforming Growth Factors; Tumor Cells, Cultured

An inhibitor of soft agar colony formation by a human breast carcinoma-derived cell line was found to be present in latent form in the majority of cytology-positive human malignant effusions. Prior to dialysis, addition of human malignant effusions resulted in less than 10% alteration in efficiency of colony formation by the BT-20 human breast carcinoma cell line (mean efficiency 1 colony/4.3 cells plated at 14 days; mean colony diameter greater than 0.8 mm). After dialysis (membrane cutoff of 3500 molecular weight), 58 of 70 malignant effusions from patients with a variety of epithelial cell carcinomas resulted in 71% mean inhibition of colony formation (range 19.1-98% inhibition). Similar inhibition of anchorage-independent growth was observed for a human colon cancer-derived cell line (HCT-15) but not for polyoma and murine sarcoma virus-transformed rodent fibroblast lines. The malignant effusion-related transformed cell growth-inhibiting factor (TGIF) was sensitive to heat, sulfhydryl reduction, and protease treatment. TGIF-containing effusion resulted in parallel inhibition of thymidine incorporation in sensitive cell types in vitro. TGIF was precipitable in 28-34% ammonium sulfate with reconstitution of activity after resolubilization. TGIF was partially purified by chromatography on Biogel A-0.5 and Biogel P-100 which yielded two peaks of inhibitory activity. The predominant species had an approximate molecular weight of 110,000 and could be recovered as a single species from DEAE-cellulose at relatively high salt concentrations (0.4 M NaCl). A smaller amount of inhibitory activity was recovered from Biogel P-100 or Biogel P-60 with an apparent molecular weight of 55,000. The higher molecular-weight TGIF which appears to be a dimer of the Mr 55,000 protein is distinguishable from previously described growth-promoting and -inhibiting factors.

Topics: Cell Division; Epidermal Growth Factor; Exudates and Transudates; Growth Inhibitors; Humans; Molecular Weight; Neoplasms; Peptides; Transforming Growth Factors; Tumor Cells, Cultured

We determined the concentrations of immunoreactive epidermal growth factor in the urine (U-irEGF) of 97 adult patients with various malignancies, including carcinomas of the urinary bladder, kidney, stomach, colon, rectum, breast, endometrium, uterine cervix, ovary, vagina, prostate, pancreas and thyroid, liposarcoma and skin melanoma. The relative U-irEGF concentrations (ng m-1 creatinine) were higher (P = 0.002) for the whole series of female patients than for healthy controls matched for sex and age. Such difference did not appear for male patients. The only specific group with a statistically supranormal U-irEGF concentration (P = 0.0005) comprised women with endometrial carcinoma of the uterus.

Topics: Adult; Aged; Aged, 80 and over; Creatinine; Epidermal Growth Factor; Female; Humans; Male; Middle Aged; Neoplasms; Sex Factors; Uterine Neoplasms

The expressions of epidermal growth factor (EGF), EGF receptor, transforming growth factor (TGF) alpha and c-Ha-ras p21 in human gastric carcinomas were studied immunohistochemically. Any of these four marker proteins from gastric tumor cells was closely correlated with the depth of tumor invasion. The incidences of cases with EGF or synchronous expression of TGF alpha and c-Ha-ras in metastatic tumors were significantly higher than those in primary tumors (p less than 0.05 or p less than 0.01). Patients with synchronous expression of EGF and its receptor or TGF alpha and ras p21 had a far poorer prognosis than those without such synchronous expression. These findings strongly suggest that the EGF-related growth factors, EGF-receptor and ras p21 mutually play an important role in tumor progression and could be useful as potential metastatic and/or prognostic markers for gastric carcinoma.

Topics: Biomarkers, Tumor; Epidermal Growth Factor; Growth Substances; Humans; Neoplasms; Oncogenes; Peptides; Transforming Growth Factors

The growth kinetics of LLC-PK1 renal epithelia is examined, particularly relating to epidermal growth factor (EGF)-induced mitogenesis in growth-arrested confluent cultures. LLC-PK1 cell proliferation is shown to be density dependent. After reaching confluence, cell number remains near constant, and addition of fresh serum-containing medium does not elicit significant increase in cell number. EGF and 12-O-tetradecanoyl-phorbol-13-acetate do however induce a marked increase in cell number, although each with different kinetics. EGF-induced mitogenesis in LLC-PK1 cells has the following characteristics: (a) DNA synthesis peaks in a defined window 10-12 h after EGF administration; (b) 10 nM EGF elicits a maximal response; (c) 10 nM EGF requires only a 30-s exposure to these cells to induce a maximal mitogenic response; (d) exposure of the basolateral membrane of these polar cells to 10 nM EGF is necessary for mitogenesis, exposure of EGF to the apical surface being ineffective. This aspect of polarity in growth factor-induced mitogenesis is a phenomenon which cannot be addressed with nonpolar fibroblastic cell cultures.

Topics: Cells, Cultured; Epidermal Growth Factor; Epithelium; Kidney; Mitosis; Neoplasms; Tetradecanoylphorbol Acetate

A radioimmunoassay for transforming growth factor alpha (TGF-alpha) using synthetic rat sarcoma transforming growth factor and its rabbit polyclonal antibody has been developed. Using radioimmunoassays, the urinary TGF-alpha and epidermal growth factor (EGF) concentrations in 31 patients with hepatocellular carcinoma (HCC), 15 probable HCC, four metastatic liver cancer, and 33 age, sex-matched healthy controls were determined. For the first time, we have shown that the average TGF-alpha concentration for HCC patients was 21.5 +/- 20.3 micrograms per g creatinine, significantly higher than that of healthy subjects, 4.9 +/- 2.8 micrograms per g creatinine (P less than 0.001). There was no statistical difference in the level of EGF between HCC patients and controls (40.9 +/- 29.3 versus 46.2 +/- 16.6 micrograms per g creatinine; P greater than 0.05). The ratio of EGF/TGF-alpha between HCC patients (3.37 +/- 4.42) and controls (15.5 +/- 13.0) was significantly different (P less than 0.001). Among patients, 65% (20 of 31) of HCC cases and 87% (13 of 15) of probable HCC cases showed a marked elevation of TGF-alpha levels. We found only 16% (five of 31) of HCC cases with increased EGF level. EGF excretion was inversely age related. Serum total protein concentration and alkaline phosphatase activity were positively correlated to EGF concentration (r = 0.522, P less than 0.01 and rt = 0.393, P less than 0.05, respectively). There was no correlation between biochemical functions of liver and TGF-alpha concentration in HCC patients. Our results also suggested that TGF-alpha may be a useful complementary tumor marker for management of patients with clinical manifestation of HCC who have low alpha-fetoprotein levels.

