Project description:The ErbB2/3 heterodimer plays a critical role in breast cancer genesis and progression. EBP1, an ErbB3 binding protein, inhibits breast cancer growth but its effects on ErbB3 ligand mediated signal transduction or ErbB receptors is not known. We report here that ectopic expression of EBP1 in MCF-7 and AU565 breast cancer cell lines inhibited HRG-induced proliferation. ErbB2 protein levels were substantially decreased in EBP1 transfectants, while ErbB3 levels were unchanged. HRG-induced AKT activation was attenuated in EBP1 stable transfectants and transfection of a constitutively activated AKT partially restored the growth response to HRG. Down-regulation of EBP1 expression in MCF-7 cells by shRNA resulted in increased cell growth in response to HRG and increased cyclin D1 and ErbB2 expression. These results suggest that EBP1, by down-regulating ErbB signal transduction, attentuates HRG-mediated growth of breast cancer cells.
Project description:As ErbB signaling is a determinant of prolactin synthesis, role of ErbB receptors was tested for prolactinoma outcomes and therapy. The objective of this study was to characterize ErbB receptor expression in prolactinomas and then perform a pilot study treating resistant prolactinomas with a targeted tyrosine kinase inhibitor (TKI). Retrospective analysis of prolactinomas and pilot study for dopamine agonist resistant prolactinomas in tertiary referral center. We performed immunofluorescent staining of a tissue array of 29 resected prolactinoma tissues for EGFR, ErbB2, ErbB3, and ErbB4 correlated with clinical features. Two patients with aggressive resistant prolactinomas enrolled and completed trial. They received lapatinib 1,250 mg daily for 6 months with tumor and hormone assessments. Main outcome measures were positive tumor staining of respective ErbB receptors, therapeutic reduction of prolactin levels and tumor shrinkage. Treated PRL levels and tumor volumes were suppressed in both subjects treated with TKI. EGFR expression was positive in 82 % of adenomas, ErbB2 in 92 %, ErbB3 in 25 %, and ErbB4 in 71 %, with ErbB2 score > EGFR > ErbB4 > ErbB3. Higher ErbB3 expression was associated with optic chiasm compression (p = 0.03), suprasellar extension (p = 0.04), and carotid artery encasement (p = 0.01). Higher DA response rates were observed in tumors with higher ErbB3 expression. Prolactinoma expression of specific ErbB receptors is associated with tumor invasion, symptoms, and response to dopamine agonists. Targeting ErbB receptors may be effective therapy in patients with resistant prolactinomas.
Project description:Epidermal growth factor receptor (EGFR) signaling is strongly implicated in glioblastoma (GBM) tumorigenesis. However, molecular agents targeting EGFR have demonstrated minimal efficacy in clinical trials, suggesting the existence of GBM resistance mechanisms. GBM cells with stem-like properties (CSCs) are highly efficient at tumor initiation and exhibit therapeutic resistance. In this study, GBMCSC lines showed sphere-forming and tumor initiation capacity after EGF withdrawal from cell culture media, compared with normal neural stem cells that rapidly perished after EGF withdrawal. Compensatory activation of related ERBB family receptors (ERBB2 and ERBB3) was observed in GBM CSCs deprived of EGFR signal (EGF deprivation or cetuximab inhibition), suggesting an intrinsic GBM resistance mechanism for EGFR-targeted therapy. Dual inhibition of EGFR and ERBB2 with lapatinib significantly reduced GBM proliferation in colony formation assays compared to cetuximab-mediated EGFR-specific inhibition. Phosphorylation of downstream ERBB signaling components (AKT, ERK1/2) and GBM CSC proliferation were inhibited by lapatinib. Collectively, these findings show that GBM therapeutic resistance to EGFR inhibitors may be explained by compensatory activation of EGFR-related family members (ERBB2, ERBB3) enabling GBM CSC proliferation, and therefore simultaneous blockade of multiple ERBB family members may be required for more efficacious GBM therapy.
