ErbB receptor tyrosine kinase network inhibition radiosensitizes carcinoma cells

PURPOSE: The expression of epidermal growth factor receptor (EGFR)-CD533, a truncation mutant of the wild-type EGFR, radiosensitizes carcinoma and malignant glioma cell lines. This deletion mutant disrupts EGFR activation and downstream signaling through the formation of inhibitory dimerizations. In this study, the effects of EGFR-CD533 on other ErbB receptor tyrosine kinase (RTK) family members were quantified to better understand the mechanism of EGFR-CD533-mediated radiosensitization. METHODS AND MATERIALS: Breast carcinoma cell lines with different ErbB RTK expression profiles were transduced with EGFR or ErbB2 deletion mutants (EGFR-CD533 and ErbB2-CD572) using an adenoviral vector. ErbB RTK activation, mitogen activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)/p70S6K signaling, and clonogenic survival were determined for expression of each deletion mutant. RESULTS: EGFR-CD533 radiosensitizes carcinoma cells with either high EGFR expression (MDA-MB231) or low EGFR expression (T47D) through significant blockade of the ErbB RTK network. Analysis of clonogenic survival demonstrate significant enhancement of the alpha/beta ratios, as determined by the linear-quadratic model. Split-dose survival experiments confirm that EGFR-CD533 reduces the repair of cellular damage after ionizing radiation. CONCLUSION: Expression of EGFR-CD533 inhibits the ErbB RTK network and radiosensitizes carcinoma cells irrespective of the ErbB RTK expression patterns, and ErbB2-CD572 does not radiosensitize cells with low EGFR expression. These studies demonstrate that the mechanism of action for EGFR-CD533-mediated radiosensitization is inhibition of the ErbB RTK network, and is an advantage for radiosensitizing multiple malignant cell types.     

mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt

Stimulation of the insulin and insulin-like growth factor I (IGF-I) receptor activates the phosphoinositide-3-kinase/Akt/mTOR pathway causing pleiotropic cellular effects including an mTOR-dependent loss in insulin receptor substrate-1 expression leading to feedback down-regulation of signaling through the pathway. In model systems, tumors exhibiting mutational activation of phosphoinositide-3-kinase/Akt kinase, a common event in cancers, are hypersensitive to mTOR inhibitors, including rapamycin. Despite the activity in model systems, in patients, mTOR inhibitors exhibit more modest antitumor activity. We now show that mTOR inhibition induces insulin receptor substrate-1 expression and abrogates feedback inhibition of the pathway, resulting in Akt activation both in cancer cell lines and in patient tumors treated with the rapamycin derivative, RAD001. IGF-I receptor inhibition prevents rapamycin-induced Akt activation and sensitizes tumor cells to inhibition of mTOR. In contrast, IGF-I reverses the antiproliferative effects of rapamycin in serum-free medium. The data suggest that feedback down-regulation of receptor tyrosine kinase signaling is a frequent event in tumor cells with constitutive mTOR activation. Reversal of this feedback loop by rapamycin may attenuate its therapeutic effects, whereas combination therapy that ablates mTOR function and prevents Akt activation may have improved antitumor activity.     

Specific inhibition of epidermal growth factor receptor tyrosine kinase by 4-anilinoquinazolines

Summary Since the mitogenic action of EGF is mediated by ligand-induced autophosphorylation of the EGF receptor (EGFR), and EGFR is commonly overexpressed in solid human tumours, inhibitors of receptor tyrosine kinase activity (RTK) could prove to be effective antitumour agents. Screening of a compound library using an EGF-RTK enzyme prepared from human tumour derived A431 cells identified a series of potent (IC₅₀<1µM) enzyme inhibitors. These inhibitors are quinazolines bearing a variety of substituted anilines at the 4-position. The most potent 4-anilinoquinazolines (IC₅₀ 20nM) have small non-polar meta substituents on the aniline ring, and are competitive with ATP and non-competitive with substrate. The growth inhibitory activity of these agents was assessed in vitro using KB cells (human oral squamous tumour) grown in the absence or presence of EGF. A selected compound, 4-(3-chloroanilino)quinazoline (CAQ), inhibited EGF-stimulated growth in a concentration dependent manner and complete blockade was observed at concentrations (1–10 µM) which had no effect on basal growth. Selectivity of growth inhibition by CAQ was further exemplified in IGF1-stimulated KB cells where no effect was detected at concentrations which completely blocked EGF-stimulated growth. Similarly, CAQ blocked TGF-stimulated growth in MCF-7 human breast cancer cells without affecting insulin-stimulated growth. These studies define a novel class of EGF-RTK inhibitors which are also potent and selective inhibitors of EGF-stimulated human tumour cell growthin vitro. Presented at the symposium "New Approaches in the Therapy of Breast Cancer", Georgetown University Medical Center, Washington DC, October 1994, generously supported by an education grant from Bristol-Myers Squibb.     

