Ligand-induced Activation Of Epidermal Growth Factor Receptor Research Articles

Suppression of Wnt/β-catenin signaling by EGF receptor is required for hair follicle development.

Mice that lack the epidermal growth factor receptor (EGFR) fail to develop a hair coat, but the mechanism responsible for this deficit is not completely understood. Here, we show that EGFR plays a critical role to attenuate wingless-type MMTV integration site family member (Wnt)/β-catenin signaling during postnatal hair follicle development. Genetic ablation of EGFR in mice resulted in increased mitotic activity in matrix cells, apoptosis in hair follicles, and impaired differentiation of epithelial lineages that form hair. EGFR is activated in wild-type hair follicle stem cells marked with SOX9 or NFATc1 and is essential to restrain proliferation and support stem cell numbers and their quiescence. We observed elevated levels of Wnt4, 6, 7b, 10a, 10b, and 16 transcripts and hyperactivation of the β-catenin pathway in EGFR knockout follicles. Using primary keratinocytes, we linked ligand-induced EGFR activation to suppression of nascent mRNA synthesis of Wnt genes. Overexpression of the Wnt antagonist sFRP1 in mice lacking EGFR demonstrated that elevated Wnts are a major cause for the hair follicle defects. Colocalization of transforming growth factor α and Wnts regulated by EGFR in stem cells and progeny indicates that EGFR autocrine loops control Wnts. Our findings define a novel mechanism that integrates EGFR and Wnt/β-catenin pathways to coordinate the delicate balance between proliferation and differentiation during development.

Type II cGMP-dependent protein kinase inhibits ligand-induced activation of EGFR in gastric cancer cells

Our previous data demonstrated that type II cGMP‑dependent protein kinase (PKG II) inhibited epidermal growth factor (EGF)-induced MAPK/ERK/JNK‑mediated signal transduction through inhibiting the phosphorylation/activation of the epidermal growth factor receptor (EGFR). Since the EGFR also binds with several other ligands as well as EGF, the present study was designed to investigate whether PKG II inhibited transforming growth factor-α (TGF-α), betacellulin (BTC) and epiregulin (EPR) induced phosphorylation/activation of the EGFR and consequent MAPK/ERK‑mediated signaling. The human gastric cancer cell line AGS, was infected with adenoviral constructs encoding cDNA of PKG II (Ad-PKG II) to increase the expression of PKG II and was treated with 8-pCPT-cGMP to activate the kinase. Western blotting was applied to detect the phosphorylation of EGFR and MAPK/ERK. The results demonstrated that treatment with EGF (100 ng/ml, 5 min), TGF-α (100 ng/ml, 5 min), BTC (100 ng/ml, 5 min) and EPR (100 ng/ml, 5 min) increased the tyrosine (tyr) 1068 phosphorylation of the EGFR and the threonine (thr) 202/tyr 204 phosphorylation of MAPK/ERK. Infecting the cells with Ad-PKG II and stimulating the kinase with 8-pCPT-cGMP efficiently inhibited the phosphorylation of the EGFR and MAPK/ERK induced by EGF, TGF-α, BTC and EPR. The results indicated that PKG II also inhibits the activation of the EGFR caused by diverse ligands of the receptor.

A bispecific transmembrane antibody simultaneously targeting intra- and extracellular epitopes of the epidermal growth factor receptor inhibits receptor activation and tumor cell growth