Project description:Although ErbB receptors have been implicated in prostate cancer progression, ErbB-directed drugs have not proven effective for prostate cancer treatment. The ErbB3-binding protein EBP1 affects both ErbB2 and androgen receptor signaling, two components of the response to ErbB-targeted therapies. We therefore examined the effects of EBP1 expression on the response to the ErbB1/2 tyrosine kinase inhibitor lapatinib. We found a negative correlation between endogenous EBP1 levels and lapatinib sensitivity in prostate cancer cell lines. We then overexpressed or inhibited expression of EBP1. Silencing EBP1 expression increased lapatinib sensitivity and overexpression of EBP1 increased resistance in androgen-containing media. Androgen depletion resulted in an increased sensitivity of androgen-dependent EBP1 expressing cells to lapatinib, but did not affect the lapatinib sensitivity of hormone resistant cells. However, EBP1 silenced cells were still more sensitive to lapatinib than EBP1-expressing cells in the absence of androgens. The increase in sensitivity to lapatinib following EBP1 silencing was associated with increased ErbB2 levels. In addition, lapatinib treatment increased ErbB2 levels in sensitive cells that express low levels of EBP1, but decreased ErbB2 levels in resistant EBP1-expressing cells. In contrast, ErbB3 and phospho ErbB3 levels were not affected by either changes in EBP1 levels or lapatinib treatment. The production of the ErbB3/4 ligand heregulin was increased in EBP1-silenced cells. EBP1-induced changes in AR levels were not associated with changes in lapatinib sensitivity. These studies suggest that the ability of EBP1 to activate ErbB2 signaling pathways results in increased lapatinib sensitivity.
Project description:Although ErbB receptors have been implicated in the progression of prostate cancer, little is known about proteins that may mediate their interactions with the androgen receptor (AR). Ebp1, a protein cloned via its association with the ErbB3 receptor, binds the AR and inhibits androgen-regulated transactivation of wild-type AR in COS cells. As the complement of coregulators in different cells are important for AR activity, we determined the effect of Ebp1 on AR function in prostate cancer cell lines. In addition, we examined the regulation of Ebp1 function by the ErbB3/4 ligand heregulin (HRG). In this study, we demonstrate, using several natural AR-regulated promoters, that Ebp1 repressed transcriptional activation of wild-type AR in prostate cancer cell lines. Downregulation of Ebp1 expression in LNCaP cells using siRNA resulted in activation of AR in the absence of androgen. Ebp1 associated with ErbB3 in LNCaP cells in the absence of HRG, but HRG induced the dissociation of Ebp1 from ErbB3. In contrast, HRG treatment enhanced both the association of Ebp1 with AR and also the ability of Ebp1 to repress AR transactivation. These studies suggest that Ebp1 is an AR corepressor whose biological activity can be regulated by the ErbB3 ligand, HRG.
Project description:The role of protein tyrosine kinase activity in ErbB3-mediated signal transduction was investigated. ErbB3 was phosphorylated in vivo in response to either heregulin (HRG) in cells expressing both ErbB3 and ErbB2, or epidermal growth factor (EGF) in cells expressing both ErbB3 and EGF receptor. A recombinant receptor protein (ErbB3-K/M, in which K/M stands for Lys-->Met amino acid substitution) containing an inactivating mutation in the putative ATP-binding site was also phosphorylated in response to HRG and EGF. Both the wild-type ErbB3 and mutant ErbB3-K/M proteins transduced signals to phosphatidylinositol 3-kinase, Shc and mitogen-activated protein kinases. Separate kinase-inactivating mutations in the EGF receptor and ErbB2 proteins abolished ErbB3 phosphorylation and signal transduction activated by EGF and HRG respectively. Hence the protein tyrosine kinase activity necessary for growth factor signalling via the ErbB3 protein seems to be provided by coexpressed EGF and ErbB2 receptor proteins.
Project description: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.
Project description:BACKGROUND: The ErbB family consists of four proteins including (EGFR)/ErbB1, ErbB2, ErbB3, and ErbB4, and plays a crucial role in the promotion of multiple tumorigenic processes. In addition to the traditional pathways of EGFR signaling, EGFR translocates to the nucleus and acts as a transcription factor in the proliferation of cancer cells. Heregulin is known as both an ErbB3 and an ErbB4 ligand. This study aimed to investigate the expression of heregulin and its relevant EGFR family members as well as their phosphorylated forms in human colorectal cancer (CRC) tissues and to determine the relationship between their expression and clinicopathological factors including patient prognosis. METHODS: We analyzed the effects of exogenous heregulin on ErbB2, ErbB3 and ErbB4 phosphorylation in Caco-2, DLD-1, and HCT 116 colon cancer cell lines by western blot analysis. We examined 155 surgical resections from colorectomy patients. Cellular localization of ErbB1-4, their phosphorylated forms and heregulin protein was analyzed in CRC surgical resections by immunohistochemical analysis. Immunohistochemical results were compared with clinicopathological factors and patient prognosis. RESULTS: Phosphorylated ErbB2 (pErbB2) and phosphorylated ErbB3 (pErbB3) were detected in both nuclear and cytosolic fractions of Caco-2 and DLD-1 cells stimulated by exogenous heregulin. Whereas, phosphorylated ErbB4 (pErbB4) was detected only in cytosolic fractions of HCT 116 cells stimulated by exogenous heregulin. Phosphorylated EGFR (pEGFR) immunoreactivity was observed in the cytoplasm and nuclei of cancer cells, whereas the pattern of EGFR staining was membranous and cytoplasmic. Subcellular localization of pErbB2, cytoplasmic, membranous, or nuclear, varied among cases. pErbB3 immunoreactivity was exclusively observed in the nuclei of cancer cells. pErbB4 immunoreactivity was observed in the cell membrane of cancer cells. Statistically, heregulin immunoreactivity correlated with pErbB2 and pErbB4 expression. In multivariate analysis for disease free survival, lymph node status, pErbB3 and pErbB4 expression retained independent prognostic significance. In multivariate analysis for overall survival, lymph node status, pEGFR and pErbB4 retained independent prognostic significance. CONCLUSIONS: ErbB2 and ErbB3 phosphorylated by heregulin localized in the nucleus of CRC cells. Phosphorylated ErbB1-4 and heregulin contribute to poorer patient prognosis in CRC. This heregulin-ErbB family member autocrine loop may be a candidate for targeted treatment of CRC.