Mer receptor tyrosine kinase inhibition impedes glioblastoma multiforme migration and alters cellular morphology

Glioblastoma multiforme (GBM) is an aggressive brain tumor, fatal within 1 year from diagnosis in most patients despite intensive multimodality therapy. The migratory and microscopically invasive nature of GBM as well as its resistance to chemotherapy renders conventional therapies inadequate in its treatment. Although Mer receptor tyrosine kinase (RTK) inhibition has been shown to decrease the long-term survival and improve the chemosensitivity of GBM in vitro, its role in malignant cellular migration has not been previously evaluated. In this study, we report for the first time a role for Mer RTK in brain tumor migration and show that Mer inhibition profoundly impedes GBM migration and alters cellular morphology. Our data demonstrate that Mer RTK inhibition results in altered signaling through focal adhesion kinase (FAK) and RhoA GTPase and a transformation of cytoskeletal organization, suggesting both molecular and structural mechanisms for the abrogation of migration. We also describe a novel and translational method of Mer RTK inhibition using a newly developed monoclonal antibody, providing proof of principle for future evaluation of Mer-targeted translational therapies in the treatment of GBM. Previous findings implicating Mer signaling in glioblastoma survival and chemotherapy resistance coupled with our discovery of the role of Mer RTK in GBM cellular migration support the development of novel Mer-targeted therapies for this devastating disease.     

抑制Src酪氨酸激酶对非小细胞肺癌细胞VEGF表达的影响

目的:探讨抑制Src酪氨酸激酶对非小细胞肺癌细胞VEGF表达的影响。方法:采用ELISA法检测非小细胞肺癌细胞株培养上清中VEGF表达以及抑制Src酪氨酸激酶对VEGF表达的影响;采用雄性严重联合免疫缺陷(SCID)小鼠,敲除NK细胞,建立非小细胞肺癌细胞诱导的皮下肿瘤和实验性肺转移瘤动物模型,采用免疫组化法研究抑制Src酪氨酸激酶对皮下肿瘤和肺转移瘤中VEGF表达的影响。结果:本实验采用的3种非小细胞肺癌细胞都表达VEGF。0.1μmol/L、0.3μmol/L、1μmol/L和3μmol/L Src酪氨酸激酶抑制剂对H226细胞VEGF表达的抑制率分别为18%、8%、24%和47%,对A549细胞VEGF表达的抑制率分别为14%、23%、50%和43%,对PC-9细胞VEGF表达的抑制率分别为25%、56%、51%和71%。抑制Src酪氨酸激酶明显抑制VEGF在肺转移瘤和皮下肿瘤中的表达。结论:抑制Src酪氨酸激酶能够抑制非小细胞肺癌细胞体外和体内VEGF的表达。     

