Novel 3-alkoxy-1H-pyrazolo[3,4-d]pyrimidines as EGFR and erbB2 receptor tyrosine kinase inhibitors

Novel 4-anilino-1H-pyrazolo[3,4-d]pyrimidines have been synthesized and evaluated in vitro for erbB2 and EGFR kinase inhibition. A representative compound displaying oral bioavailability in rat and dog illustrates the potential of this series to provide orally active erbB2 inhibitors.     

Abundance and localization of skeletal muscle-related erbB2 may stimulate tumour growth during initial stages of oral oncogenesis

ErbB2 and erbB3 transmembrane receptors, known to be associated with neuronal and skeletal muscle developmental function, seem to play an important role in human oral oncogenesis. This study was designed to determine gradual erbB2 and erbB3 expression in an experimental animal system of induced oral carcinogenesis in Syrian golden hamsters. Thirty-seven animals were divided into one control group (N=7) and three experimental groups (N=10 each one), which were treated with carcinogen 9,10-dimethyl-1,2-benzanthracene and sacrificed at 10, 14 and 19 weeks after treatment. The histological status of observed lesions in the three experimental groups corresponded well with tumour advancement (from oral mucosal dysplasia to moderately differentiated squamous cell carcinoma). Tissue sections ranging from normal mucosa to squamous cell carcinoma were studied using monoclonal antibodies against erbB2 and erbB3 proteins. Cytoplasmic erbB2 expression was gradually increased in pre-cancerous stages, remained stable in initial tumour stages and substantially decreased in moderately-differentiated carcinomas, suggesting that it may be useful as an early prognostic factor. On the contrary, erbB3 was not expressed at all either in normal or tumour tissue.     

Metformin inhibits breast cancer cell growth,colony formation and induces cell cycle arrest in vitro

The anti-diabetic drug metformin reduces human cancer incidence and improves the survival of cancer patients, including those with breast cancer. We studied the activity of metformin against diverse molecular subtypes of breast cancer cell lines in vitro. Metformin showed biological activity against all estrogen receptor (ER) positive and negative, erbB2 normal and abnormal breast cancer cell lines tested. It inhibited cellular proliferation, reduced colony formation and caused partial cell cycle arrest at the G1 checkpoint. Metformin did not induce apoptosis (as measured by DNA fragmentation and PARP cleavage) in luminal A, B or erbB2 subtype breast cancer cell lines. At the molecular level, metformin treatment was associated with a reduction of cyclin D1 and E2F1 expression with no changes in p27kip1 or p21waf1. It inhibited mitogen activated protein kinase (MAPK) and Akt activity, as well as the mammalian target of rapamycin (mTOR) in both ER positive and negative, erbB2-overexpressing and erbB2-normal expressing breast cancer cells. In erbB2-overexpressing breast cancer cell lines, metformin reduced erbB2 expression at higher concentrations, and at lower concentrations within the therapeutic range, it inhibited erbB2 tyrosine kinase activity evidenced by a reduction of phosphorylated erbB2 (P-erbB2) at both auto- and Src- phosphorylation sites. These data suggest that metformin may have potential therapeutic utility against ER positive and negative, erbB2-overexpressing and erbB2-normal expressing breast cancer cells.     

c-erbB_2对大鼠黄体细胞hCG诱导的孕酮分泌的影响

采用离体细胞体外孵育法 ,研究反义c erbB2 寡脱氧核苷酸 (antisensec erbB2 ODN)对大鼠黄体细胞hCG诱导的孕酮分泌的影响 ,及其与外源性cAMP和Ca2 以及蛋白抑制剂放线菌酮 (CYX)之间的关系。结果表明 ,反义c erbB2 以剂量相关方式抑制黄体细胞hCG诱导的孕酮的产生 ,同时使c erbB2 蛋白染色阳性的黄体细胞百分数下降 ,无义tatODN没有相应的作用。10 -4 mol/L的二丁酰cAMP能明显反转反义c erbB2 ODN对孕酮产生和c erbB2 表达的抑制作用 ,钙离子通道阻断剂维拉帕米和蛋白抑制剂CYX对此抑制作用有协同效应。该实验说明c erbB2 参于hCG诱导黄体细胞生孕酮作用     