Project description:Dimerization or the formation of higher-order oligomers is required for the activation of ErbB receptor tyrosine kinases. The heregulin (HRG) receptor, ErbB3, must heterodimerize with other members of the family, preferentially ErbB2, to form a functional signal transducing complex. Here, we applied single molecule imaging capable of detecting long-lived and mobile associations to measure their stoichiometry and mobility and analyzed data from experiments globally, taking the different lateral mobility of monomeric and dimeric molecular species into account. Although ErbB3 was largely monomeric in the absence of stimulation and ErbB2 co-expression, a small fraction was present as constitutive homodimers exhibiting a ?40% lower mobility than monomers. HRG stimulation increased the homodimeric fraction of ErbB3 significantly and reduced the mobility of homodimers fourfold compared to constitutive homodimers. Expression of ErbB2 elevated the homodimeric fraction of ErbB3 even in unstimulated cells and induced a ?2-fold reduction in the lateral mobility of ErbB3 homodimers. The mobility of ErbB2 was significantly lower than that of ErbB3, and HRG induced a less pronounced decrease in the diffusion coefficient of all ErbB2 molecules and ErbB3/ErbB2 heterodimers than in the mobility of ErbB3. The slower diffusion of ErbB2 compared to ErbB3 was abolished by depolymerizing actin filaments, whereas ErbB2 expression induced a substantial rearrangement of microfilaments, implying a bidirectional interaction between ErbB2 and actin. HRG stimulation of cells co-expressing ErbB3 and ErbB2 led to the formation of ErbB3 homodimers and ErbB3/ErbB2 heterodimers in a competitive fashion. Although pertuzumab, an antibody binding to the dimerization arm of ErbB2, completely abolished the formation of constitutive and HRG-induced ErbB3/ErbB2 heterodimers, it only slightly blocked ErbB3 homodimerization. The results imply that a dynamic equilibrium exists between constitutive and ligand-induced homo- and heterodimers capable of shaping transmembrane signaling.
Project description:Both erbB3 and IGF-1 receptor (IGF-1R) have been shown to play an important role in trastuzumab resistance. However, it remains unclear whether erbB3- and IGF-1R-initiated signaling pathways possess distinct effects on the sensitivity of lapatinib, a dual tyrosine kinase inhibitor against both EGFR and erbB2, in trastuzumab-resistant breast cancer. Here, we show that the trastuzumab-resistant SKBR3-pool2 and BT474-HR20 breast cancer sublines, as compared the parental SKBR3 and BT474 cells, respectively, exhibit refractoriness to lapatinib. Knockdown of erbB3 inhibited Akt in SKBR3-pool2 and BT474-HR20 cells, significantly increased lapatinib efficacy, and dramatically re-sensitized the cells to lapatinib-induced apoptosis. In contrast, specific knockdown of IGF-1R did not alter the cells' responsiveness to lapatinib. While the levels of phosphorylated Src (P-Src) were reduced upon IGF-1R downregulation, the P-Akt levels remained unchanged. Furthermore, a specific inhibitor of Akt, but not Src, significantly enhanced lapatinib-mediated anti-proliferative/anti-survival effects on SKBR3-pool2 and BT474-HR20 cells. These data indicate that erbB3 signaling is critical for both trastuzumab and lapatinib resistances mainly through the PI-3K/Akt pathway, whereas IGF-1R-initiated Src activation results in trastuzumab resistance without affecting lapatinib sensitivity. Our findings may facilitate the development of precision therapeutic regimens for erbB2-positive breast cancer patients who become resistant to erbB2-targeted therapy.