Topics: Adult; alpha-Fetoproteins; Carcinoma, Hepatocellular; Creatinine; Epidermal Growth Factor; Female; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Peptides; Reference Values; Transforming Growth Factors

The effect of epidermal growth factor (EGF) on the in vitro growth of 186 malignant human tumor specimens (45 melanomas, 32 sarcomas, and 56 lung, 16 gynecological, 14 breast, 12 genitourinary, and 11 gastrointestinal carcinomas) was evaluated in the cellular adhesive matrix human tumor culture system supplemented with transferrin, insulin, hydrocortisone, and estradiol. EGF increased tumor growth by at least 50% in 81% of the 186 tumors and by over 100% in 54%. The enhanced growth induced by EGF was related to an accelerated cellular division independent of tumor type and not to an increase in the actual number of clonogenic units. The drug concentrations of cell cycle-independent Adriamycin and cisplatin needed to achieve a 90% tumor cell kill were not altered by the responsiveness of the tumor to EGF.

Topics: Antineoplastic Agents; Breast Neoplasms; Carcinoma; Cell Division; Cell Line; Cell Survival; Cells, Cultured; Culture Media; Epidermal Growth Factor; Extracellular Matrix; Gastrointestinal Neoplasms; Humans; Lung Neoplasms; Melanoma; Neoplasms; Sarcoma; Urogenital Neoplasms

The mechanisms of ectopic enzyme and enzyme inhibitor-production in neoplastic tissues were investigated. No evidence was obtained to suggest any difference in the structural genes of amylase-producing tumors and normal lymphocytes. mRNA sequence coding for amylase precursor in tumor tissues was identical to that of salivary amylase, suggesting that the amylase in amylase-producing tumors was identical to, or closely resembled the amylase in salivary gland. High incidence of elevation of serum pancreatic secretory trypsin inhibitor (PSTI) was observed in patients with various malignant tumors. PSTI-positive malignant cells were also frequently found in malignant tissues. A comparison of human PSTI mRNA sequence with mouse epidermal growth factor (EGF) mRNA sequence showed that they were 46% homologous. Human PSTI stimulated [3H]thymidine incorporation into DNA in human fibroblasts at concentrations present in human serum.

Topics: Amylases; Animals; Base Sequence; Epidermal Growth Factor; Female; Humans; Lung Neoplasms; Mice; Mice, Nude; Neoplasms; Ovarian Neoplasms; RNA, Messenger; Salivary Glands; Thymoma; Thymus Neoplasms; Trypsin Inhibitor, Kazal Pancreatic; Trypsin Inhibitors

Topics: Colony-Stimulating Factors; Epidermal Growth Factor; Growth Inhibitors; Growth Substances; Humans; Neoplasms; Oncogenes; Peptides; Receptors, Cell Surface; Transforming Growth Factors

Growth factors are proteins or peptides which evoke the replication of the nuclear DNA and thus cell division. A survey is given of the structure, the importance and the mechanism of action of the biochemically exactly characterized growth factors. The epidermal growth factor consists of 53 amino acids and is made over outwardly by proteolysis from a large precursor molecule in the membrane: it is attached to a receptor of the cell membrane consisting of three large parts, by means of which the intracellularly lying part of the receptor becomes effective as proteinkinase and phosphorylizes the parts of the receptor as well as adjacently lying proteins. This acts as a signal for the beginning of the phase of the DNA-synthesis (replication) in the chromosomes. The receptor for the epidermal growth factor has a high degree of structure similarity with the insulin receptor which is also active as proteinkinase after insulin binding. The importance of the decomposition of phosphatidylinosite and of its phosphate esters in binding of certain ligands to receptors is shown.

Topics: Aged; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Interleukin-1; Interleukin-2; Neoplasms; Nerve Growth Factors; Phosphatidylinositol Phosphates; Phosphatidylinositols; Platelet-Derived Growth Factor; Receptors, Cell Surface; Receptors, Nerve Growth Factor; Receptors, Platelet-Derived Growth Factor; Somatomedins

Activity of transforming growth factor (TGF) in urine was examined by the use of cloned BALB/3T3 A31-1-1 indicator cells. When urine was extracted by an acid-ethanol method, fractionated on a Bio-Gel P-100 column, and analyzed for TGF activity, samples from healthy persons showed no activity, but 80% of the samples from advanced cancer patients had the activity. Activity observed in tumor-bearing patients was eliminated after therapy. Further analysis was carried out in a much simpler way by the fractionation of urine samples after only dialysis and lyophilization. An extensive survey of TGF activity in urine using BALB/3T3 indicator cells revealed weak or intermediate (19%) activity in healthy persons and high (56%) or intermediate (31%) activity in advanced cancer patients. When urine samples were analyzed and compared using both BALB/3T3 cells and NRK 49F cells, the BALB/3T3 cells were shown to form colonies in response to beta-type TGF but not to alpha-type TGF. The present results are the first demonstration of an elevated level of beta-type TGF in urine from cancer patients.

Topics: Biological Assay; Epidermal Growth Factor; Female; Humans; Male; Molecular Weight; Neoplasms; Peptides; Pregnancy; Time Factors; Transforming Growth Factors

