Endocytic downregulation of ErbB receptors: mechanisms and relevance in cancer

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

Overexpression of ErbB2 receptor inhibits IGF-I-induced Shc-MAPK signaling pathway in breast cancer cells

Overexpression of the ErbB2 receptor in one-third of human breast cancers contributes to the transformation of epithelial cells and predicts poor prognosis for breast cancer patients. We report that the overexpression of ErbB2 inhibits IGF-I-induced MAPK signaling. IGF-I-induced MAPK phosphorylation and MAPK kinase activity are reduced in ErbB2 overexpressing MCF-7/HER2-18 cells relative to control MCF-7/neo cells. In SKBR3/IGF-IR cells, reduction of ErbB2 by antisense methodology restores the IGF-I-induced MAPK activation. The inhibition of IGF-I-induced MAP kinase activation in ErbB2 overexpressing breast cancer cells is correlated with decreased IGF-I-induced Shc tyrosine-phosphorylation, leading to a decreased association of Grb2 with Shc and decreased Raf phosphorylation. However, IGF-I-induced tyrosine-phosphorylation of IGF-I receptor and IRS-I and AKT phosphorylation were unaffected by ErbB2 overexpression. Consistent with these results, we observed that the proportion of IGF-I-stimulated proliferation blocked by the MAPK inhibitor PD98059 fell from 82.6% in MCF-7/neo cells to 41.2% in MCF-7/HER2-18 cells. These data provide evidence for interplay between the IGF-IR and ErbB2 signaling pathways. They are consistent with the view that the IGF-IR mediated attenuation of trastuzumab-induced growth inhibition we recently described is dependent on IGF-I-induced PI3K signaling rather than IGF-I-induced MAPK signaling.     

Flotillin depletion affects ErbB protein levels in different human breast cancer cells

The ErbB3 receptor is an important regulator of cell growth and carcinogenesis. Among breast cancer patients, up to 50–70% have ErbB3 overexpression and 20–30% show overexpressed or amplified ErbB2. ErbB3 has also been implicated in the development of resistance to several drugs used against cancers driven by ErbB1 or ErbB2. One of the main challenges in ErbB-targeting therapy is to inactivate signaling mediated by ErbB2–ErbB3 oncogenic receptor complexes. We analyzed the regulatory role of flotillins on ErbB3 levels and ErbB2–ErbB3 complexes in SKBR3, MCF7 and MDA-MB-134-VI human breast cancer cells. Recently, we described a mechanism for interfering with ErbB2 signaling in breast cancer and demonstrated a molecular complex of flotillin scaffolding proteins with ErbB2 and Hsp90. In the present study, flotillins were found to be in a molecular complex with ErbB3, even in cells without the presence of ErbB2 or other ErbB receptors. Depletion of either flotillin-1 or flotillin-2 resulted in downregulation of ErbB3 and a selective reduction of ErbB2–ErbB3 receptor complexes. Moreover, flotillin-2 depletion resulted in reduced activation of Akt and MAPK signaling cascades, and as a functional consequence of flotillin depletion, breast cancer cells showed an impaired cell migration.     

A novel gene STYK1/NOK is upregulated in estrogen receptor-alpha negative estrogen receptor-beta positive breast cancer cells following estrogen treatment

The human STYK1/NOK protein is approximately 30–35% similar to mouse fibroblast growth factor receptor 3 and a kinase homologue in D. melanogaster in the tyrosine protein kinase region. STYK1/NOK was identified as being up regulated in MDA-MB-231, an estrogen receptor-alpha negative breast cancer cell line, following 12 h of estrogen treatment at 1 × 10⁻⁹ M. On further investigation of STYK1/NOK in estrogen treated cell line MDA-MB-231, STYK1/NOK was up regulated at 6 h post treatment when compared to untreated cells. We also investigated the expression levels of STYK1/NOK in other breast cancer cell lines MCF-7, MDA-MB-231, BT-549, and MDA-MB-435S using QRT-PCR. In addition, the analysis of message accumulation was increased with other synthetic estrogen response modifiers. We propose that the regulation of STYK1/NOK is achieved independent of ERα and suggests further investigation to the relevance of this kinase in breast cancer progression.     