Epidermal growth factor receptor (EGFR) plays an important role in essential cellular processes such as proliferation, survival and migration. Aberrant activation of EGFR is frequently found in human cancers of various origins and has been implicated in cancer pathogenesis. The therapeutic antibody cetuximab (Erbitux) inhibits tumor growth by binding to the extracellular domain of EGFR, thereby preventing ligand binding and receptor activation. This activity is shared by the single chain antibody fragment scFv(225) that contains the same antigen binding domain. The unrelated EGFR-specific antibody fragment scFv(30) binds to the intracellular domain of the receptor and retains antigen binding upon expression as an intrabody in the reducing environment of the cytosol. Here, we used scFv(225) and scFv(30) domains to generate a novel type of bispecific transmembrane antibody termed 225.TM.30, that simultaneously targets intra- and extracellular EGFR epitopes. Bispecific 225.TM.30 and related membrane-anchored monospecific 225.TM and TM.30 proteins carrying extracellular scFv(225) or intracellular scFv(30) antibody fragments linked to a transmembrane domain were expressed in EGFR-overexpressing tumor cells using a doxycycline-inducible retroviral system. Induced expression of 225.TM.30 and 225.TM, but not TM.30 reduced EGFR surface levels and ligand-induced EGFR activation, while all three molecules markedly inhibited tumor cell growth. Co-localization of 225.TM with EGFR was predominantly found on the cell surface, while interaction with 225.TM.30 and TM.30 proteins resulted in the redistribution of EGFR to perinuclear compartments. Our data demonstrate functionality of this novel type of membrane-anchored intrabodies in tumor cells and suggest distinct modes of action of mono- and bispecific variants.

Structural Evaluation of EGFR Inhibition Mechanisms for Nanobodies/VHH Domains

The epidermal growth factor receptor (EGFR) is implicated in human cancers and is the target of several classes of therapeutic agents, including antibody-based drugs. Here, we describe X-ray crystal structures of the extracellular region of EGFR in complex with three inhibitory nanobodies, the variable domains of heavy chain only antibodies (VHH). VHH domains, the smallest natural antigen-binding modules, are readily engineered for diagnostic and therapeutic applications. All three VHH domains prevent ligand-induced EGFR activation, but use two distinct mechanisms. 7D12 sterically blocks ligand binding to EGFR in a manner similar to that of cetuximab. EgA1 and 9G8bind an epitope near the EGFR domain II/III junction, preventing receptor conformational changes required for high-affinity ligand binding and dimerization. This epitope is accessible to the convex VHH paratope but inaccessible to theflatter paratope of monoclonal antibodies. Appreciating the modes of binding and inhibition of these VHH domains will aid in developing them for tumor imaging and/or cancer therapy.

Differentiation between the EGFR antibodies necitumumab, cetuximab, and panitumumab: In vitro biological and binding activities.

e13030 Background: The monoclonal antibodies cetuximab (chimeric IgG1), necitumumab (human IgG1), and panitumumab (human IgG2) competitively inhibit ligand binding to epidermal growth factor receptor (EGFR) in domain 3. These antibodies block ligand induced EGFR activation and downstream molecular signaling. Beyond protein backbone differences, further differentiation of these EGFR antibodies is an important area of investigation. Methods: The effect on the in vitro proliferation of DiFi human colorectal cancer cells was assessed by ATP luminescence. The binding of the antibodies to the soluble extracellular domain of EGFR was evaluated by ELISA and surface plasmon resonance (SPR). Inhibition of ligand induced EGFR activation and downstream signaling was evaluated utilizing electrochemiluminescence method. Finally, binding kinetics was analyzed by SPR using a capture method. Results: The necitumumab concentration producing a half-maximal anti proliferative response (IC50) in DIFI cells was comparable to that of cetuximab. In contrast, a 1.6 fold greater concentration of panitumumab was required to produce the similar level of anti- proliferative response. This observation is supported by in vitro binding assays, which indicated that cetuximab and necitumumab binds to EGFR more effectively than panitumumab. Furthermore, the IC50 of nectitumab for the inhibition of EGFR phosphorylation was at least two fold greater than that of panitumumab. When EGFR binding kinetics were analyzed by SPR, panitumumab showed a much higher affinity (Kd) to EGFR than necitumumab and cetuximab, with a lower binding rate (kon) and a much slower dissociation rate (koff). Given the results in functional assays and the SPR data indicates that the high binding affinity (Kd) of panitumumab does not correlate with improved ability to block EGFR activation, downstream pathway activation and tumor cell proliferation. Conclusions: Although, as published, panitumumab has a higher affinity (Kd) than cetuximab and necitumumab, in vitro functional assays indicate that necitumumab and cetuximab have equal or greater ability to impact EGFR signaling and effect downstream activation of EGFR.