The limitations of the agar suspension culture method for primary culturing of human tumor cells prompted development of a monolayer system optimized for cell adhesion and growth. This method grew 83% of fresh human tumor cell biopsy specimens, cultured and not contaminated, from a heterogeneous group of 396 tumors including lung cancer (93 of 114, 82%); melanoma (54 of 72, 75%); sarcoma (46 of 59, 78%); breast cancer (35 of 39, 90%); ovarian cancer (16 of 21, 76%); and a miscellaneous group consisting of gastrointestinal, genitourinary, mesothelioma, and unknown primaries (78 of 91, 86%). Cell growth was characterized morphologically with Papanicolaoustained coverslip cultures and cytogenetically with Giemsastained metaphase spreads. Morphological features such as nuclear pleomorphism, chromatin condensation, basophilic cytoplasm, and melanin pigmentation were routinely seen. Aneuploid metaphases were seen in 90% of evaluable cultures, with 15 of 28 showing 70% or more aneuploid metaphases. Colony-forming efficiency ranged between 0.01 and 1% of viable tumor cells, with a median efficiency of 0.2%. This culture system uses a low inoculum of 25,000 viable cells per well which permitted chemosensitivity testing of nine drugs at four doses in duplicate from 2.2 X 10(6) viable tumor cells and radiation sensitivity testing at five doses in quadruplicate from 0.6 X 10(6) cells. Cultures were analyzed for survival by computerized image analysis of crystal violet-stained cells. Drug sensitivity studies showed variability in sensitivity and in survival curve shape with exponential cell killing for cisplatin, Adriamycin, and etoposide, and shouldered survival curves for 5-fluorouracil frequently seen. Radiation sensitivity studies also showed variability in both sensitivity and survival curve shape. Many cultures showed exponential cell killing, although others had shouldered survival curves. This method for growing cells from primary human biopsy specimens is more efficient than the agar culture method, enables easier and better biological analysis of the actual cells grown, and permits improved characterization of drug and radiation survival curves.

Topics: Biopsy; Cell Adhesion; Cell Cycle; Cell Survival; Cells, Cultured; Culture Media; Dose-Response Relationship, Radiation; Epidermal Growth Factor; Humans; Karyotyping; Neoplasms; Neoplastic Stem Cells; Radiography

Transforming growth factor type beta (TGF beta) has been purified from serum-free culture fluids of transformed mouse L-929 cells which are capable of continual growth in serum-free medium in the absence of any exogenously added polypeptide growth factors. TGF beta has been purified to homogeneity as judged by NH2-terminal amino acid sequence analysis. Analysis of the purified polypeptide by gel electrophoresis indicates that TGF beta is composed of two polypeptide chains of Mr 12,500 cross-linked by disulfide bonds. TGF beta was characterized by its ability to induce anchorage-dependent normal rat kidney (NRK) cells to grow in soft agar in the presence of epidermal growth factor (EGF). TGF beta was also able to enhance both EGF-induced DNA synthesis and cell proliferation on growth-arrested NRK cells in monolayer cultures under serum-free conditions. We also show that in mouse melanoma B-16 cells under serum-free conditions TGF beta stimulates both DNA synthesis in monolayer cultures and anchorage-independent growth in soft agar. Paradoxically, the anchorage-independent growth in the presence of serum of many human cell lines, including melanomas, and mammary, prostatic, vulvar, and lung carcinomas is inhibited by TGF beta at saturating concentrations similar to those that stimulate colony formation of the rodent cell lines L-929 and B-16 under serum-free conditions. The peculiar action of TGF beta is further revealed by the observations that while EGF and TGF beta synergize to induce inhibition of anchorage-independent growth of A-431 human vulvar carcinoma cells, their effects on the anchorage-independent growth of one human lung carcinoma cell line (A-549) and two human prostatic carcinoma cell lines (PC-3 and DU-145) are antagonistic. Moreover, we show that in the rodent and human cell lines TGF beta interacts with specific cellular receptors which may mediate the actions of TGF beta. We conclude that the expression of both TGF beta and TGF beta receptors by L-929 cells and the stimulation of growth of L-929 cells in serum-free medium by TGF beta suggests that TGF beta may be important for maintaining the transformed state of this tumor cell line, and the way in which a cell responds to TGF beta is dependent on the presence or absence of growth factors contained in the serum.

Topics: Amino Acid Sequence; Breast; Cell Division; Cell Line; Cell Transformation, Neoplastic; DNA; Epidermal Growth Factor; Growth Substances; Humans; Kidney; Melanoma; Molecular Weight; Neoplasm Proteins; Neoplasms; Peptides; Transforming Growth Factors

Topics: Animals; Epidermal Growth Factor; Growth Substances; Humans; Neoplasms; Neoplasms, Experimental; Nerve Growth Factors; Oncogenes; Platelet-Derived Growth Factor

Topics: Cell Fractionation; Endosomes; Enzymes; Epidermal Growth Factor; Humans; KB Cells; Lipid Metabolism; Neoplasms

Tyrosyl kinase activity was detected in 1.0 microliter or less of human serum with the substrates angiotensin II, polyamino acid polymer Glu-Tyr (4:1), anti-pp60src IgG, and endogenous serum proteins. Most (about 84%) of the tyrosyl kinase activity was in the 100,000 X g soluble fraction from serum and a similar level of activity was found in the soluble fraction from plasma. Expression of tyrosyl kinase activity in individual serum samples differed more than 15-fold. The different levels of tyrosyl kinase activity were not likely due to phosphatases, proteases, ATPases, or kinase inhibitors and activators in serum. The normal serum and plasma tyrosyl kinase activities were not stimulated by epidermal growth factor, platelet-derived growth factor, insulin, or growth factors from fetal calf serum. Investigations of samples from patients with malignant disorders indicated that those with malignant melanoma contained the highest levels of serum tyrosyl kinase activity.

Topics: Adenosine Triphosphate; Angiotensin II; Animals; Endopeptidases; Epidermal Growth Factor; Humans; Immunoglobulin G; Insulin; Intercellular Signaling Peptides and Proteins; Neoplasms; Oncogene Protein pp60(v-src); Peptides; Phosphoprotein Phosphatases; Phosphoproteins; Platelet-Derived Growth Factor; Protein Kinases; Protein-Tyrosine Kinases; Rabbits; Viral Proteins

The cytotoxic activity against human tumor cells of toxic conjugates of Pseudomonas exotoxin with anti-transferrin receptor antibody or epidermal growth factor was potentiated up to 10 to 20-fold by the calcium antagonists verapamil, D-600, and diltiazem and by the lysosomotropic agent beta-glycylphenyl-naphthylamide. The potentiating activity of these agents could be predicted by measuring the inhibition of protein synthesis by the immunotoxins on various cell lines. The use of potentiating agents such as these in combination with immunotoxins may prove useful in the treatment of some human cancers.