抑制Src酪氨酸激酶对非小细胞肺癌细胞VEGF表达的影响

目的：探讨抑制Src酪氨酸激酶对非小细胞肺癌细胞VEGF表达的影响。方法：采用ELISA法检测非小细胞肺癌细胞株培养上清中VEGF表达以及抑制Src酪氨酸激酶对VEGF表达的影响;采用雄性严重联合免疫缺陷（SCID）小鼠,敲除NK细胞,建立非小细胞肺癌细胞诱导的皮下肿瘤和实验性肺转移瘤动物模型,采用免疫组化法研究抑制Src酪氨酸激酶对皮下肿瘤和肺转移瘤中VEGF表达的影响。结果：本实验采用的3种非小细胞肺癌细胞都表达VEGF。0.1μmol/L、0.3μmol/L、1μmol/L和3μmol/L Src酪氨酸激酶抑制剂对H226细胞VEGF表达的抑制率分别为18%、8%、24%和47%,对A549细胞VEGF表达的抑制率分别为14%、23%、50%和43%,对PC-9细胞VEGF表达的抑制率分别为25%、56%、51%和71%。抑制Src酪氨酸激酶明显抑制VEGF在肺转移瘤和皮下肿瘤中的表达。结论：抑制Src酪氨酸激酶能够抑制非小细胞肺癌细胞体外和体内VEGF的表达。     

CEP-751 inhibits trk receptor tyrosine kinase activity in vitro and exhibits anti-tumor activity

The present report describes the in vitro and in vivo profile of CEP-751, a novel receptor tyrosine kinase inhibitor. CEP-751 at 100 nM inhibits the receptor tyrosine kinase activity of the neurotrophin receptors trkA, trkB and trkC. CEP-751 has no effect on activity of receptors for EGF, IGF-I, insulin or on erbB2; inhibition of receptors for PDGF and bFGF was observed but occurred with lesser potency than inhibition of trk. CEP-751 exhibited anti-tumor efficacy against tumors derived from NIH3T3 cells transfected with trkA. Inhibition of trk phosphorylation could also be measured in these tumors, suggesting that anti-tumor efficacy of CEP-751 is related to inhibition of trk receptor tyrosine kinase activity. CEP-751 was found to be without effect when administered to nude mice bearing SK-OV-3 tumors, which overexpress erbB2 receptors, providing further evidence that inhibition of tumor growth may be related to inhibition of trk receptor tyrosine kinase activity. Our data indicate that CEP-751 is a potent trk inhibitor which possesses anti-tumor activity. Int. J. Cancer 72:673–679, 1997. © 1997 Wiley-Liss, Inc.     

N-甲基-N＇-硝基-N-亚硝胍抑制细胞表皮生长因子受体酪氨酸激酶活性

目的：研究低浓度N-甲基-N′-硝基-N-亚硝胍（MNNG）诱发细胞表皮生长因子受体（EGFR）形成的二聚体,干扰了EGFR介导的下游信号转导功能的分子机制。方法：构建EGFR胞内域真核表达的载体,转染Lec-1细胞,纯化EGFR胞内域重组蛋白质后,分别以不同浓度的MNNG（0.25、0.5和1.0μmol/L MNNG）处理EGFR1h,应用酶联免疫吸附测定法检测MNNG各浓度对重组EGFR胞内域蛋白质酪氨酸激酶活性的影响。结果：MNNG浓度大于0.5μmol/L即可显著下调EGFR胞内域酪氨酸活性（P〈0.01）。结论：MNNG通过抑制EGFR胞内域的酪氨酸激酶活性而阻断其对下游信号的传递。     

Vertical VEGF targeting: A combination of ligand blockade with receptor tyrosine kinase inhibition

The aim of this study was to examine the anti-tumour effects of dual vertical VEGF targeting consisting in the association between bevacizumab, a VEGF-depleting drug, and the VEGF receptor antityrosine kinase AZD2171. Mice bearing human head and neck CAL33 xenografted tumours were treated once daily for 11 d with either vehicle (controls), AZD2171 (2.5mg/kg/day, p.o.), bevacizumab (5mg/kg/day, i.p.) or the bevacizumab-AZD2171 combination. The AZD2171-bevacizumab combination produced additive effects on tumour growth and reduced the number of proliferating cells relative to control. Bevacizumab did not influence tumour vascular necrosis whilst AZD2171 (p=0.01) and the combination (p=0.01) increased it. The number of mature tumour cells decreased significantly with the combination treatment only (p=0.001), which induced the largest increase in the Bax/Bcl2 ratio (up to 25-fold) and a progressive 3-fold decrease in HIF1-alpha expression between 24h and 192h. The present data indicate that there is no redundancy in targeting the same angiogenic pathway with the presently tested clinically applicable drugs. The study provides a strong rationale for future clinical trials.     