The effect of cell–ECM adhesion on signalling via the ErbB family of growth factor receptors

Integrins and growth factor receptors of the ErbB family are involved in the regulation of cellular interactions with the extracellular microenvironment. Cross-talk between these two groups of transmembrane receptors is essential for cellular responses and can be regulated through the formation of multimolecular complexes. Tetraspanins as facilitators and building blocks of specialized microdomains may be involved in this process. In the present study, we demonstrated that, in contrast with previous reports, integrin-mediated adhesion did not stimulate ligand-independent activation of ErbB receptors in epithelial cells. However, integrin-dependent adhesion potentiated ligand-induced activation of EGFR (epidermal growth factor receptor) and ErbB2 and facilitated receptor homo- and hetero-dimerization. The actin cytoskeleton appeared to play a critical role in this phenomenon.

Abstract 1058: E-cadherin mutations contribute to gastric carcinogenesis by modulating multiple EGFR-dependent downstream signaling pathways

Abstract Mutations in the gene encoding the cell adhesion molecule E-cadherin and overexpression of the epidermal growth factor receptor (EGFR) represent fundamental genetic alterations associated with diffuse-type gastric cancer. Mutations, e.g. in frame deletion of exon 8 (del 8), lead to loss of putative extracellular Ca2+-binding sites of E-cadherin, thereby impairing its functionality. Several studies have shown a bidirectional crosstalk between E-cadherin and EGFR. Functional wild-type (wt) E-cadherin inhibits ligand-induced EGFR activation, but mutations lead to a loss of its suppressive function, resulting in increased EGFR activation and recruitment of downstream signalling components. The phenotype of cells harboring such mutations is characterized by increased proliferation and elevated migratory and invasive potential. Our project aims at studying the impact of somatic mutations of E-cadherin on EGFR-mediated signalling pathways, in order to identify signalling molecules of these pathways as molecular targets for selective therapies in gastric cancer. Cell lines expressing wt or mutant E-cadherin (del 8) were EGF-stimulated and cellular lysates were analyzed by commercial proteome profiler antibody array (R&D Systems). The array allowed detection of the phosphorylation status of 48 signalling molecules and facilitated identification of differential activation. We found differential response to EGF stimulation of proteins involved in DNA damage and repair, such as p53 and CHK2, in both cell lines. As both proteins are tumorsuppressors, their dysfunction could contribute to tumor progression. Furthermore, Src-family kinases (SFKs) like Src, Yes, Fgr, Lck and Lyn exhibited different activation profiles in response to EGF-stimulation in both cell lines. As SFKs are key players in multiple cellular processes like cell growth, differentiation, cellular adhesion, migration and invasion, the impact of their differential activation on the cellular phenotype is subject of ongoing experiments. To determine the role of the identified targets in gastric carcinogenesis and the mechanisms of crosstalk between E-cadherin and EGFR a protein microarray platform was established, allowing analysis of multiple samples under the same experimental conditions. This approach will reveal new insights in EGFR-mediated signalling pathways contributing to gastric carcinogenesis and complete our data describing how tumor associated mutations of E-cadherin affect cellular signalling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1058.