8-Prenylnaringenin, inhibits estrogen receptor-alpha mediated cell growth and induces apoptosis in MCF-7 breast cancer cells

The discovery that the hop constituent 8-prenylnaringenin (8PN) shows potent estrogenic activity, higher than that of the known phytoestrogens coumestrol, genistein and daidzein, has spurred an intense activity aimed at elucidating its biological profile and its dietary relevance connected with the consumption of beer. We have investigated if 8PN can induce signal transduction pathways via rapid estrogen receptor (ER) activation. Under conditions of estrogen-dependent growth, treatment of MCF-7 human breast cancer cells with 8PN induced a rapid and transient activation of the MAP kinase Erk-1 and Erk-2, with kinetics similar to those induced by 17beta-estradiol (E2). 8PN could trigger the MAP kinase pathway via dual c-Src kinase activation and association with ERalpha. Co-treatment with the ER antagonist ICI 182,780 blocked each step of this transduction pathway, confirming its ER dependence. However, and in striking contrast with E2, 8PN could not induce the PI3K/Akt pathway, resulting in altered kinetics and levels of cyclin D1 expression. In accordance with these observations, flow cytometric and biochemical analysis showed that 8PN inhibited cell cycle progression and induced apoptosis in MCF-7 cells. Interference with an ER associated PI3K pathway is proposed as a possible mechanism underlying the inhibition of survival and proliferation of estrogen responsive cells by 8PN. Taken together, our finding show that 8PN is an interesting new chemotype to explore the biology of ERs.     

The rat ErbB2 tyrosine kinase receptor produced in plants is immunogenic in mice and confers protective immunity against ErbB2+ mammary cancer

The rat ErbB2 (rErbB2) protein is a 185‐kDa glycoprotein belonging to the epidermal growth factor‐related proteins (ErbB) of receptor tyrosine kinases. Overexpression and mutations of ErbB proteins lead to several malignancies including breast, lung, pancreatic, bladder and ovary carcinomas. ErbB2 is immunogenic and is an ideal candidate for cancer immunotherapy. We investigated the possibility of expressing the extracellular (EC) domain of rErbB2 (653 amino acids, aa) in Nicotiana benthamiana plants, testing the influence of the 23 aa transmembrane (TM) sequence on protein accumulation. Synthetic variants of the rErbB2 gene portion encoding the EC domain, optimized with a human codon usage and either linked to the full TM domain (rErbB2_TM, 676 aa), to a portion of it (rErbB2‐pTM, 662 aa), or deprived of it (rErbB2_noTM, 653 aa) were cloned in the pEAQ‐HT expression vector as 6X His tag fusions. All rErbB2 variants (72–74.5 kDa) were transiently expressed, but the TM was detrimental for rErbB2 EC accumulation. rERbB2_noTM was the most expressed protein; it was solubilized and purified with Nickel affinity resin. When crude soluble extracts expressing rErbB2_noTM were administered to BALB/c mice, specific rErbB2 immune responses were triggered. A potent antitumour activity was induced when vaccinated mice were challenged with syngeneic transplantable ErbB2⁺ mammary carcinoma cells. To our knowledge, this is the first report of expression of rErbB2 in plants and of its efficacy in inducing a protective antitumour immune response, opening interesting perspectives for further immunological testing.     

