Expression of the ErbB4 receptor causes reversal regulation of PP2A in the Shc signal transduction pathway in human cancer cells

Expression of ErbB4 receptor is correlated with the incidence of non-metastatic types of human cancers, whereas the overexpression of other ErbB receptor families (ErbB1/EGFR, ErbB2 and ErbB3) is correlated to the formation of metastatic tumors. However, the molecular mechanism underlying this phenomenon has been unclear. Earlier, we demonstrated that okadaic acid (OA), an inhibitor of a serine/threonine phosphatase PP2A, stimulated the growth hormone-induced ERK phosphorylation in the wild type Chinese hamster ovary (CHO) cells and the cells expressing ErbB1 receptor, but suppressed ERK activation in CHO cells that express ErbB4 receptor. PP2A had been understood as a negative regulator of the growth hormone-stimulated signal transduction pathways, however, this observation suggested that expression of ErbB4 receptor reversed the regulation of PP2A in the ErbB4 signalling pathway. In this study, we found that OA suppressed phosphorylation of Shc at Tyr317, therefore it down-regulated ERK phosphorylation in the ErbB4 expressing CHO cells. Accordingly, basal PP2A contributed to the phosphorylation of Shc Tyr317 in ErbB4 expressing CHO cells, nevertheless it had been reported that PP2A negatively regulates Shc tyrosine phosphorylation in the EGF- or IGF-I-induced signalling pathways. By testing OA for human cancer cell lines that express different types of ErbB receptors, we found that ErbB4 receptor expression was accompanied with positive regulation of PP2A for phosphorylation of Shc Tyr317 and its downstream ERK phosphorylation in MCF-7 and SK-OV-3 cell lines, but not in LNCaP and PC-3 cells. Thus, PP2A regulates the ERK activity in a cell-specific manner, and it is speculated that distinct regulation of PP2A in the ErbB4 receptor signalling pathway may cause a difference in progression of cancer phenotypes.     

Identification of prolidase as a high affinity ligand of the ErbB2 receptor and its regulation of ErbB2 signaling and cell growth

ErbB2, an important membrane-bound receptor tyrosine kinase, was discovered nearly 30 years ago, but a natural ligand has never been found previously. ErbB2 is also an important oncogene and anticancer target, and its overexpression in cancer is associated with poor disease prognosis. Here, we report that human prolidase (PEPD) is a high affinity ligand of ErbB2 and binds as a homodimer to subdomain 3 in the extracellular domain of this receptor. In ErbB2-overexpressing cells, both ErbB2 monomers and activated dimers exist. PEPD bound to ErbB2 monomers relatively slowly but caused ErbB2 dimerization, ErbB2 phosphorylation and downstream signaling. In contrast, PEPD bound rapidly to ErbB2 homodimers and rapidly silenced ErbB2 dimer-Src signaling, a key oncogenic pathway of ErbB2, by disrupting the association of Src with ErbB2. PEPD also caused pronounced ErbB2 depletion, resulting from ErbB2 internalization and degradation. Moreover, PEPD strongly inhibited the DNA synthesis, anchorage-independent growth and invasion and migration of cells that overexpressed ErbB2 but had no effect on cells without ErbB2 overexpression. Cells became sensitized to PEPD upon achieving stable ErbB2 overexpression. Thus, the impact of PEPD on ErbB2 is predominantly inhibitory, and PEPD targets cells addicted to ErbB2. PEPD is also a dipeptidase, but its enzymatic function is not involved in ErbB2 modulation. These findings revise our understanding of ErbB2 and PEPD and may be especially important for combating ErbB2-positive cancers.     

ErbB2 down-regulates microRNA-205 in breast cancer

Gene amplification and protein overexpression of erbB2 (Her2/neu) has been observed in approximately 20–30% of breast cancers. ErbB2-positive breast cancer is tend to be more aggressive than other types of breast cancer and therefore further investigation on the signaling pathways of erbB2 is needed for the therapeutic target for breast cancer treatment. Here we report that microRNA-205 (miR-205), a molecule also reported to be associated with breast cancer, is negatively regulated by erbB2 overexpression. Breast epithelial cells exogenously overexpressed with erbB2 decreased the expression of miR-205, whereas increased the expression of cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), and cyclin-dependent kinase 6 (CDK6). The decreased expression of miR-205 slightly increased by the transfection of erbB2 siRNA into the erbB2-overexpressing breast cancer epithelial cells. Overexpression of erbB2 enabled breast epithelial cells to grow anchorage-independently in soft agar, and the transfection of the precursor of miR-205 into the cells leaded to the decrease in the ability to grow in soft agar. These results suggest that down-regulation of miR-205 in erbB2-overexpressing breast epithelial cells is essential for erbB2-induced tumorigenesis, and miR-205 may have the potential to be a novel important alternative therapeutic target for erbB2-positive breast cancer.     