Topics: Antibodies, Neoplasm; Calcium Channel Blockers; Cell Survival; Cells, Cultured; Cytotoxins; Dipeptides; Drug Synergism; Epidermal Growth Factor; Humans; Neoplasm Proteins; Neoplasms; Receptors, Cell Surface; Receptors, Transferrin

Radiation inactivation with high energy electrons from a linear accelerator was used to determine the functional molecular size of the epidermal growth factor (EGF) binding site and the tyrosine-specific protein kinase activity in A-431 membranes. The target size of the protein portion of the EGF receptor glycoprotein was 147,000 daltons when the radiation-dependent decrease in maximal binding capacity was measured. Since the target size is in good agreement with the molecular size of the protein portion of the EGF receptor determined by denaturing biochemical methods, it appears that the monomeric receptor is the functional binding site in situ. The target size of the EGF-stimulated kinase activity associated with the affinity-purified EGF receptor/kinase was 133,000 and 144,000 daltons when assayed for the ability to autophosphorylate or to phosphorylate a tyrosine-containing peptide, respectively. However, the target size of the kinase activity that did not adhere to an EGF-affinity column was 54,000 and 69,000 daltons when assayed for phosphorylation of endogenous and exogenous substrates, respectively. Intermediate target sizes were obtained when kinase assays were performed on membranes prior to fractionation by affinity chromatography. These results, taken with other biochemical data, indicate that A-431 membranes contain a kinase activity that is a domain of the glycoprotein that contains the EGF binding site and that the membranes also contain another tyrosine-specific kinase or kinases that have an average size of approximately 60,000 daltons.

Topics: Animals; Binding Sites; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Mice; Molecular Weight; Neoplasms; Phosphorylation; Protein Kinases; Protein-Tyrosine Kinases; Receptors, Cell Surface

We have examined the effect of epidermal growth factor(EGF) on three kinds of kinases activities, phosphatidylinositol(PI) kinase, phosphatidylinositol 4-phosphate[PI(4)P] kinase and diacylglycerol(DG) kinase that make important roles in the regulation of inositol phospholipids metabolism. When isolated plasma membrane vesicles from A431 cells were incubated at 30 degrees C with [gamma-32P]ATP and exogenously added DG, EGF enhanced the activity of DG kinase approximately 2-fold. This stimulation is found to be dose-dependent with a half maximal activation at 1 nM. In this case, EGF increased Vmax without changing Km Value for ATP or DG. Although this activation was observed in the absence of detergent, it was more evident when membrane vesicles were treated with 1 mM deoxycholate. Interestingly, the effect of EGF was only detected in magnesium containing medium. The use of manganese instead of magnesium diminished the stimulatory effect in either condition, presence or absence of deoxycholate. On the other hand, the stimulation of PI kinase or PI(4)P kinase activity was not caused by EGF. These results suggest that DG kinase activation by EGF makes important roles in cellular responses leading to cell growth.

Topics: Cell Membrane; Cells, Cultured; Diacylglycerol Kinase; Epidermal Growth Factor; Humans; In Vitro Techniques; Kinetics; Neoplasms; Phosphatidic Acids; Phosphatidylinositols; Phosphotransferases

Recent advances in protein chemistry and genetic engineering have revealed new information about the molecular lesions involved in the induction and maintenance of cancer cells. It is now known that a single base change in the DNA of human cells leads to cancer. The normal pathway of proliferation and differentiation is perturbed by changes to molecules involved in the intracellular biochemical pathways controlled by growth factors. Some cancer cells appear to produce their own growth factor, others have higher concentrations of growth factor receptors on their surface and others have mutated versions of the intracellular proteins linked to the growth factor receptors. This increased understanding of growth control in normal and neoplastic cell populations is gradually providing a foundation for new approaches to cancer therapy.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA, Neoplasm; Epidermal Growth Factor; Growth Substances; Hematopoietic Stem Cells; Humans; Neoplasms; Neoplastic Stem Cells; Nerve Growth Factors; Platelet-Derived Growth Factor; Receptors, Cell Surface

The current understanding in biology and function of 4 growth factors is reviewed. PDGF suggests functions for proto-oncogens in normal cells, which may interact in tightly linked hierachies to induce malignant growth. PDGF-requirement of normal fibroblast cell-lines is lost when the cells are infected with tumor viruses. TGF is able to stimulate growth of normally anchorage dependent cells in an anchorage independent manner in soft agar. This ability is thought to be the best in-vitro correlate of neoplastic transformation. The peptide hormones bombesin/gastrin releasing factor and EGF can act as autocrine growth factors in various lung cancer cell-lines and stimulate clonal tumor cell growth in-vitro. The potential clinical application of these types of growth factors may enable the in-vitro growth from any lung cancer patient and allow individual drug testing. TCGF produced by T-cells to activate T-cells, is central to immune stimulation and immune response. Models for potential indirect anticancer effects either by in-vivo administration or by in-vivo incubation plus passive transfer of T-cells are presented to be initiated in future clinical trials.

Topics: Animals; Bombesin; Cell Line; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Interleukin-2; Neoplasms; Oncogenes; Peptides; Platelet-Derived Growth Factor; Receptors, Cell Surface; Transforming Growth Factors

As many as 40 distinct oncogenes of viral and cellular origin have been identified to date. Many of these genes can be grouped into functional classes on the basis of their effects on cellular phenotype. These groupings suggest a small number of mechanisms of action of the oncogene-encoded proteins. Some data suggest that, in the cytoplasm, these proteins may regulate levels of critical second messenger molecules; in the nucleus, these proteins may modulate the activity of the cell's transcriptional machinery. Many of the gene products can also be related to a signaling pathway that determines the cell's response to growth-stimulating factors. Because some of these genes are expressed in nongrowing, differentiated cells, the encoded proteins may in certain tissues mediate functions that are unrelated to cellular growth control.

Topics: Animals; Birds; Cell Nucleus; Cell Transformation, Neoplastic; Chickens; Cytoplasm; Deltaretrovirus; DNA Tumor Viruses; Drosophila; Epidermal Growth Factor; Growth Substances; Guanosine Triphosphate; Humans; Mutation; Neoplasms; Oncogenes; Platelet-Derived Growth Factor; Polyomavirus; Proto-Oncogenes; Rats; Repetitive Sequences, Nucleic Acid; Retroviridae; Simian virus 40; Transcription, Genetic

Cellular genes which encode proteins involved in the response of cells to stimulation by growth factors may be potential oncogenes. The factors involved in the signal transmission from growth factor-receptor interaction to DNA synthesis constitute a cascade system which we call the 'growth factor-proto-oncogene pathway(s) of mitogenesis'. For each growth factor, all the responsive cells, regardless of cell types and tissue source, have specific growth factor receptors which are similar, if not identical, in molecular weight and biological activity. Thus, we believe that the growth factor-proto-oncogene pathway(s) functions in the same manner in all responsive cells. Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and brain-derived growth factor (BDGF) are major growth factors for connective tissue cells and do not share a common pathway in mitogenesis in responsive cells. The gene product of c-myc may be involved in the cellular response of cells stimulated by PDGF or FGF, but not directly in the signal transmission which leads to DNA synthesis.