NDV‐D90 inhibits 17β‐estradiol‐mediated resistance to apoptosis by differentially modulating classic and nonclassic estrogen receptors in breast cancer cells

Newcastle disease virus (NDV) is endowed with the oncolytic ability to kill tumor cells, while rarely causing side effects in normal cells. Both estrogen receptor α (ERα) and the G protein estrogen receptor (GPER) modulate multiple biological activities in response to estrogen, including apoptosis in breast cancer (BC) cells. Here, we investigated whether NDV‐D90, a novel strain isolated from natural sources in China, promoted apoptosis by modulating the expression of ERα or the GPER in BC cells exposed to 17β‐estradiol (E2). We found that NDV‐D90 significantly killed the tumor cell lines MCF‐7 and BT549 in a time‐ and dose‐dependent manner. We also found that NDV‐D90 exerted its effects on the two cell lines mainly by inducing apoptosis but not necrosis. NDV‐D90 induced apoptosis via the intrinsic and extrinsic signaling pathways in MCF‐7 cells (ER‐positive cells) during E2 exposure not only by disrupting the E2/ERα axis and enhancing GPER expression but also by modulating the expression of several apoptosis‐related proteins through ERα‐and GPER‐independent processes. NDV‐D90 promoted apoptosis via the intrinsic signaling pathway in BT549 cells (ER‐negative cells), possibly by impairing E2‐mediated GPER expression. Furthermore, NDV‐D90 exerted its antitumor effects in vivo by inducing apoptosis. Overall, these results demonstrated that NDV‐D90 promotes apoptosis by differentially modulating the expression of ERα and the GPER in ER‐positive and negative BC cells exposed to estrogen, respectively, and can be utilized as an effective approach to treating BC.     

Association of ErbB2 Ser1113 phosphorylation with epidermal growth factor receptor co-expression and poor prognosis in human breast cancer

The carboxyterminal domain of the epidermal growth factor receptor (EGFR) – a putative binding site for the ubiquitin ligase Cbl – is the site of serine phosphorylation events which are essential for ligand-dependent EGFR desensitization and degradation. Using a monoclonal antibody (aPS¹¹¹³) which selectively recognizes the homologous phosphorylated domain in the ErbB2 oncoprotein, we show here that wild-type ErbB2 becomes Ser¹¹¹³-phosphorylated following treatment of 3T3 cells with growth factors or tyrosine phosphatase inhibitors. In EGFR-overexpressing A431 cells, ligand-inducible aPS¹¹¹³ immunoreactivity declines more rapidly than other detectable phosphorylation events and is followed by EGFR downregulation. Analysis of 65 ErbB2-expressing primary breast cancers reveals a highly significant relationship between Ser¹¹¹³ phosphorylation and EGFR overexpression (p < 0.0001) as well as an association with poor prognosis (p = 0.005). We submit that ErbB2 Ser¹¹¹³ phosphorylation status represents a novel and informative biomarker of cancer cell biology and tumor behavior.     

Up-regulation of PI3K/Akt signaling by 17beta-estradiol through activation of estrogen receptor-alpha, but not estrogen receptor-beta, and stimulates cell growth in breast cancer cells

Estrogen stimulates cell proliferation in breast cancer. The biological effects of estrogen are mediated through two intracellular receptors, estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta). However, the role of ERs in the proliferative action of estrogen is not well established. Recently, it has been known that ER activates phosphatidylinositol-3-OH kinase (PI3K) through binding with the p85 regulatory subunit of PI3K. Therefore, possible mechanisms may include ER-mediated phosphoinositide metabolism with subsequent formation of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)), which is generated from phosphatidylinositol 4,5-bisphosphate via PI3K activation. The present study demonstrates that 17beta-estradiol (E2) up-regulates PI3K in an ERalpha-dependent manner, but not ERbeta, and stimulates cell growth in breast cancer cells. In order to study this phenomenon, we have treated ERalpha-positive MCF-7 cells and ERalpha-negative MDA-MB-231 cells with 10nM E2. Treatment of MCF-7 cells with E2 resulted in a marked increase in PI3K (p85) expression, which paralleled an increase in phospho-Akt (Ser-473) and PIP(3) level. These observations also correlated with an increased activity to E2-induced cell proliferation. However, these effects of E2 on breast cancer cells were not observed in the MDA-MB-231 cell line, indicating that the E2-mediated up-regulation of PI3K/Akt pathway is ERalpha-dependent. These results suggest that estrogen activates PI3K/Akt signaling through ERalpha-dependent mechanism in MCF-7 cells.     