Expression of Mona (monocytic adapter) in myeloid progenitor cells results in increased and prolonged MAP kinase activation upon macrophage colony-stimulating factor stimulation

Mona is an SH3 and SH2 domain-containing adapter molecule that is induced during monocytic differentiation. Here we have first shown that M-CSFR is the major Mona partner in M-CSF signaling, the interaction being mediated through tyrosine 697 of the receptor. Next we asked whether Mona expression would alter the Ras/MAP kinase pathway since Mona is a likely competitor of Grb2 for binding to M-CSFR. We found that M-CSF induced late and massive phosphorylation of ERK molecules in Mona-expressing myeloid cells compared to non-expressing cells. These results suggest that Mona expression might modify M-CSF signaling during monocytic differentiation.     

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.     

Crosstalk between the extracellular domain of the ErbB2 receptor and IGF-1 receptor signaling

Insulin-like growth factor 1 receptor (IGF-1R) plays an important role in cell growth and malignant transformation. To investigate IGF-1R-dependent signaling events and its effects on apoptosis induction and cellular proliferation, we generated a constitutively active, ligand-independent IGF-1R variant. We fused the cytoplasmic domain of the IGF-1R to the extracellular and transmembrane domains of the oncogenic ErbB2 receptor (ErbB2^V→E/IGF-1). A fusion protein in which the wild-type sequence of the ErbB2 receptor was used, served as a control (ErbB2^V/IGF-1R). ErbB2^V/IGF-1R, ErbB2^V→E/IGF-1R and IGF-1R were stably transfected into interleukin 3 (IL-3)-dependent BaF/3 cells. ErbB2^V→E/IGF-1R expressing cells exhibited ligand-independent, constitutive tyrosine phosphorylation of the receptor fusion protein. Constitutively, activated ErbB2^V→E/IGF-1R conferred IL-3 independence for growth and survival to the transfected BaF/3 cells. Constitutive activation of the IGF-1R results in cellular growth and protection against apoptosis upon IL-3 withdrawal in BaF/3 cells.     

Ubiquitination and downregulation of ErbB2 and estrogen receptor-alpha by kinase inhibitor MP-412 in human breast cancer cells

ErbB2 has been proven to be an important target for breast cancer therapy. MP-412 is a dual ErbB2 and epidermal growth factor receptor tyrosine kinase inhibitor belonging to an irreversible-type anilinoquinazoline derivative. We demonstrate herein that along with the kinase inhibition, MP-412 has the ability to induce ubiquitination, internalization, and degradation of ErbB2 in several human breast cancer cell lines at concentrations relatively higher than those required for kinase inhibition. Another irreversible inhibitor, CI-1033, showed similar activity, while the reversible compounds were ineffective, suggesting a crucial role of covalent bonding functionality in these effects. In MCF7 cells, MP-412 depleted not only ErbB2 but also estrogen receptor (ER)-α, and to some extent, affected Raf-1, while MP-412 activated Hsp70 expression. Moreover, we observed that MP-412 increased immunocomplexing of Hsp70 with ErbB2 and ER-α, with simultaneous induction of ubiquitination of these client proteins. Furthermore, in combination with proteasome inhibitor, MP-412 resulted in the noticeable accumulation of ErbB2 and ER-α in the detergent insoluble fraction of cell lysates. These results suggest that MP-412 acts as an inhibitor of Hsp90 function, whereas MP-412 did not bind directly to ATP-binding site of Hsp90, unlike geldanamycin. We also found that new protein synthesis was involved in the activity of MP-412 on Hsp90 modulation. Since downregulation of ErbB2 and ER-α by accelerating the ubiquitin-proteolysis system will become an attractive approach for breast cancer therapy, we expect MP-412 to be a lead compound for the drug design and the development of such agents.     

Interaction of antibodies with ErbB receptor extracellular regions

Antibodies to the extracellular region of the ErbB receptors have played key roles in the development of a mechanistic understanding of this family of receptor tyrosine kinases. An extensively studied class of such antibodies inhibits activation of ErbB receptors, and these antibodies have been the focus of intense development as anti-cancer agents. In this review we consider the properties of ErbB receptors antibodies in light of the current structure-based model for ErbB receptor homo- and hetero-dimerization and activation. Crystal structures of the Fab fragments from five different inhibitory antibodies in complex with the extracellular regions of EGFR and ErbB2 have been determined. These structures highlight several different modes of binding and mechanisms of receptor inhibition. Information about antibody interactions with the structurally well-characterized soluble extracellular regions of ErbB receptors can be combined with the rich knowledge of the effects of these antibodies in cultured cells, and in vivo, to provide insights into the conformation and activation of ErbB receptors at the cell surface.     

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.     