Cross interference with TNF-α-induced TAK1 activation via EGFR-mediated p38 phosphorylation of TAK1-binding protein 1

Transforming growth factor-α-activated kinase 1 (TAK1) has been widely recognized as a kinase that regulates multiple intracellular signaling pathways evoked by cytokines and immune receptor activation. We have recently reported that tumor necrosis factor-α (TNF-α) triggers internalization of epidermal growth factor receptor (EGFR) through a TAK1-p38α signaling pathway, which results in a transient suppression of the EGFR. In the present study, we investigated the pathway of intracellular signaling in the opposite direction. Ligand-induced activation of EGFR caused phosphorylation of the TAK1-binding proteins TAB1 and TAB2 in a TAK1-independent manner. EGFR-mediated phosphorylation of TAB1 was completely inhibited by a chemical inhibitor and siRNA of p38α. The phosphorylation of TAB1 was occurred at Ser-423 and Thr-431, the residues underlying the p38-mediated feedback inhibition of TAK1. In contrast, phosphorylation of TAB2 was sustained, and largely resistant to p38 inhibition. The inducible phosphorylation of TAB1 interfered with a response of EGF-treated cells to TNF-α-induced TAK1 activation, which led to the reduction of NF-κB activation. Collectively, these results demonstrated that EGFR activation interfered with TNF-α-induced TAK1 activation via p38-mediated phosphorylation of TAB1.

Reduced Expression of Epidermal Growth Factor Receptors in Rat Liver During Aging

Proliferative responsiveness of hepatocytes to epidermal growth factor (EGF) declines during aging. The role of EGF receptors in mediating age-dependent changes of EGF-induced mitogenic signaling in liver remains incompletely understood. We assessed EGF receptor expression levels in whole liver specimens as well as in freshly isolated and cultured hepatocytes from young adult and senescent Fischer 344 male rats. Hepatic EGF receptor messenger RNA and protein levels, and the number of high- and low-affinity receptor binding sites, decreased with aging. Ligand-induced EGF receptor activation, determined by receptor dimerization and tyrosine phosphorylation, was reduced in old animals in parallel with the age-related decline in receptor expression. Stimulation of the extracellular signal-regulated kinase pathway by EGF was also attenuated in hepatocytes from old animals. Our results implicate decreased expression of EGF receptors as a key determinant of reduced mitogenic signaling responsive to EGF stimulation of liver during aging.

Association of tetraspanin CD9 with transmembrane TGFα confers alterations in cell-surface presentation of TGFα and cytoskeletal organization

Ligand presentation is a major determinant of receptor activation. The epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, is activated by growth factors of the transforming growth factor alpha (TGFalpha) family. The tetraspanin CD9 interacts with transmembrane TGFalpha and decreases its ectodomain shedding to release soluble TGFalpha. Here we report that CD9 has a role in the maturation of transmembrane TGFalpha and its stabilization at the cell surface, and in the cell-surface distribution in polarized epithelial cells. Furthermore, coexpression of CD9 and TGFalpha confers changes in cytoskeletal organization with a decrease in actin stress fibers and focal adhesions, and changes in RhoA and Rac1 GTPase activity. These alterations are reversed by blocking EGFR signaling. Finally, we demonstrate changes in cell adhesion and migration resulting from coexpression of TGFalpha with CD9. These results provide insight into the role of CD9 in the presentation of TGFalpha in epithelial and carcinoma cells, whose physiology is driven by ligand-induced EGFR activation.

RET/PTC-Induced Cell Growth Is Mediated in Part by Epidermal Growth Factor Receptor (EGFR) Activation: Evidence for Molecular and Functional Interactions between RET and EGFR

RET/PTC rearrangements are one of the genetic hallmarks of papillary thyroid carcinomas. RET/PTC oncoproteins lack extracellular or transmembrane domains, and activation takes place through constitutive dimerization mediated through coiled-coil motifs in the NH(2) terminus of the chimeric protein. Based on the observation that the epidermal growth factor receptor (EGFR) kinase inhibitor PKI166 decreased RET/PTC kinase autophosphorylation and activation of downstream effectors in thyroid cells, despite lacking activity on the purified RET kinase, we proceeded to examine possible functional interactions between RET/PTC and EGFR. Conditional activation of RET/PTC oncoproteins in thyroid PCCL3 cells markedly induced expression and phosphorylation of EGFR, which was mediated in part through mitogen-activated protein kinase signaling. RET and EGFR were found to coimmunoprecipitate. The ability of RET to form a complex with EGFR was not dependent on recruitment of Shc or on their respective kinase activities. Ligand-induced activation of EGFR resulted in phosphorylation of a kinase-dead RET, an effect that was entirely blocked by PKI166. These effects were biologically relevant, as the EGFR kinase inhibitors PKI166, gefitinib, and AEE788 inhibited cell growth induced by various constitutively active mutants of RET in thyroid cancer cells as well as NIH3T3 cells. These data indicate that EGFR contributes to RET kinase activation, signaling, and growth stimulation and may therefore be an attractive therapeutic target in RET-induced neoplasms.