A trivalent anti-erbB2/anti-CD16 bispecific antibody retargeting NK cells against human breast cancer cells

Bispecific antibody (BsAb) can physically cross-link immune cells to tumor cells, circumventing the proper structures for tumor cell-immune cell interactions and activating the cellular cytotoxic mechanisms. The optimal BsAb should target tumor cells with high affinity, but activate trigger molecules on cytotoxic cells by monovalent binding of Fab fragments. In the present study, a trivalent anti-erbB2/anti-CD16 BsAb was produced. This BsAb possesses bivalent arms specifically binding to the extracellular domain of erbB2 and monovalent Fab fragment redirecting NK cells. The recombinant protein could be expressed and purified from Escherichia coli as native proteins without refolding. It was fully functional in bispecific binding to SKBR3 and NK cells. The molecular size of this trivalent BsAb protein is larger than diabody and smaller than whole antibody and expected to have advantages for both high penetration of small antibody fragments and the slow circulation clearance of whole antibody. This novel protein may be an attractive target for further improvement and evaluation.     

Activation of ErbB2 receptor tyrosine kinase supports invasion of endothelial cells by Neisseria meningitidis

ErbB2 is a receptor tyrosine kinase belonging to the family of epidermal growth factor (EGF) receptors which is generally involved in cell differentiation, proliferation, and tumor growth, and activated by heterodimerization with the other members of the family. We show here that type IV pilus-mediated adhesion of Neisseria meningitidis onto endothelial cells induces tyrosyl phosphorylation and massive recruitment of ErbB2 underneath the bacterial colonies. However, neither the phosphorylation status nor the cellular localization of the EGF receptors, ErbB3 or ErbB4, were affected in infected cells. ErbB2 phosphorylation induced by N. meningitidis provides docking sites for the kinase src and leads to its subsequent activation. Specific inhibition of either ErbB2 and/or src activity reduces bacterial internalization into endothelial cells without affecting bacteria-induced actin cytoskeleton reorganization or ErbB2 recruitment. Moreover, inhibition of both actin polymerization and the ErbB2/src pathway totally prevents bacterial entry. Altogether, our results provide new insight into ErbB2 function by bringing evidence of a bacteria-induced ErbB2 clustering leading to src kinase phosphorylation and activation. This pathway, in cooperation with the bacteria-induced reorganization of the actin cytoskeleton, is required for the efficient internalization of N. meningitidis into endothelial cells, an essential process enabling this pathogen to cross host cell barriers.     

Estrogen signals via an extra-nuclear pathway involving IGF-1R and EGFR in tamoxifen-sensitive and -resistant breast cancer cells

Activation of IGF-1R can activate metalloproteinases which release heparin-binding EGF (Hb-EGF) and lead to EGFR-dependent MAPK activation in certain tissues. We postulated that this pathway is operative in E₂-induced MAPK activation in breast cancer tissues. As evidence, we showed that E₂ rapidly induced the phosphorylation of both IGF-1R and EGFR and that siRNA knockdown or selective inhibitors against either growth factor receptor inhibited E₂-induced MAPK activation. The selective inhibitors or knockdown of either IGF-1R or EGFR significantly inhibited cell growth and reversed cell death protection induced by E₂ in MCF-7 cells. Our data support the conclusion that the IGF-1R acts upstream of EGFR in a linear pathway which mediates E₂ action on MAPK activation, cell growth stimulation and anti-apoptosis in breast cancer cells. During the process of development of tamoxifen resistance this pathway is up-regulated with increased sensitivity to activate EGFR for cell growth and protection against apoptosis. Surprisingly, translocation of ERα out of the nucleus into the cytoplasm, mediated by c-Src, occurs during development of resistance. This effect can be abrogated by administration of the c-Src inhibitor, PP2 which also restores sensitivity to tamoxifen.