Epidermal growth factor receptor tyrosine kinase inhibition augments a murine model of pulmonary fibrosis

The inappropriate regeneration of sequentially injured epithelium is an important process leading to pulmonary fibrosis. Previous studies have shown that the epithelial expression of epidermal growth factor receptor (EGFR) is increased in fibrotic lung tissue, compared with normal lung tissue, suggesting that EGFR-mediated signaling is involved in epithelial regeneration in fibrotic lung diseases. We examined the effect of EGFR inhibition using ZD1839, a selective EGFR tyrosine kinase inhibitor (TKI), on bleomycin-induced pulmonary fibrosis in mice. ICR mice were administered a single intratracheal injection of bleomycin (5 units/kg) on day 1. ZD1839 (200 mg/kg) or vehicle alone were administered p.o. 1 h before this injection and on days 1-5 each week for 3 weeks. Lung tissue was harvested on day 21. Lung histology and collagen analysis performed on day 21 showed more severe fibrosis in the mice receiving both bleomycin and the EGFR-TKI than in the mice receiving bleomycin and the vehicle. An immunohistochemistry analysis showed that phosphorylated EGFR and proliferation cell nuclear antigen were highly expressed by the regenerated epithelial cells in the mice treated with bleomycin and the vehicle. In contrast, the expression of these antigens was attenuated in the mice treated with bleomycin and the EGFR-TKI. In vitro studies also demonstrated that the addition of ZD1839 at a concentration of < or =1 microM suppressed the proliferation of type II-like epithelial cells (A549) but not that of lung fibroblasts (IMR90). These results suggest that the inhibition of EGFR phosphorylation augments bleomycin-induced pulmonary fibrosis by reducing regenerative epithelial proliferation. Our data suggest that EGFR-TKIs should be used with caution in cancer patients with pulmonary fibrosis.     

Simultaneous targeting of Src kinase and receptor tyrosine kinase results in synergistic inhibition of renal cell carcinoma proliferation and migration

There have been recent improvements in the treatment for metastatic renal cell carcinoma (RCC) with receptor tyrosine kinase (RTK) inhibitors being one of newer treatment options. We hypothesized that simultaneous targeting of Src kinase and the RTK may have synergistic effects to further improve therapies on metastatic RCC. The effects of Src kinase inhibitor saracatinib and multiple RTK inhibitor sunitinib on RCC cell line (ACHN) and Caki-1 were studied. Saracatinib alone or in combination with sunitinib inhibited the migration of ACHN and Caki-1 cells in vitro. Activation of migration related components FAK, P130Cas and Paxillin were blocked by saracatinib at 0.05- to 3-μM concentrations. Combined treatment resulted in improved growth inhibition, greater loss of the S phase cell population and decreased clonogenic colony formation compared to sunitinib alone in the metastatic Caki-1 line. Molecular studies in Caki-1 showed that saracatinib alone and in combination with sunitinib inhibited phosphorylation of the cell progression regulator c-Myc in a dose-dependent manner. Sunitinib alone or in combination suppressed cyclin-D1 expression with the combination showing greater dose-dependent effect. Sunitinib inhibited vascular endothelial growth factor (VEGF) secretion through the inhibition of STAT3 signaling and VEGF biosynthesis. HIF1-α expression in normoxic and hypoxic conditions in Caki-1 cells was inhibited by either saracatinib or sunitinib when administered alone, however, a greater reduction occurred when these compounds were given in combination. Targeting Src kinase and RTK simultaneously with saracatinib and sunitinib resulted in 70-80% blockade of RCC cell migration, synergistic inhibition of cell growth and reduction of acquired drug resistance in Caki-1 cells. The results show promise for combination targeted therapy of RCC.     