Epidermal growth factor receptor and cancer: control of oncogenic signalling by endocytosis

The epidermal growth factor receptor (EGFR) and other members of the EGFR/ErbB receptor family of receptor tyrosine kinases (RTKs) are important regulators of proliferation, angiogenesis, migration, tumorigenesis and metastasis. Overexpression, mutations, deletions and production of autocrine ligands contribute to aberrant activation of the ErbB proteins. The signalling output from EGFR is complicated given that other ErbB proteins are often additionally expressed and activated in the same cell, resulting in formation of homo-and/or heterodimers. In particular, association of EGFR with ErbB2 prevents its down-regulation, underscoring the importance of the cellular background for EGFR effects. Signalling from ErbB proteins can either be terminated by dissociation of ligand resulting in dephosphorylation, or blunted by degradation of the receptors. Although proteasomal targeting of ErbB proteins has been described, lysosomal degradation upon ligand-induced endocytosis seems to play the major role in EGFR down-regulation. Preclinical and clinical data have demonstrated that EGFR is a central player in cancer, especially in carcinomas, some brain tumours and in non-small cell lung cancer. Such studies have further validated EGFR as an important molecular target in cancer treatment. This review focuses on mechanisms involved in ligand-induced EGFR activation and endocytic down-regulation. A better understanding of EGFR biology should allow development of more tumour-selective therapeutic approaches targeting EGFR-induced signalling.

UBPY-mediated Epidermal Growth Factor Receptor (EGFR) De-ubiquitination Promotes EGFR Degradation

Whereas poly-ubiquitination targets protein substrates for proteasomal degradation, mono-ubiquitination is known to regulate protein trafficking in the endosomal system and to target cargo proteins for lysosomal degradation. The role of the de-ubiquitinating enzymes AMSH and UBPY in endosomal trafficking of cargo proteins such as the epidermal growth factor receptor (EGFR) has only very recently been the subject of study and is already a matter of debate. Although one report (Mizuno, E., Iura, T., Mukai, A., Yoshimori, T., Kitamura, N., and Komada, M. (2005) Mol. Biol. Cell 16, 5163-5174) concludes that UBPY negatively regulates EGFR degradation by de-ubiquitinating the EGFR on endosomes, another report (Row, P. E., Prior, I. A., McCullough, J., Clague, M. J., and Urbe, S. (2006) J. Biol. Chem. 281, 12618-12624) concludes that UBPY-mediated EGFR de-ubiquitination is essential for EGFR degradation. Here, we demonstrate that Usp8/UBPY, the mammalian ortholog of budding yeast Ubp4/Doa4, constitutively co-precipitates in a bivalent manner with the EGFR. Moreover, UBPY is a substrate for Src-family tyrosine kinases that are activated after ligand-induced EGFR activation. Using overexpression of three different recombinant dominant negative UBPY mutants (UBPY C748A mutant, UBPY 1-505, and UBPY 640-1080) in NIH3T3 and HEK293 cells, we demonstrate that UBPY affects both constitutive and ligand-induced (i) EGFR ubiquitination, (ii) EGFR expression levels, and (iii) the appearance of intermediate EGFR degradation products as well as (iv) downstream mitogen-activated protein kinase signal transduction. Our findings provide further evidence in favor of the model that UBPY-mediated EGFR de-ubiquitination promotes EGFR degradation.