Topics: Animals; Cattle; DNA; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Neoplasms; Platelet-Derived Growth Factor; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogenes; Receptors, Cell Surface

A survey of human tumor cell lines for increased PDGF or EGF receptors identified 5 lines which bind from 6 to 13 times more EGF than normal human fibroblasts. Immunoprecipitation analysis links the elevated binding activity to increased quantities of the EGF receptor protein. EGF receptor gene amplification was detected in 2 of the cell lines. No evidence for EGF receptor gene rearrangements was found at the level of DNA or RNA structure. The results suggest that elevated levels of EGF receptor can be associated with at least three distinct mechanisms. These include gene amplification accompanied by rearrangement, gene amplification without accompanied alteration of mRNA transcripts, and extensive expression without gene amplification.

Topics: Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Gene Expression Regulation; Humans; Immunologic Techniques; Membrane Proteins; Molecular Weight; Neoplasms; Receptors, Cell Surface; RNA, Messenger

Topics: Calcitriol; Epidermal Growth Factor; Humans; Hypercalcemia; Lymphokines; Neoplasms; Parathyroid Hormone; Prostaglandins

Partial proteolysis with trypsin has been used to map the sites of phorbol ester-induced phosphorylation of the epidermal growth factor (EGF) receptor. Both 12-O-tetradecanoylphorbol 13-acetate (TPA) and EGF stimulate phosphorylation of the EGF receptor in intact human carcinoma cells. Under the conditions examined, EGF is more effective than TPA in stimulating phosphorylation of a 45 kDa intracellular receptor domain, while TPA is more effective than EGF in inducing phosphorylation of a 120 kDa transmembrane EGF-binding domain. The phosphorylation of the 120 kDa peptide occurs primarily on threonine residues. Two-dimensional peptide mapping indicates that the two major phosphopeptides found in the 120 kDa receptor fragment correspond to the major new phosphopeptides found in intact EGF receptor following treatment with TPA. Thus, the major sites of TPA-induced threonine phosphorylation reside in the 120 kDa binding domain of the EGF receptor.

Topics: Binding Sites; Cell Line; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Humans; Models, Biological; Neoplasms; Peptide Fragments; Phorbols; Phosphorylation; Receptors, Cell Surface; Tetradecanoylphorbol Acetate; Threonine; Trypsin

To investigate the possibility that the epidermal growth factor (EGF) receptor functions as an oncogene product, we have determined the levels of EGF receptor protein and RNA in a variety of malignant and normal human cells, using a specific polyclonal antibody to the EGF receptor and a cDNA clone (plasmid pE7) that encodes the EGF receptor, respectively. Besides A431 epidermoid carcinoma cells, which are known to make large amounts of EGF receptor, cell lines from two ovarian cancers, two cervical cancers, and one kidney cancer were found to contain substantial amounts of receptor protein (11-22% of A431). Normal human fibroblasts (Detroit 551), a human lymphocyte line (IM-9), and a leukemic lymphocyte line (CEM) contained low or undetectable levels of EGF receptor. RNA blot analysis showed that among the human cell lines examined the levels of a 10- and a 5.6-kilobase species of pE7-specific RNA generally correlated with the amount of the EGF receptor protein. Genomic DNA blot analysis revealed that except for A431 none of these cell lines expressing high levels of EGF receptor protein possessed amplified receptor gene sequences. A431 cells are known to secrete a truncated form of the EGF receptor. An abundant 2.9-kilobase RNA is found only in A431 cells; it could encode the truncated form of the EGF receptor.

Topics: Cell Line; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblasts; Genes; Humans; Neoplasms; Nucleic Acid Hybridization; Receptors, Cell Surface

We have measured the production of a basic-somatomedin-like activity (SLA) by a variety of human tumor-derived, transformed, and normal postnatal cell cultures; and we have compared the production of SLA by these cell types with the production of SLA by adult rat hepatocytes cultured in serum-free medium. Cells derived from a human epidermoid carcinoma (KB), a pancreatic carcinoma (Panc-1), a Simian virus 40 transformed adult human skin-derived cell line (SV40 fibroblasts), and a normal adult human skin-derived fibroblast line released SLA when cultured in a serum-free growth medium. No SLA was recovered from the culture medium of human choriocarcinoma-derived cells (BeWo) or of a human lymphoblastoid cell line (IM-9). The production of SLA by rat hepatocytes cultured in serum-free medium appeared to exceed the production of SLA by the other cell cultures. In cultures of KB cells, SV40 fibroblasts, and rat hepatocytes, the production of SLA depended on the frequency with which the growth medium was renewed; in general, the highest rates of SLA production were observed when the medium was renewed every 48-72 h. The presence of mouse epidermal growth factor (urogastrone) (EGF-URO) in the serum-free culture medium stimulated the production of SLA by KB cells and by rat hepatocytes, but did not increase SLA production by normal or by SV40-transformed human skin-derived fibroblasts. We conclude that tumor-derived cells are capable of producing somatomedin-like activity and that the production of SLA by such cells can be subject to controls (nutrient availability, EGF-URO stimulation) that regulate SLA production, either by normal adult tissues, like liver, or by a variety of normal embryonic tissues.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Epidermal Growth Factor; Humans; Liver; Mice; Neoplasms; Simian virus 40; Somatomedins