A new series of neutral 5-substituted 4-anilinoquinazolines as potent, orally active inhibitors of erbB2 receptor tyrosine kinase

Starting from initial lead 1 containing a basic 5-substituent, optimisation of the glycolamide-derived neutral 5-substituent led to potent inhibitors of erbB2 with good pharmacokinetics. Representative compounds 19 and 21 inhibited phosphorylation of erbB2 in a mouse BT474C xenograft model after oral administration.     

erbB-2与胃癌细胞恶性表型及其增殖调控的关系

应用基因重组、基因转染、Ｓｏｕｔｈｅｒｎ杂交、Ｎｏｒｔｈｅｒｎ杂交、细胞生长曲线测定及裸鼠成瘤观察了反义ｅｒｂＢ－２逆转录病毒重组载体的转染对人胃癌细胞中ｅｒｂＢ－２过度表达的影响及其对ＥＧＦ的反应性．研究结果显示,ｅｒｂＢ－２的表达被其反义重组子特异抑制,伴有细胞增殖能力的下降及致瘤性的下降;在ＥＧＦ的刺激下,ｅｒｂＢ－２高表达肿瘤细胞生长速度提高的幅度显著大于ｅｒｂＢ－２反义重组载体转染细胞;ＥＧＦ促细胞增殖及促基因表达的功能在ｅｒｂＢ－２反义重组子转染后受到抑制,提示ｅｒｂＢ－２在细胞增殖调控中具有重要功能．     

Skeletal muscle stem cells express anti-apoptotic ErbB receptors during activation from quiescence

To be effective for tissue repair, satellite cells (the stem cells of adult muscle) must survive the initial activation from quiescence. Using an in vitro model of satellite cell activation, we show that erbB1, erbB2 and erbB3, members of the EGF receptor tyrosine kinase family, appear on satellite cells within 6 h of activation. We show that signalling via erbB2 provides an anti-apoptotic survival mechanism for satellite cells during the first 24 h, as they progress to a proliferative state. Inhibition of erbB2 signalling with AG825 reduced satellite cell numbers, concomitant with elevated caspase-8 activation and TUNEL labelling of apoptotic satellite cells. In serum-free conditions, satellite cell apoptosis could be largely prevented by a mixture of erbB1, erbB3 and erbB4 ligand growth factors, but not by neuregulin alone (erbB3/erbB4 ligand). Furthermore, using inhibitors specific to discrete intracellular signalling pathways, we identify MEK as a pro-apoptotic mediator, and the erbB-regulated factor STAT3 as an anti-apoptotic mediator during satellite cell activation. These results implicate erbB2 signalling in the preservation of a full compliment of satellite cells as they activate in the context of a damaged muscle.     

CD44 enhances neuregulin signaling by Schwann cells

We describe a key role for the CD44 transmembrane glycoprotein in Schwann cell-neuron interactions. CD44 proteins have been implicated in cell adhesion and in the presentation of growth factors to high affinity receptors. We observed high CD44 expression in early rat neonatal nerves at times when Schwann cells proliferate but low expression in adult nerves, where CD44 was found in some nonmyelinating Schwann cells and to varying extents in some myelinating fibers. CD44 constitutively associated with erbB2 and erbB3, receptor tyrosine kinases that heterodimerize and signal in Schwann cells in response to neuregulins. Moreover, CD44 significantly enhanced neuregulin-induced erbB2 phosphorylation and erbB2-erbB3 heterodimerization. Reduction of CD44 expression in vitro resulted in loss of Schwann cell-neurite adhesion and Schwann cell apoptosis. CD44 is therefore crucial for maintaining neuron-Schwann cell interactions at least partly by facilitating neuregulin-induced erbB2-erbB3 activation.     

Synthesis and SAR of potent EGFR/erbB2 dual inhibitors

A series of 6-alkoxy-4-anilinoquinazoline compounds was prepared and evaluated for in vitro inhibition of the erbB2 and EGFR kinase activity. The IC(50) values of the best compounds were below 0.10 uM. Further, several of these compounds inhibit the growth of erbB2 and EGFR over-expressing tumor cell lines at concentrations below 1 uM.     