Antitumor Activity of Epidermal Growth Factor Receptor–Related Protein Is Mediated by Inactivation of ErbB Receptors and Nuclear Factor-κB in Pancreatic Cancer

The erbB family of receptor tyrosine kinases plays critical roles in human cancers, including pancreatic cancer. Discovering a specific agent, which targets multiple members of the erbB family, would be important in pancreatic cancer therapy. Recently, we isolated a novel negative regulator of epidermal growth factor receptor (EGFR), termed EGFR-related protein (ERRP), whose expression attenuates EGFR activation. In the current study, we examined the effects of recombinant ERRP on the growth and ligand-induced activation of multiple members of erbB family in three pancreatic cancer cell lines that express varying levels of EGFR and other member(s) of its family, specifically HER-2. Additionally, we compared the growth inhibitory effect of ERRP with that of Erbitux or Herceptin. Our results showed that ERRP is most effective in inhibiting proliferation of BxPC-3, HPAC, and PANC-1 pancreatic cancer cells. ERRP also inhibited ligand-induced activation of EGFR, HER-2, and HER-3 (ErbB3). In contrast, Erbitux and Herceptin only partially or modestly inhibited activation of EGFR, HER-2, and HER-3. Most importantly, ERRP was found to inhibit pancreatic tumor growth in a severe combined immunodeficient mouse xenograft model. The antitumor activity of ERRP correlates well with tumor differentiation and down-regulation of nuclear factor-kappaB activity. In summary, our results suggest that ERRP is an effective pan-erbB inhibitor, which could be a potential therapeutic agent for pancreatic cancers expressing different levels and subclasses of erbB family of proteins.

Epidermal growth factor receptor dimerization status determines skin toxicity to HER-kinase targeted therapies

Skin toxicity, a common drug-related adverse event observed in cancer patients treated with epidermal growth factor receptor (EGFR)-directed therapies is rarely seen with therapies targeting HER2. This study reports the significance of the EGFR and HER2 dimerization status in skin with regard to these dermatologic side effects. We demonstrate the differential effect of HER-directed therapies on the ligand driven activation status of EGFR, HER2 and MAPK in normal human epidermal keratinocytes. EGFR-directed therapies, such as gefitinib and cetuximab, inhibited ligand-induced activation of EGFR and MAPK in human keratinocytes. Pertuzumab, an antibody interfering with functional HER2 heterodimerization, failed to block ligand-induced HER signaling in primary keratinocytes. Using a novel proximity-based dimerization assay (eTag™) we show that EGFR homodimers are the predominant HER dimer pair in normal primary kertinocytes and in normal skin tissue from 16 patients with solid malignancies. The presence of [p]EGFR and [p]MAPK, but the absence of [p]HER2, demonstrates productive signaling via EGFR but not HER2 in human skin. These data illustrate the importance of the EGFR dimerization partner in human skin and suggests that inhibition of EGFR homodimer signaling rather than EGFR/HER2 heterodimer signaling maybe the key molecular event determining dermatologic toxicity discrepancies observed between EGFR-targeted versus HER2-targeted therapies.

A critical role for enhanced TGF-α and EGFR expression in the initiation of parathyroid hyperplasia in experimental kidney disease