Reverse-phase high-performance liquid chromatography (HPLC) performed on urine from cancer patients and normal controls revealed the presence of seven chromatographically distinct peaks of transforming growth factor (TGF) activity, as measured by colony formation of normal rat kidney cells in soft agar. Comparison of urines from normal donors and cancer patients showed no differences in EGF (epidermal growth factor)-dependent beta-TGF-like activity but did reveal distinct patterns of EGF-related, EGF-independent alpha-TGF-like activity. All urine samples contained at least two chromatographically distinguishable forms of EGF-dependent TGF activity, eluting from HPLC as broad peaks with 30 and 43% acetonitrile. The remaining five TGFs eluted as sharp peaks with 32, 34, 35, 37, and 38% acetonitrile, demonstrated EGF-competing activity, and thus were functionally related to EGF. Two of the five EGF-related TGFs were consistently elevated only in the urine of cancer patients and eluted with 32% (TGFA) and 37% (TGFD) acetonitrile Two of the other EGF-related TGFs, eluting with 34% (TGFB) and 35% (TGFC) acetonitrile, were commonly found in both normals and cancer patients. The fifth EGF-related TGF, TGFE, eluting with 38% acetonitrile, was found only in normal donor specimens. TGFA corresponded to the unique Mr 30,000 TGF activity previously identified only in the urine of cancer patients. These observations demonstrate that cancer patients produce high levels of EGF-related TGF activities which can be readily distinguished, using reverse-phase HPLC, from EGF-related TGFs produced by normal individuals. Using a solid-phase competitive radioreceptor binding assay for EGF, we demonstrated that quantitation of EGF-competing activity is as sensitive and effective as the soft-agar colony formation assay for distinguishing HPLC profiles of urinary TGF from cancer patients versus that from normal individuals.

Topics: Adolescent; Adult; Carcinoma, Squamous Cell; Cell Line; Chromatography, High Pressure Liquid; Epidermal Growth Factor; ErbB Receptors; Female; Growth Substances; Humans; Kinetics; Male; Middle Aged; Neoplasm Proteins; Neoplasms; Peptides; Receptors, Cell Surface; Reference Values; Transforming Growth Factors

Responses of human diploid cells, TIG-1, were examined with respect to their ability to initiate DNA synthesis under the influence of various growth factors and their combinations. The following agents stimulated DNA synthesis in quiescent TIG-1 cells at 37-49 PDL (population doubling level) (66-79% of lifespan completed): fetal bovine serum; tumor-derived DNA synthesis factors such as those from rat rhodamine fibrosarcoma, human adenoma and from the conditioned medium of cultured human pituitary cells; human and mouse epidermal growth factors; tumor promotors such as 12-O-tetradecanoylphorbol 13-acetate and teleocidin; microtubule-disrupting agents as colchicine, vinblastine, podophyllotoxin and TN-16; melittin; and dexamethasone. Cells at 58-60 PDL (94-97% of lifespan completed) were stimulated to synthesize DNA by fetal bovine serum, tumor-derived DNA synthesis factors and epidermal growth factors, but not by other agents. Finally, in senescent cells at 62 PDL (100% of lifespan completed), any of these growth factors and of their combinations failed to induce DNA synthesis at all. These senescent cells, however, still retained the ability to initiate DNA synthesis following infection with SV40 as reported previously [Exp. Cell Res., 143 (1983) 343-349].

Topics: Carcinogens; Cell Survival; Cells, Cultured; Colchicine; DNA; Epidermal Growth Factor; Growth Substances; Hormones; Humans; Melitten; Neoplasms

Topics: Animals; Cell Transformation, Neoplastic; Epidermal Growth Factor; Growth Substances; Humans; Insulin; Mitosis; Neoplasms; Oncogenes; Platelet-Derived Growth Factor; Protein Kinases; Protein-Tyrosine Kinases

Topics: Animals; Cell Communication; Cell Differentiation; Cell Transformation, Neoplastic; Epidermal Growth Factor; Growth Inhibitors; Humans; Neoplasms

Transforming growth factors are known to induce anchorage-independent growth of non-transformed cells, and are released by a variety of cells, including MSV-transformed cells. This study demonstrates that the differentiated cells derived from F9 and PC-13 embryonal carcinoma cells, but not the parental cells themselves, respond by increased growth to several factors released by MSV-transformed cells, including partially purified sarcoma growth factor. The chemical properties of the growth-promoting activity are shown to match the chemical properties of the transforming growth factors released by MSV-transformed cells. Furthermore, F9 and PC-13 embryonal carcinoma cells, which do not respond to factors released by MSV-transformed cells, are shown to release factors with transforming growth factor activity. Based on the close relationship between mouse embryonal carcinoma cells and cells of early mouse embryos, it is suggested that molecules with transforming growth factor activity may play a role during the early stages of mammalian development.

Topics: Animals; Binding, Competitive; Cell Differentiation; Cell Division; Cell Line; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Mice; Neoplasms; Peptides; Receptors, Cell Surface; Transforming Growth Factors

Surgically removed solid human benign and malignant neoplasms and nonneoplastic tissues were examined for the presence of transforming growth factors (TGFs). TGFs are polypeptide growth factor-like substances which cause the appearance of a reversible neoplastic phenotype in nontransformed, anchorage-dependent cells in culture, including the induction of the ability to grow while suspended in semisolid medium. Acid-ethanol extracts from adenocarcinomas of the breast, colon, kidney, and ovary; fibrosarcoma and leiomyosarcoma; Hodgkin's lymphoma; fibroadenoma of the breast; uterine leiomyoma; and nonneoplastic kidney and lung were found to cause growth in soft agar of both nontransformed mouse AKR-2B and rat NRK cells. This colony-stimulating activity, where tested, was heat and acid stable but was destroyed by trypsin and dithiothreitol treatment, indicating that the activity is due to a polypeptide with disulfide bonds. Extracts from several of the tumors provided sufficient material for purification by molecular sieve chromatography. Peaks of colony-stimulating activity from a Bio-Gel P-60 column eluted with 1 M acetic acid were detected in the M, 3,000 to 25,000 range with the apparent molecular weight varying depending on the type of tumor being studied and the indicator cells used. The data suggest that at least three TGFs are present in human tumors. Evidence is presented differentiating these TGFs into TGFa, which has selective activity for stimulating AKR-2B cells, and TGFn, which has selective activity for stimulating NRK cells. The NGFn activity was further subdivided into a TGFns fraction and TGFnl fraction, denoting small (less than 6,000) and large (12,000 to 20,00) apparent molecular weights, respectively. The TGFa and TGFnl activities were present in malignant and nonneoplastic (kidney and lung) tissue, whereas the TGFns activity predominated in benign neoplasms. These TGFs exhibited no competition with epidermal growth factor for binding to the epidermal growth factor receptor, and the TGFnl activity was potentiated by epidermal growth factor.