Analysis of a tyrosine-specific protein kinase activity associated with the retroviral erbB oncogene product

The transforming protein erbB of avian erythroblastosis virus (AEV) has considerable sequence homology with the epidermal growth factor (EGF) and appears to represent a truncated form of this receptor. The sequence of the erbB gene is furthermore related to that of other viral transforming genes such as src, fps, yes or abl. The transforming proteins of these src-related oncogenes as well as receptors for EGF, platelet-derived growth factor (PDGF), and insulin are associated with tyrosine-specific protein kinases. It has been difficult to demonstrate this activity for the erbB protein. To analyze the erbB gene product, we prepared polyclonal antibodies against a bacterially expressed erbB DNA restriction fragment (BamHI/BamHI). The antiserum is shown to immunoprecipitate the erbB protein from AEV-transformed chicken fibroblasts and also recognizes the EGF receptor protein. Both proteins become phosphorylated in vitro on tyrosine residues upon the addition of [gamma-32P]ATP. The protein kinase activity is low compared to other oncogene-specific kinases. This is not due to kinase blocking by the serum, because erbB carboxyterminal synthetic peptide antibodies give rise to low levels of protein kinase activity as well indicating that this may be a characteristic property of erbB in vitro.     

Signal transduction of erbB receptors in trastuzumab (Herceptin) sensitive and resistant cell lines: local stimulation using magnetic microspheres as assessed by quantitative digital microscopy.

BACKGROUND: ErbB2 (HER-2), a member of the epidermal growth factor (EGF) receptor family, is a class I transmembrane receptor tyrosine kinase. Although erbB2 has no known physiologic ligand, it can form complexes with other members of the family and undergo transactivation of its very potent kinase activity, thereby initiating downstream signaling and cell proliferation. ErbB2 is a frequent pathologic marker in ductal invasive breast carcinomas and is targeted by using a specific humanized monoclonal antibody, trastuzumab (Herceptin). The antibody is effective in only 20% to 50% of erbB2-positive tumors, and this resistance, as yet poorly understood, constitutes a major therapeutic challenge. METHODS: Magnetic microspheres coated with ligands or antibodies are widely used for separation of proteins and cells and allow localized, high intensity, and precisely timed stimulation of cells. We used EGF- and trastuzumab-covered paramagnetic microspheres, quantitative confocal laser scanning microscopy, and digital image processing to investigate the (trans)activation of and local signal propagation from erbB1 and erbB2 on trastuzumab sensitive and resistant carcinoma cell lines expressing these receptors at high levels. RESULTS: On A431 cells expressing high levels of endogenous erbB1 and transfected erbB2-mYFP (A4-erbB2-mYFP F4 cell line), EGF-coupled-microspheres activated erbB1 and transactivated erbB2-mYFP. In two other cell lines with comparable erbB2 expression but lower levels of erbB1, EGF microspheres transactivated erbB2 less efficiently. Trastuzumab in solution activated erbB2 on A4-erbB2-mYFP and the trastuzumab sensitive SKBR-3 cells, but only negligibly on the resistant JIMT-1 cells that showed a 10 times higher K(d) for the antibody. Nevertheless, pronounced erbB2 activation and tyrosine phosphorylation could be detected after stimulation with trastuzumab-coupled microspheres in all cell lines, although transactivation of erbB1 was negligible. Receptor phosphorylation was restricted to the immediate proximity of the microspheres, i.e., receptor clusters external to these locations remained inactive. CONCLUSION: ErbB1 ligand and erbB2 specific antibody attached to magnetic microspheres are efficient tools in assessing erbB activation, localized signal propagation, and erbB heterodimer formation. Trastuzumab coupled to microspheres is more efficient at accessing erbB2 and activating it than trastuzumab in solution.     