The parathyroid hyperplasia secondary to kidney disease is associated with enhanced expression of the growth promoter transforming growth factor-alpha (TGF-alpha). TGF-alpha stimulates growth through activation of its receptor, the epidermal growth factor receptor (EGFR), normally expressed in the parathyroid glands. Because enhanced coexpression of TGF-alpha and EGFR causes aggressive cellular growth, these studies utilized highly specific inhibitors of EGFR tyrosine kinase, a step mandatory for TGF-alpha-induced EGFR activation, to assess the contribution of growth signals from enhanced expression of TGF-alpha exclusively or both TGF-alpha and EGFR to the rapid parathyroid growth induced by kidney disease and exacerbated by high-phosphorus (P) and low-calcium (Ca) diets in rats. The enhancement in parathyroid gland weight and proliferating activity (proliferating cell nuclear antigen/Ki67) induced by kidney disease and aggravated by either high P or low Ca intake, within the first week after 5/6 nephrectomy, in rats, coincided with simultaneous increases (2- to 3-fold) in TGF-alpha and EGFR content. Conversely, prevention of the increases in both TGF-alpha and EGFR paralleled the efficacy of either P restriction or high-Ca intake in ameliorating uremia-induced parathyroid hyperplasia. More importantly, suppression of TGF-alpha/EGFR signaling, through prophylactic administration of potent and highly selective inhibitors of ligand-induced EGFR activation, completely prevented both high-P- and low-Ca-induced parathyroid hyperplasia as well as TGF-alpha self-upregulation. Thus enhanced parathyroid TGF-alpha/EGFR expression, self-upregulation, and growth signals occur early in kidney disease, are aggravated by low-Ca and high-P intake, and constitute the main pathogenic mechanism of the severity of parathyroid hyperplasia.

Epidermal growth factor receptor (EGFR)–related protein inhibits multiple members of the EGFR family in colon and breast cancer cells

Inactivation of epidermal growth factor receptor (EGFR) family members represents a promising strategy for the development of selective therapies against epithelial cancers. Current anti-EGFR therapies, such as cetuximab (Erbitux), gefitinib (Iressa), or trastuzumab (Herceptin), target EGFR or HER-2 but not both. Because solid tumors express different EGFRs, identification of inhibitor(s), targeting multiple EGFR family members may provide a therapeutic benefit to a broader patient population. We have identified a natural inhibitor of EGFRs called EGFR-related protein (ERRP), a 53 to 55 kDa protein that is present in most, if not all, normal human epithelial cells. The growth of colon (HCT-116, Caco2, and HT-29) and breast (MDA-MB-468 and SKBR-3) cancer cells expressing varying levels of EGFR, HER-2, and/or HER-4 was inhibited by recombinant ERRP in a dose-dependent manner. In contrast, ERRP caused no inhibition of growth of normal mouse fibroblast cell lines (NIH-3T3, NIH-3T3/P67), and the growth of nontransformed rat small intestinal IEC-6 cells expressing relatively low levels of EGFRs was inhibited only at high doses of ERRP. Transforming growth factor-alpha or heparin-binding epidermal growth factor-induced activation of EGFR and HER-2 was inhibited by ERRP in colon and breast cancer cells expressing high levels of EGFR or HER-2. In contrast, cetuximab inhibited the growth- and ligand-induced activation of EGFR in cell lines expressing high levels of EGFR, whereas trastuzumab was effective only in HER-2-overexpressing cells. ERRP and trastuzumab, but not cetuximab, attenuated heregulin-alpha-induced activation of colon and breast cancer cells that expressed high levels of HER-2. Furthermore, ERRP, but not cetuximab or trastuzumab, significantly induced apoptosis of colon and breast cancer cells. None of these agents induced apoptosis of either NIH-3T3 mouse fibroblast or normal rat small intestinal IEC cells. Our results suggest that ERRP is an effective pan-erbB inhibitor and, thus, may be a potential therapeutic agent for a wide variety of epithelial cancers expressing different levels and subclasses of EGFRs.

Ligand-induced, p38-dependent Apoptosis in Cells Expressing High Levels of Epidermal Growth Factor Receptor and ErbB-2