Topics: Animals; Cell Division; Cells, Cultured; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Humans; Kidney; Lung; Mice; Mice, Inbred AKR; Molecular Weight; Neoplasms; Peptides; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Transforming Growth Factors

Transforming growth factors (TGFs) are a heterogeneous family of polypeptides that induce anchorage-independent growth in nonneoplastic anchorage-dependent cells. They have been found in many tissues, both neoplastic and nonneoplastic. All TGFs isolated thus far are of low molecular weight (6000-25,000), are acid and heat stable, and are inactivated by reagents that reduce disulfide bonds. TGFs have been classified as type alpha or type beta based on their interactions with the receptor for epidermal growth factor (EGF) and their requirement for EGF (or an EGF-like polypeptide) for functional activity. TGF-alpha and TGF-beta act synergistically. TGF-alpha induces phosphorylation of tyrosine in the EGF receptor. TGF-beta, isolated from bovine sources, accelerates experimental wound healing in rats.

Topics: Animals; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Cell Transformation, Viral; Drug Stability; Epidermal Growth Factor; ErbB Receptors; Humans; Mice; Molecular Weight; Neoplasms; Neoplasms, Experimental; Peptides; Rats; Receptors, Cell Surface; Transforming Growth Factors

Using seventeen human tumor cell lines derived from a variety of tissues, specific binding sites for epidermal growth factor (EGF), a mouse submandibular gland-derived growth factor, has been characterized. A significant amount of membrane-bound EGF receptors, although considerably varied, was demonstrated in all the tumor cell lines studied. Epidermoid carcinoma appeared to have more EGF receptors than adenocarcinoma. One small cell carcinoma of the lung, one choriocarcinoma of the stomach and three bone tumors also possessed EGF receptors comparable to those of epidermoid carcinoma, while one adenoacanthoma of the stomach had less EGF receptors comparable to adenocarcinoma. Among a variety of phorbol esters tested, tetradecanoyl phorbol acetate, a potent tumor promotor, was shown to be the most effective compound in inhibiting 125I-labeled EGF binding to its receptors. Our results indicate that human tumor cells contain varying amounts of membrane-bound receptors for EGF and that phorbol esters interact with these EGF receptor sites. However, the relationship between EGF receptor sites on tumor cells and cellular proliferation and/or differentiation awaits further study.

Topics: Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Phorbol Esters; Radioligand Assay; Receptors, Cell Surface

Urine from 22 patients with a variety of disseminated cancers and from an equivalent number of nonmalignant controls of similar age and sex was tested for the presence of transforming growth factor (TGF) activity as measured by the ability to promote the growth in soft agar of nontransformed indicator cells. Cancer patients included those with carcinomas of the lung, breast, colon, and ovary, as well as melanomas and sarcomas. The nonmalignant controls included both normals and individuals with a variety of inflammatory and infectious disorders. Aliquots of unfrozen urine were acid extracted, chromatographed on a Bio-Gel P-30 column, and then tested for TGF activity using normal rat kidney fibroblasts and epidermal growth factor (EGF)-competing activity with human carcinoma A431 cells. These assays revealed that a high-molecular-weight TGF activity (Mr 30,000 to 35,000) which coelutes with EGF-competing activity was present in 18 of 22 cancer patients but present in only five of 22 nonmalignant controls (p less than 0.01). In contrast, a low-molecular-weight TGF activity (Mr 6000 to 8000) which does not coelute with EGF-competing activity was found in all urines tested. These results indicate that an EGF-related, high-molecular-weight TGF activity is found in the urine of cancer patients and may be a useful tumor marker. Unlike other tumor markers described previously, high-molecular-weight TGF activity has a biological activity which is related to the expression of the transformed phenotype.

Topics: Binding, Competitive; Carcinoma; Cell Transformation, Neoplastic; Chromatography, Gel; Epidermal Growth Factor; Humans; Molecular Weight; Neoplasms; Peptides; Transforming Growth Factors

Topics: Epidermal Growth Factor; Female; Humans; Male; Neoplasms

The purpose of the present study was to determine whether skin fibroblasts from individuals, either with an inherited predisposition to cancer or with genetic disorders usually associated with a high risk of cancer, can be oncogenically transformed in vitro by a tumor promoter alone. The effects of chronic and limited applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) on several properties that are associated with transformation were examined using skin fibroblasts from individuals with polyposis coli, a familial cancer syndrome, xeroderma pigmentosum, Fanconi's anemia, and trisomy 21. The results of this study show that TPA treatment induces similar changes on cellular morphology, growth rate, saturation density, epidermal growth factor binding, and cytoskeleton in fibroblasts from both normal and genetically predisposed individuals. None of these cell lines, however, acquired anchorage-independent growth or unlimited growth potential in culture after chronic application of TPA. These observations suggest clearly that skin fibroblasts from individuals with either a genetic predisposition to or a high risk of cancer may not exist in a preneoplastic or "initiated" state susceptible to oncogenic transformation by TPA alone and that the mechanism of genetically determined cancer induction may be different from that of chemical carcinogenesis.

Topics: Cell Adhesion; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cytoskeleton; Disease Susceptibility; Down Syndrome; Epidermal Growth Factor; Fanconi Anemia; Female; Fibroblasts; Humans; Intestinal Polyps; Male; Neoplasms; Phorbols; Tetradecanoylphorbol Acetate; Trypsin; Xeroderma Pigmentosum

Epidermal growth factor (EGF), a mitogenic polypeptide hormone, stimulates the phosphorylation of certain endogenous proteins in membrane preparations derived from A431 cells, a human tumor cell line. Membrane vesicles prepared from A431 cells were reacted with 5'-p-fluorosulfonylbenzoyl adenosine (5'-p-FSO2BzAdo). Reaction of the vesicles with 5'-p-FSO2BzAdo results in a time-dependent inhibition of EGF-stimulable protein kinase activity which parallels an increase in incorporation into the vesicles of the 5'-p-sulfonylbenzoyl-[8-14C]adenosine moiety from 5'-p-FSO2Bz[14C]Ado. The primary bands labeled have Mr = 170,000 and 150,000. Labeling of these bands by 5'-p-FSO2Bz[14C]Ado is inhibited by incubation of the membrane vesicles with adenyl-5'-yl imidodiphosphate, an ATP analog. Inactivation of the kinase with N-ethylmaleimide or by heating results in a sharply decreased labeling of the proteins with Mr = 170,000 and 150,000. Proteins of these molecular weights have previously been identified in these cells as the EGF receptor and a degradation product of the receptor. These experiments provide chemical evidence that the EGF receptor and the EGF-stimulable kinase are the same protein.