Vitamin E analogs trigger apoptosis in HER2/erbB2-overexpressing breast cancer cells by signaling via the mitochondrial pathway

Alpha-tocopheryl succinate (alpha-TOS) is a redox-silent vitamin E (VE) analog with high pro-apoptotic and anti-neoplastic activity. Here we investigated whether alpha-TOS and several novel VE analogs kill breast cancer cells over-expressing the anti-apoptotic receptor protein HER2/erbB2. The agents induced apoptosis at comparable levels in both erbB2-low and -high cells. Generation of reactive oxygen species (ROS) preceded mitochondrial destabilization and execution of apoptosis, as evidenced by the anti-apoptotic effects of exogenous superoxide dismutase and mitochondrially targeted coenzyme Q. Dissipation of DeltaPsi(m) was followed by cytochrome c and Smac/Diablo re-localization and caspase-dependent cleavage of death substrate. A resistance to apoptosis for the corresponding rho(0) counterparts confirmed a critical dependency for mitochondria during the induction of apoptosis in breast cancer cells mediated by VE analogs and linked apoptosis to generation of radicals as judged by the delayed accumulation of ROS in the cybrid cell types. We conclude that alpha-TOS causes efficient apoptosis in breast cancer cells independent of their erbB2 status. Since erbB2 is frequently over-expressed in breast cancers and renders the neoplastic disease resistant to established treatment, our findings are of clinical interest.     

MUC4 involvement in ErbB2/ErbB3 phosphorylation and signaling in response to airway cell mechanical injury

The receptor tyrosine kinases ErbB2 and ErbB3 are phosphorylated in response to injury of the airway epithelium. Since we have shown that the membrane mucin MUC4 can act as a ligand/modulator for ErbB2, affecting its localization in polarized epithelial cells and its phosphorylation, we questioned whether Muc4 was involved, along with ErbB2 and ErbB3, in the damage response of airway epithelia. To test this hypothesis, we first examined the localization of MUC4 in human airway samples. Both immunocytochemistry and immunofluorescence showed a co‐localization of MUC4 and ErbB2 at the airway luminal surface. Sequential immunoprecipitation and immunoblotting from airway cells demonstrated that the MUC4 and ErbB2 are present as a complex in airway epithelial cells. To assess the participation of MUC4 in the damage response, cultures of NCI‐H292 or airway cells were scratch‐wounded, then analyzed for association of phospho‐ErbB2 and ‐ErbB3 with MUC4 by sequential immunoprecipitation and immunoblotting. Wounded cultures exhibited increased phosphorylation of both receptors in complex with MUC4. Scratch wounding also increased activation of the downstream pathway through Akt, as predicted from our previous studies on Muc4 effects on ErbB2 and ErbB3. The participation of MUC4 in the phosphorylation response was also indicated by siRNA repression of MUC4 expression, which resulted in diminution of the phosphorylation of ErbB2 and ErbB3. These studies provide a new model for the airway epithelial damage response, in which the MUC4–ErbB2 complex is a key element in the sensor mechanism and phosphorylation of the receptors. J. Cell. Biochem. 107: 112–122, 2009. © 2009 Wiley‐Liss, Inc.     

A signaling role for the uncleaved form of alpha 6 integrin in differentiating lens fiber cells

Many alpha integrin subunits are cleaved during their processing to yield heavy and light chains, which remain associated by disulfide bonds. While uncleaved alpha integrin subunits can form functional receptors that sometimes have distinct signaling roles from their better-characterized endoproteolytically cleaved counterparts, their expression at the cell surface and their association with signaling complexes have yet to be determined in vivo. In this study, we demonstrate that, in differentiating lens fiber cells, the uncleaved form of alpha 6 integrin was expressed at the cell surface. This form of alpha 6 integrin coimmunoprecipitated with both the signaling adaptor molecule Shc and its downstream effector Grb2, suggesting that, in lens fiber cells, uncleaved alpha 6 integrin was associated with a Shc-mediated signaling complex. We show that expression of the cleaved form of alpha 6 integrin progressively decreased relative to uncleaved alpha 6 integrin as the state of lens cell differentiation increased, resulting in the predominance of uncleaved alpha 6 integrin in the lens fiber cell zones. Interestingly, we previously have shown that alpha 6 integrin is localized principally along the extensive cell-cell interfaces of these lens fiber cells, in the absence of its extracellular matrix ligand laminin. While we found that the cleaved form of alpha 6 integrin contained both high mannose and complex sugars, the uncleaved form of alpha 6 integrin contained only high mannose sugars. These properties suggest that the uncleaved form of alpha 6 integrin may have a unique role in the embryonic lens. Its high association with Shc and Grb2 in the differentiating cortical fiber cell zone indicates that alpha 6 integrin may provide a cell survival signal in the presence of the apoptotic-like processes that are initiated in this region of the embryonic lens to clear the lens cells of their organelles.     