Increased expression of the epidermal growth factor (EGF) receptor (EGFR) and ErbB-2 is implicated into the development and progression of breast cancer. Constant ligand-induced activation of EGFR and ErbB-2 receptor-tyrosine kinases is thought to be involved in the transformation of fibroblasts and mammary epithelial cells. Data herein show that ligand stimulation of cells that express both the EGFR and the ErbB-2 may result either in cell proliferation or apoptosis depending on the expression levels of EGFR and ErbB-2. Mammary tumor cells that express low levels of both receptors or high levels of ErbB-2 and low levels of EGFR survive and proliferate in the presence of EGF. In contrast, fibroblastic cells or mammary tumor cells, which co-express high levels of EGFR and ErbB-2 invariably undergo apoptosis in response to EGF. In these cells persistent activation of p38 MAPK is an essential element of the apoptotic mechanism. Also, the data implicate a p38-dependent change in mitochondrial membrane permeability as a downstream effector of apoptosis. Ligand-dependent apoptosis in cells co-expressing high levels of EGFR and ErbB-2 could be a natural mechanism that protects tissues from unrestricted proliferation in response to the sustained activation of receptor-tyrosine kinases.

Skin Cancer Chemopreventive Effects of a Flavonoid Antioxidant Silymarin Are Mediated via Impairment of Receptor Tyrosine Kinase Signaling and Perturbation in Cell Cycle Progression

Enhanced tyrosine kinase activity due to aberrant or overexpression of receptor and/or non-receptor tyrosine kinases has been implicated in a variety of human malignancies including cutaneous neoplasms. Epidermal growth factor receptor (EGFR)-mediated tyrosine phosphorylation may be a primary indicator of signal transduction regulating cell growth and proliferation. Recent studies have shown that skin tumor promoters such as phorbol ester and ultraviolet B radiation activate EGFR in mouse skin as well as in cell culture. Similarly, oxidative stress, which is implicated in skin tumor promotion, also activates EGFR-mediated cell signaling. Since this signaling pathway has been suggested to be involved in skin tumor promotion, its impairment by antioxidants may lead to an efficient way for skin cancer prevention and therapy. Recently, we showed that silymarin, a flavonoid antioxidant, affords exceptionally high to complete protection against several skin tumor promoters caused tumor promotion in mouse skin. Employing human epidermoid carcinoma cells A431 that contain overexpressed EGFR, in this study, we assessed whether the anti-skin tumor promoting effects of silymarin are due to its inhibitory effect on EGFR activation and down stream signaling pathway leading to perturbations in cell cycle progression. Treatment of cells with silymarin resulted in a significant inhibition of ligand-induced activation of EGFR with no change in its protein levels. Silymarin treatment also resulted in a significant decrease in tyrosine phosphorylation of Shc, an immediate downstream target of EGFR, but no change in the protein levels of Shc. The inhibition of EGFR activation by silymarin was associated with a highly significant to complete inhibition of EGFR intrinsic kinase activity. Cells treated with silymarin also showed a significant G2-M arrest in cell cycle progression, and a highly significant inhibition of DNA synthesis and cell growth in a dose-dependent manner. Taken together, these results suggest that skin cancer chemopreventive effects of silymarin are mediated via impairment of EGFR signaling which ultimately leads to perturbation in cell cycle progression resulting in the inhibition of proliferation and induction of growth arrest.

Inhibition of ligand-induced activation of epidermal growth factor receptor tyrosine phosphorylation by curcumin.

We explored the regulation of epidermal growth factor (EGF)-mediated activation of EGF receptor (EGF-R) phosphorylation by curcumin (diferuloyl-methane), a recently identified kinase inhibitor, in cultured NIH 3T3 cells expressing human EGF-R. Treatment of cells with a saturating concentration of EGF for 5-15 min induced increased EGF-R tyrosine phosphorylation by 4- to 11-fold and this was inhibited in a dose- and time-dependent manner by up to 90% by curcumin, which also inhibited the growth of EGF-stimulated cells. There was no effect of curcumin treatment on the amount of surface expression of labeled EGF-R and inhibition of EGF-mediated tyrosine phosphorylation of EGF-R by curcumin was mediated by a reversible mechanism. In addition, curcumin also inhibited EGF-induced, but not bradykinin-induced, calcium release. These findings demonstrate that curcumin is a potent inhibitor of a growth stimulatory pathway, the ligand-induced activation of EGF-R, and may potentially be useful in developing anti-proliferative strategies to control tumor cell growth.