Differentiation of human airway epithelia is dependent on erbB2

A clinical case documented a reversible change in airway epithelial differentiation that coincided with the initiation and discontinuation of trastuzumab, an anti-erbB2 antibody. This prompted the investigation into whether blocking the erbB2 receptor alters differentiation of the airway epithelium. To test this hypothesis, we treated an in vitro model of well-differentiated human airway epithelia with trastuzumab or heregulin-alpha, an erbB ligand. In addition, coculturing with human lung fibroblasts tested whether in vivo subepithelial fibroblasts function as an endogenous source of ligands able to activate erbB receptors expressed by the overlying epithelial cells. Epithelia were stained with hematoxylin and eosin and used for morphometric analysis. Trastuzumab treatment decreased the ciliated cell number by 49% and increased the metaplastic, flat cell number by 640%. Heregulin-alpha treatment increased epithelial height and decreased the number of metaplastic and nonciliated columnar cells, whereas it increased the goblet cell number. We found that normal human lung fibroblasts express transforming growth factor-alpha, heparin-binding epidermal-like growth factor, epiregulin, heregulin-alpha, and amphiregulin, all of which are erbB ligands. Cocultures of airway epithelia with primary fibroblasts increased epithelial height comparable to that achieved following heregulin-alpha treatment. These data show that erbB2 stimulation is required for maintaining epithelial differentiation. Furthermore, the mesenchyme underlying the airway epithelium secretes a variety of erbB ligands that may direct various pathways of epithelial differentiation.     

人胚鼻咽上皮发育分化与c—erbB—2的表达变化

应用免疫组织化学方法对人胚鼻咽的ｃ－ｅｒｂＢ－２表达情况进行了研究．结果表明,人胚鼻咽上皮的ｃ－ｅｒｂＢ－２表达没有发育阶段性与鼻咽部位置的差异,而与鼻咽上皮的种类密切相关．在假复层纤毛柱状上皮中,以纤毛层的ｃ－ｅｒｂＢ－２表达阳性信号最强;在典型的过渡型上皮中,ｃ－ｅｒｂＢ－２阳性反应细胞主要分布于上皮的下五分之四左右的区域,表层细胞无阳性信号出现;而在复层鳞状上皮中,ｃ－ｅｒｂＢ－２阳性细胞的位置进一步下移,主要分布于上皮的下三分之二左右的区域．这些结果提示,ｃ－ｅｒｂＢ－２在人胚鼻咽上皮中的表达随细胞分化程度的增加而降低直至完全没有表达．     

ErbB2 and EGFR are downmodulated during the differentiation of 3T3-L1 preadipocytes

The expression of receptors belonging to the epidermal growth factor receptor subfamily has been largely studied these last years in epithelial cells mainly as involved in cell proliferation and malignant progression. Although much work has focused on the role of these growth factor receptors in the differentiation of a variety of tissues, there is little information in regards to normal stromal cells. We investigated erbB2 expression in the murine fibroblast cell line Swiss 3T3L1, which naturally or hormonally induced undergoes adipocyte differentiation. We found that the Swiss 3T3-L1 fibroblasts express erbB2, in addition to EGFR, and in a quantity comparable to or even greater than the breast cancer cell line T47D. Proliferating cells increased erbB2 and EGFR levels when reaching confluence up to 4- and 10-fold, respectively. This expression showed a significant decrease when growth-arrested cells were stimulated to differentiate with dexamethasone and isobutyl-methylxanthine. Differentiated cells presented a decreased expression of both erbB2 and EGFR regardless of whether the cells were hormonally or spontaneously differentiated. EGF stimulation of serum-starved cells increased erbB2 tyrosine phosphorylation and retarded erbB2 migration in SDS-PAGE, suggesting receptor association and activation. Heregulin-alpha1 and -beta1, two EGF related factors, had no effect on erbB2 or EGFR phosphorylation. Although 3T3-L1 cells expressed heregulin, its specific receptors, erbB3 and erbB4, were not found. This is the first time in which erbB2 is reported to be expressed in an adipocytic cell line which does not depend on non EGF family growth factors (thyroid hormone, growth hormone, etc.) to accomplish adipose differentiation. Since erbB2 and EGFR expression were downmodulated as differentiation progressed it is conceivable that a mechanism of switching from a mitogenic to a differentiating signaling pathway may be involved, through regulation of the expression of these growth factor receptors.     