Multitargeted receptor tyrosine kinase inhibition: an antiangiogenic strategy in non-small cell lung cancer

In the United States, the leading cause of cancer-related deaths is lung cancer, of which more than 85% of cases are categorized as non-small cell lung cancer. The process of angiogenesis, which results in the formation of vasculature, is a complex and coordinated process that is required for cancer growth and metastasis. Pathways that promote angiogenesis have been targeted as a therapeutic approach in multiple types of cancer, including non-small cell lung cancer. Of these, the vascular endothelial growth factor pathway has been the most well studied, but more recently, the platelet-derived growth factor and fibroblast growth factor pathways have been identified as regulators of angiogenesis and potential mediators of resistance to vascular endothelial growth factor inhibition. Bevacizumab, a monoclonal antibody that binds to vascular endothelial growth factor, is currently the only antiangiogenic drug approved for the treatment of non-small cell lung cancer; however, several tyrosine kinase inhibitors that target vascular endothelial growth factor receptors as well as platelet-derived growth factor receptors and/or fibroblast growth factor receptors are being developed. This article reviews the role of the fibroblast growth factor and platelet-derived growth factor pathways in angiogenesis and provides a summary of dual (e.g., sorafenib, sunitinib) and triple (e.g., BIBF 1120, pazopanib) antiangiogenic tyrosine kinase inhibitors currently in development for the treatment of non-small cell lung cancer.     

Molecular mechanisms of cardiotoxicity of tyrosine kinase inhibition

Cancer therapy has progressed remarkably in recent years. In no area has this been more apparent than in the development of "targeted therapies", particularly those using drugs that inhibit the activity of certain tyrosine kinases, activating mutations or amplifications of which are causal, or strongly contributory, to tumorigenesis. However, some of these therapies have been associated with toxicity to the heart. Here we summarize what is known about the cardiotoxicity of cancer drugs that target tyrosine kinases. We focus on basic mechanisms through which interruption of specific signalling pathways leads to cardiomyocyte dysfunction and/or death, and contrast this with therapeutic responses in cancer cells.     

Prognostic relevance of receptor tyrosine kinase expression in breast cancer: A meta-analysis

Background

Receptor tyrosine kinases (RTKs) may facilitate tumor progression if activated aberrantly. The prognostic impact of human epidermal growth factor receptor 2 (HER2) overexpression and effectiveness of its therapeutic targeting is well established, but the effects on prognosis of overexpression of other RTKs is unknown. Here we evaluate the association of RTK expression and survival in breast cancer.

Methods

PubMed was searched to identify studies evaluating the association between expression of RTKs other than HER2 and survival of women with breast cancer. Published data were extracted and computed into odds ratios (OR) for death at 5 years with 95% confidence intervals (CI). Data were pooled in a meta-analysis using the Mantel–Haenszel random-effect model. For studies reporting data for more than one RTK the lowest and highest OR were used for separate analyses.

Results

Sixteen studies comprising 11,056 patients were included in the analysis. There was an association between overexpression of RTKs and decreased 5-year OS and this was highly significant when using highest ORs from studies reporting more than one RTK (OR = 2.42; 95% CI = 1.92–3.06, P < 0.001). Similar results were observed for 5-year BCSS. Worse OS was seen with overexpression of fibroblast growth factor receptor 2/3 (FGFR) (OR = 3.81; 95% CI = 1.79–8.11) and epidermal growth factor receptor (EGFR)/HER1 (OR = 2.45; 95% CI = 1.90–3.15).

Conclusion

Overexpression of various RTKs is associated with poor outcomes. This data suggests the clinical evaluation of combination of agents against RTKs or relevant oncogenic nodes.     

mTOR kinase inhibition causes feedback-dependent biphasic regulation of AKT signaling