The role of Neuregulin-1beta/ErbB signaling in the heart

Products of the Neuregulin-1 (Nrg-1) gene, along with the ErbB family of receptor tyrosine kinases through which Nrg-1 ligands signal, play a critical role during cardiovascular development. Through studies of genetically manipulated mice, as well as studies in cells isolated from adult hearts, it appears that Nrg-1/ErbB signaling is an essential paracrine mediator of cell-cell interactions that not only regulates tissue organization during development, but also helps to maintain cardiac function throughout an organism's life. Studies in cells isolated from the heart demonstrate that Nrg-1 can activate a number of signaling pathways, which mediate cellular adaptations to stress in the myocardium. These observations provide insight as to why ErbB2-targeted cancer treatments have deleterious effects on cardiac function in some cancer patients. Moreover emerging data suggest that Nrg-1 ligands might be useful clinically to restore cardiac function after cardiac injury. In this review we will attempt to synthesize the literature behind this rapidly growing and exciting area of research.     

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.     

MicroRNA-188-5p promotes apoptosis and inhibits cell proliferation of breast cancer cells via the MAPK signaling pathway by targeting Rap2c

Breast cancer is a common malignancy that is highly lethal with poor survival rates and immature therapeutics that urgently needs more effective and efficient therapies. MicroRNAs are intrinsically involved in different cancer remedies, but their mechanism in breast cancer has not been elucidated for prospective treatment. The function and mechanism of microRNA-188-5p (miR-188) have not been thoroughly investigated in breast cancer. In our study, we found that the expression of miR-188 in breast cancer tissues was obviously reduced. Our findings also revealed the abnormal overexpression of miR-188 in 4T1 and MCF-7 cells significantly suppressed cell proliferation and migration and also enhanced apoptosis. miR-188 induced cell cycle arrest in the G1 phase. To illuminate the molecular mechanism of miR-188, Rap2c was screened as a single target gene by bioinformatics database analysis and was further confirmed by dual-luciferase assay. Moreover, Rap2c was found to be a vital molecular switch for the mitogen-activated protein kinase signaling pathway in tumor progression by decreasing apoptosis and promoting proliferation and migration. In conclusion, our results revealed that miR-188 is a cancer progression suppressor and a promising future target for breast cancer therapy.     

A novel mouse monoclonal antibody targeting ErbB2 suppresses breast cancer growth

Overexpression of ErbB2 in breast cancer is associated with increased recurrence and worse prognosis. Accumulating evidences suggest that molecular targeted therapy is a promising anticancer strategy. In this study, we produced a novel anti-ErbB2 monoclonal antibody, 6G10, that recognized an epitope distinct from the trastuzumab binding site. 6G10 induced aggregation of BT474 breast cancer cells and inhibited proliferation of various breast cancer cell lines including BT474. A growth inhibition assay showed that 6G10 had EC₅₀ values comparable to trastuzumab, indicating that the drugs have a similar level of potency. Furthermore, intraperitoneal administration of 6G10 completely inhibited the growth of xenografted tumors derived from BT474 and SK-BR-3 cells. These data suggested that 6G10 has great therapeutic potential and could be administered to patients alternatively, or synergistically, with trastuzumab.     

EGF receptor inhibitors increase ErbB3 mRNA and protein levels in breast cancer cells

The potential benefits of drugs directly targeting the ErbB receptors for cancer therapy have led to an extensive development within this field. However, the clinical effects of ErbB receptor-targeting drugs in cancer treatment are limited due to a high frequency of resistance. It has been reported that, when inhibiting the epidermal growth factor receptor (EGFR) with the tyrosine kinase inhibitor gefitinib, increased activation of ErbB3 via MET, or by re-localization of ErbB3 mediates cell survival. Here we show further evidence that members of the ErbB receptor family facilitate resistance to EGFR inhibitor treatment in ErbB2 overexpressing breast cancer cells. We found that gefitinib treatment increased ErbB3 expression, both at protein and mRNA levels. ErbB3 expression was upregulated not only by gefitinib but also by a panel of different EGFR inhibitors, suggesting that inhibition of EGFR in general affects ErbB3 expression. In addition, we found that gefitinib treatment increased ErbB2 expression levels while EGFR inhibitors decreased the activity of ErbB2. Concentrations of gefitinib that decreased phospho-ErbB2 reversely increased ErbB3 levels. We further examined changes induced by gefitinib treatment on mRNA levels of the most common genes known to be involved in breast cancer. As expected, we found that gefitinib downregulated genes whose functions were linked to cellular proliferation, such as Ki-67, topoisomerase II alpha and cyclins, and surprisingly downregulated gene expression of FAS which is involved in apoptotic signaling. Together, our data strongly suggest that resistance to EGFR inhibitors may result from the compensation of other family members and that combinations of anti-cancer drugs are required to increase the sensitivity of these treatments.