erbB3 Is an Active Tyrosine Kinase Capable of Homo- and Heterointeractions

Often considered to be a “dead” kinase, erbB3 is implicated in escape from erbB-targeted cancer therapies. Here, heregulin stimulation is shown to markedly upregulate kinase activity in erbB3 immunoprecipitates. Intact, activated erbB3 phosphorylates tyrosine sites in an exogenous peptide substrate, and this activity is abolished by mutagenesis of lysine 723 in the catalytic domain. Enhanced erbB3 kinase activity is linked to heterointeractions with catalytically active erbB2, since it is largely blocked in cells pretreated with lapatinib or pertuzumab. erbB2 activation of erbB3 is not dependent on equal surface levels of these receptors, since it occurs even in erbB3-transfected CHO cells with disproportionally small amounts of erbB2. We tested a model in which transient erbB3/erbB2 heterointeractions set the stage for erbB3 homodimers to be signaling competent. erbB3 homo- and heterodimerization events were captured in real time on live cells using single-particle tracking of quantum dot probes bound to ligand or hemagglutinin tags on recombinant receptors.     

ErbB receptors in fetal endothelium--a potential linkage point for inflammation-associated neonatal disorders

Objective: ErbB receptors and their ligands play crucial roles in development. During late gestation, they might also be involved in the pathogenesis of prematurity-associated disorders. ErbB receptor dimerization leads to a diversity of biologic signals. We studied the expression and localization patterns of erbB receptors in the developing human umbilical endothelial cell system. It is still unclear, whether expression patterns might be developmentally regulated and depend on the cell type studied. Methods: Primary human umbilical venous endothelial cells (HUVEC) and arterial endothelial cells (HUAEC) were isolated between 24 and 42 weeks of gestation and used for immunoprecipitation, Western blotting, and confocal microscopy. Results: All four erbB receptors were present in HUVEC and HUAEC. Expression patterns were similar for cell types at gestational ages examined. ErbB4 always co-precipitated with erbB1 in both cell types independent of the gestational age. Confocal microscopy revealed that all erbB receptors were localized in the nucleus, erbB1 and erbB3 in the nucleoli, while erbB2 and erbB4 spared the nucleolar region. All receptors showed a tendency to co-localize. Growth factor stimulation altered localization patterns. Cellular subfractionation experiments for erbB4 largely confirmed microscopy results. Pretreatment with lipopolysaccharide enhanced this nuclear localization of erbB4, particularly of its intracellular domain. Conclusions: All erbB receptors are present in both HUVEC and HUAEC at all gestational ages tested. ErbB receptor expression patterns were independent of the developmental stage of the endothelial cell, at least in the third trimester. We speculate that endothelial erbB receptors might play a role in normal development in mid and late gestation. We also speculate that these findings, together with the known involvement of erbB receptors in development, inflammation, and angiogenesis, will open new avenues for erbB receptor-related research in the pathogenesis of fetal and neonatal inflammation-associated disorders.     

Cell type dependent endocytic internalization of ErbB2 with an artificial peptide ligand that binds to ErbB2

ErbB2, which is a member of the epidermal growth factor (erbB) receptor family, is frequently overexpressed in breast and ovarian cancers. Antibody and small molecule anti-tyrosine kinase inhibitors have been developed for targeted therapies for cancers overexpressing erbB2. Internalization and downregulation of erbB2, which is induced by a ligand, may be important for efficacious therapeutic effects. However, ligand-dependent erbB2 internalization has not been well characterized. Here we investigated the internalization of erbB2 in SKBr3 and SKOv3 cells, both overexpressing erbB2, using an EC-1 peptide fused to eGFP (EC-eGFP), which specifically binds to erbB2. ErbB2 was internalized in SKOv3 cells when the cells were treated with EC-eGFP. The accumulation of endosomal erbB2 was EC-eGFP dependent, which colocalized with transferrin implying endocytosis via clathrin-coated pits. In contrast, internalization of erbB2 was not observed in SKBr3 cells. As a result, two different mechanisms, which are cell type dependent for the internalization of erbB2, are proposed.