Targeting the epidermal growth factor receptor in non-small cell lung cancer

The epidermal growth factor receptor (EGFR) has been implicated in a multiplicity of cancer-related signal transduction pathways like cellular proliferation, adhesion, migration, neoangiogenesis and apoptosis inhibition, all of them important features of cancerogenesis and tumour progression. The inhibition of this receptor has been discovered as a suitable pharmaceutical intervention aimed at interrupting tumour activity. In cancer, both monoclonal antibodies and small molecules with anti-tyrosine kinase activity have been assessed in several trials with significant efficacy in clinical applications. The current review focuses in particular on the clinical data of EGFR inhibition in non-small cell lung cancer with emphasis on tyrosine kinase inhibition.     

非小细胞肺癌组织化疗前后表皮生长因子受体基因的突变

目的:探讨非小细胞肺癌(non-small cell lung cancer,NSCLC)患者化疗前、后肿瘤组织中表皮生长因子受体(epidermal growth factor receptor,EGFR)基因外显子19和21的突变状况。方法:提取31例NSCLC患者化疗前、后肿瘤组织标本中的基因组DNA,采用巢式PCR技术扩增EGFR基因外显子19和21,并进行测序分析。结果:6例患者化疗前、后EGFR基因发生突变,其中4例为19号外显子发生缺失突变,2例为2l号外显子发生替代突变,且化疗前、后的突变状况一致。女性患者突变率(2/3)高于男性(4/28)(P=0.029),非吸烟者的突变率(4/9)高于吸烟者(2/22)(P=0.043)。结论:NSCLC组织EGFR基因外显子19和21突变在化疗前、后无明显改变。     

Up-regulation of E-cadherin by an anti-epidermal growth factor receptor monoclonal antibody in lung cancer cell lines.

Many human epithelial carcinomas are characterized by the overexpression and constitutive activation of the epidermal growth factor receptor (EGF-R) via an autocrine signaling loop. We have investigated the effects of a ligand-blocking monoclonal antibody (mAb) against the EGF-R LA1 on selected parameters of human lung cancer cell lines (H322 and H661) and normal human bronchial epithelial (NHBE) cells. Using Western blot analysis, we show that H322 and NHBE cell lines express comparable levels of EGF-R/p170erbB-1. The LA1 mAb against EGF-R inhibits growth, induces differentiation to a more epithelial phenotype, reduces the constitutive activation of EGF-R, and upregulates epithelial cadherin glycoprotein expression in H322 and NHBE cells. In contrast, LA1 had no effect on either growth, differentiation, receptor tyrosine phosphorylation, or the expression of adhesion molecules in H661 cells, which is consistent with our finding that this cell line does not express detectable levels of EGF-R. These studies demonstrate that a blocking anti-EGF-R mAb can regulate proliferation, differentiation, and the expression of cell adhesion molecules in human bronchial epithelial cells. Our findings suggest possible therapeutic avenues for the treatment of invasive carcinomas via the blockade of EGF-R with antibodies.     

Erlotinib-induced autophagy in epidermal growth factor receptor mutated non-small cell lung cancer

Purpose

Erlotinib is a commonly used tyrosine kinase inhibitor (TKI) in non-small cell lung cancer (NSCLC). Autophagy is a catabolic process in response to stress and deprivation of nutrients. This study aims to investigate whether autophagy confers acquired resistance to erlotinib treatment in NSCLC.

Methods

Four NSCLC cell lines (HCC827, HCC4006, H358 and H1975) with different epidermal growth factor receptor (EGFR) mutation status (exon 19 deletion, exon 19 deletion, wild-type and L858R/T790M respectively) were selected. MTT assay, crystal violet staining and Annexin-V assay were performed to determine cell viability and apoptosis. Autophagic proteins were detected by Western blot. Acidic vesicular organelle (AVO) formation was determined by acridine orange staining. Autophagy inhibitor (chloroquine) and RNA interference were used to demonstrate the biological effect of erlotinib-induced autophagy.

Results

In line with EGFR mutation status, it was shown that both HCC827 and HCC4006 cells were sensitive to erlotinib, while H358 and H1975 cell lines were resistant. Erlotinib treatment at clinically relevant concentrations induced autophagy (increased LC3II expression, Atg-5/Atg12 conjugation, formation of AVO and p62 degradation) in sensitive NSCLC cell lines, via p53 nuclear translocation, AMPK activation and mTOR suppression. Addition of chloroquine, as an autophagy inhibitor, enhanced erlotinib sensitivity in sensitive cells. Similarly, silencing of Atg5 or Beclin-1 significantly increased sensitivity to erlotinib in both sensitive cell lines. In contrast, there was no induction of autophagy in resistant H358 and H1975 cell lines upon erlotinib exposure.