Topics: Adenosine; Affinity Labels; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Molecular Weight; Neoplasms; Protein Kinase Inhibitors; Protein Kinases; Receptors, Cell Surface

Transforming growth factors (TGF) are growth-promoting polypeptides that cause phenotypic transformation and anchorage-independent growth of normal cells. They have been isolated from several human and animal carcinoma and sarcoma cells. One TGF is sarcoma growth factor (SGF) which is released by murine sarcoma virus-transformed cells. Whereas the TGF interacts with epidermal growth factor (EGF) cell membrane receptors, it is not detectable in culture fluids from cells which contain high numbers of free EGF cell membrane receptors. The SGF acts as a tumor promoter in cell culture systems, and its effect on the transformed phenotype is blocked by retinoids (vitamin A and synthetic analogs). The production of TGF by transformed cells and the responses of normal cells to the addition of TGF to the culture medium raise the possibility that cells "autostimulate" their growth by releasing factors that rebind at the cell surface. The term "autocrine secretion" has been proposed for this type of situation in which a cell secretes a hormone-like substance for which it has external cell membrane receptors. The autocrine concept may provide a partial explanation for some aspects of tumor cell progression.

Topics: Animals; Binding Sites; Carcinogens; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; In Vitro Techniques; Neoplasms; Peptides; Receptors, Cell Surface; Sarcoma Viruses, Murine; Transforming Growth Factors

The effect of EGF on the soft agar colony-forming ability of fresh human tumor cells was assessed in 40 specimens obtained from various types of carcinoma including those of the breast, endometrium ovary, and other sites. Cells from four established human tumor cell lines (three breast and one endometrial) were also included in this study. The results showed that addition of EGF at a concentration of 50 ng/ml resulted in a 50% higher cloning efficiency in soft agar in 40% of the samples of fresh human tumors. When cells from tumor cell lines were plated in semi-solid medium containing EGF, the number of colonies formed was at least twice as high as controls. Cells from fresh tumor biopsies were assayed for EGF receptors to determine whether the correlation between the proliferative response in EGF-supplemented semi-solid medium as compared to control could be related to the number of EGF receptors present on the cells. Specific receptors for EGF were detected by using radioiodinated EGF in early-passage cell cultures from some of the tumors tested for clonogenicity. The number of receptors ranged from 0.3 to 3.27 X 10(5) per cell. Cells from two melanoma specimens possessed less than 0.3 X 10(3) EGF receptors per cell. We found no correlation between the number of EGF receptor on a cell surface and the mitogenic effect of EGF on the same tumor cells grown in semi-solid medium.

Topics: Biopsy; Cell Line; Clone Cells; Culture Media; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Receptors, Cell Surface

The ability of high-density lipoprotein (HDL) to support the growth of an established tumor cell line exposed to defined medium supplemented with transferrin has been examined. Low-density A-431 carcinoma cells maintained on extracellular matrix- or fibronectin-coated dishes proliferated actively when exposed to a synthetic medium supplemented with HDL, 500 micrograms protein per ml. Epidermal growth factor added at concentrations above 0.5 ng/ml inhibited cell growth, while at concentrations above 5 ng/ml it was cytotoxic. Among the various substrata tested for their ability to support the active proliferation of low-density A-431 cells when exposed to transferrin and HDL, plastic was the least efficient. On fibronectin-coated dishes, cells ceased to proliferate after 8 population doublings, while on extracellular matrix-coated dishes cells could be passaged for 50 population doublings. In the case of colon carcinoma, rhabdomyosarcoma, and Ewing's sarcoma cells exposed to medium supplemented with transferrin, the addition to the cultures of HDL alone resulted in a growth rate and final cell density which were similar to those observed when cells were exposed to serum-supplemented medium. In the case of the mammary carcinoma cell lines MCF-7 and ZR-75-1, HDL also supported cell growth, although to a lesser extent than did serum. The present study therefore indicates that HDL is capable of supporting, either totally or partially, the in vitro proliferation of tumor cells.

Topics: Blood; Breast Neoplasms; Cell Division; Cell Line; Colonic Neoplasms; Culture Media; Epidermal Growth Factor; Fibronectins; Humans; Insulin; Lipoproteins, HDL; Neoplasms; Rhabdomyosarcoma; Sarcoma, Ewing; Transferrin

The effect of epidermal growth factor (EGF) and fibroblast feeder layers on the proliferation of human tumor cells in soft agar was examined. The addition of EGF to medium supporting growth of tumor cells significantly increased (P less than or equal to 0.02) the number of colonies grown from the cells of 36 to 58 patients (62%) with a variety of neoplasms. The increase in the number of colonies was dependent on the concentration of EGF and was maximal at a concentration of 50 ng EGF/ml. The addition of glucocorticoids did not potentiate the effect of EGF. Lethally irradiated fibroblast feeder layers alone only slightly increased the number of colonies. However, the addition of fibroblasts to cultures significantly increased the mitogenic effect of EGF. The results suggest that a proportion of epithelial-derived tumors retain responsiveness in vitro to physiologic growth regulators such as EGF.

Topics: Agar; Cell Division; Cells, Cultured; Culture Media; Epidermal Growth Factor; Epithelium; Fibroblasts; Glucocorticoids; Humans; Neoplasms

It is suggested that a common proliferative factor is required for the growth of clones of both antigen-triggered B lymphocytes and carcinogen-triggered epithelial cells, and that the common growth-promoting substance is secreted by macrophages which have originally differentiated in support of the immune response. This macrophage-derived factor seems to be antigenically distinct from granulocyte colony-stimulating factor. Identification of the macrophage-derived clonal proliferative factor could provide new understanding of the proliferation of clones of immunocytes and neoplastic cells.

Topics: Animals; Antibody-Producing Cells; B-Lymphocytes; Cell Division; Clone Cells; Epidermal Growth Factor; Growth Substances; Humans; In Vitro Techniques; Macrophages; Mice; Models, Biological; Neoplasm Metastasis; Neoplasms

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Epidermal Growth Factor; Humans; Neoplasms; Peptides; Protein Biosynthesis; Receptors, Cell Surface

Topics: Animals; Carcinogens; Cell Transformation, Neoplastic; Cocarcinogenesis; Dietary Fats; Epidermal Growth Factor; Humans; Neoplasms; Ornithine Decarboxylase; Phenobarbital; Plasminogen Activators; Receptors, Drug; Saccharin; Smoking; Tetradecanoylphorbol Acetate