mTOR kinase inhibitors block mTORC1 and mTORC2 and thus do not cause the mTORC2 activation of AKT observed with rapamycin. We now show, however, that these drugs have a biphasic effect on AKT. Inhibition of mTORC2 leads to AKT serine 473 (S473) dephosphorylation and a rapid but transient inhibition of AKT T308 phosphorylation and AKT signaling. However, inhibition of mTOR kinase also relieves feedback inhibition of receptor tyrosine kinases (RTK), leading to subsequent phosphoinositide 3-kinase activation and rephosphorylation of AKT T308 sufficient to reactivate AKT activity and signaling. Thus, catalytic inhibition of mTOR kinase leads to a new steady state characterized by profound suppression of mTORC1 and accumulation of activated AKT phosphorylated on T308, but not S473. Combined inhibition of mTOR kinase and the induced RTKs fully abolishes AKT signaling and results in substantial cell death and tumor regression in vivo. These findings reveal the adaptive capabilities of oncogenic signaling networks and the limitations of monotherapy for inhibiting feedback-regulated pathways. SIGNIFICANCE: The results of this study show the adaptive capabilities of oncogenic signaling networks, as AKT signaling becomes reactivated through a feedback-induced AKT species phosphorylated on T308 but lacking S473. The addition of RTK inhibitors can prevent this reactivation of AKT signaling and cause profound cell death and tumor regression in vivo, highlighting the possible need for combinatorial approaches to block feedback-regulated pathways.     

Enhanced susceptibility of irradiated tumor vessels to vascular endothelial growth factor receptor tyrosine kinase inhibition

Previous experiments with PTK787/ZK222584, a specific inhibitor of vascular endothelial growth factor receptor (VEGFR) tyrosine kinases, using irradiated human FaDu squamous cell carcinoma in nude mice, suggested that radiation-damaged tumor vessels are more sensitive to VEGFR inhibition. To test this hypothesis, the tumor transplantation site (i.e., the right hind leg of nude mice) was irradiated 10 days before transplantation of FaDu to induce radiation damage in the host tissue. FaDu tumors vascularized by radiation-damaged blood vessels appeared later, grew at a slower rate, and showed more necrosis and a smaller vessel area per central tumor section than controls. PTK787/ZK222584 at a daily dose of 50 mg/kg body weight had no impact on growth of control tumors. In contrast, tumors vascularized by radiation-damaged vessels responded to PTK787/ZK222584 with longer latency and slower growth rate than controls, and a trend toward further increase in necrosis, indicating that irradiated tumor vessels are more susceptible to VEGFR inhibition than unirradiated vessels. Although not proving causality, expression analysis of VEGF and VEGFR2 shows that enhanced sensitivity of irradiated vessels to a specific inhibitor of VEGFR tyrosine kinases correlates with increased expression of the molecular target.     

Enhancement of doxorubicin cytotoxicity of human cancer cells by tyrosine kinase inhibition of insulin receptor and type I IGF receptor

The type I insulin-like growth factor receptor (IGF1R) contributes to cancer cell biology. Disruption of IGF1R signaling alone or in combination with cytotoxic agents has emerged as a new therapeutic strategy. Our laboratory has shown that sequential treatment with doxorubicin (DOX) and anti-IGF1R antibodies significantly enhanced the response to chemotherapy. In this study, we examined whether inhibition of the tyrosine kinase activity of this receptor family would also enhance chemotherapy response. Cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP) inhibited IGF1R and insulin receptor (InsR) kinase activity and downstream activation of ERK1/2 and Akt in MCF-7 and LCC6 cancer cells. PQIP inhibited both monolayer growth and anchorage-independent growth in a dose-dependent manner. PQIP did not induce apoptosis, but rather, PQIP treatment was associated with an increase in autophagy. We examined whether sequential or combination therapy of PQIP with DOX could enhance growth inhibition. PQIP treatment together with DOX or DOX followed by PQIP significantly inhibited anchorage-independent growth in MCF-7 and LCC6 cells compared to single agent alone. In contrast, pre-treatment with PQIP followed by DOX did not enhance the cytotoxicity of DOX in vitro. Furthermore, OSI-906, a PQIP derivative, inhibited IGF-I signaling in LCC6 xenograft tumors in vivo. When given once a week, simultaneous administration of OSI-906 and DOX significantly enhanced the anti-tumor effect of DOX. In summary, these results suggest that timing and duration of the IGF1R/InsR tyrosine kinase inhibitors with chemotherapeutic agents should be evaluated in clinical trials. Long-term disruption of IGF1R/InsR may not be necessary when combined with cytotoxic chemotherapy.