Conclusions

Erlotinib can induce both apoptosis and autophagy in sensitive NSCLC cell lines with activating EGFR mutation (exon 19 del). Inhibition of autophagy can further enhance sensitivity to erlotinib in EGFR-mutated NSCLC, suggesting that autophagy may serve as a protective mechanism.     

Characterization of epidermal growth factor receptor in primary human non-small cell lung cancer

Membrane preparations from 36 human non-small cell lung cancers were examined for the expression of epidermal growth factor (EGF) receptors, and comparisons were made between tumor types and stage. Eight normal lung membrane preparations were also examined. The concentrations of EGF receptors were assessed by ligand binding studies using 125I-radiolabeled EGF. In two point saturation experiments using 0.3 nM 125I-EGF incubated with membranes from 35 primary lung tumors, a median of 18 fmol/mg of protein (range, 1.1 to 530) was found. This was significantly greater than binding to eight lung membranes: median, 6.1 fmol/mg of protein (range, 1.0 to 14.5) (P less than 0.02). Scatchard binding curves obtained in 21 of the 36 tumors and seven of eight of the normal lung preparations showed high and low affinity sites for EGF receptors on all but two tumor membranes. The dissociation constant of the high affinity sites was similar on tumor and normal lung membranes: range, 0.75 to 30 x 10(-10) M/liter. However, the tumors had a significantly higher concentration of these receptor sites: median, 30.4 fmol/mg of protein versus a median of 6.2 fmol/mg of protein on normal lung membranes (P less than 0.01). Likewise, there were significantly more low affinity sites on tumors: median, 237 fmol/mg compared to 60.2 fmol/mg on normal lung (P less than 0.01). No differences were found in this analysis between tumors of different histological subtypes or clinical stage. It is possible that the high level of expression of high affinity sites on lung tumors could be used as a target for ligand-complexed drugs.     

Update on epidermal growth factor receptor mutations in non-small cell lung cancer.

In 2004, several investigators reported that somatic mutations in the epidermal growth factor receptor gene were associated with clinical responses to erlotinib and gefitinib in patients with non-small cell lung cancer. Since then, multiple groups have examined the biological properties that such mutations confer as well as the clinical relevance of these mutations in patients with non-small cell lung cancer. Although a tremendous amount of knowledge has been gained in the past 2 years, there remain a number of important epidemiologic, biological, and clinical questions.     

Second-generation epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer

Inhibiting epidermal growth factor receptor (EGFR) signaling has proven to be an effective strategy for treating non-small cell lung cancer (NSCLC) patients and the first generation of agents developed for this purpose, gefitinib and erlotinib, stimulated a unique escalation in both biologic and clinical research within the field. Second-generation EGFR-targeted agents that aim to further improve patient outcomes are now in preclinical and clinical trials. This review discusses four promising agents that are currently being studied in NSCLC: EKB-569, HKI-272, CI-1033, and ZD6474.     

表皮生长因子受体与非小细胞肺癌预后之间的关系

目的探讨细胞膜及细胞核内表皮生长因子受体(EGFR)水平与非小细胞肺癌(NSCLC)预后之间的关系。方法选取2010年4月至2013年4月间南京市溧水区人民医院保存的60例非小细胞肺癌患者的病理档案,采用免疫组化的方法分析其病理组织切片中细胞膜及细胞核内EGFR及细胞周期蛋白D1(Cyclin D1)的表达。结果 39例(65.0%)样本细胞膜EGFR呈阳性,7例(11.7%)样本细胞膜EGFR呈强阳性;18例(30.0%)样本细胞核EGFR呈阳性和5例(8.3%)样本细胞核EGFR呈强阳性。Kaplan-Meier生存分析和Log-rank检验发现非小细胞肺癌患者细胞膜EGFR强阳性与总体生存率(OS)无明显相关,差异无统计学意义(P>0.05),细胞核EGFR强阳性组与总体生存率(OS)呈负相关,差异有统计学意义(P<0.01)。细胞核EGFR表达水平与Cyclin D1之间正相关,差异有统计学意义(P<0.05),细胞膜EGFR表达水平与Cyclin D1之间无明显相关,差异无统计学意义(P>0.05)。结论细胞核内EGFR水平与非小细胞肺癌患者的预后相关,核内EGFR强阳性者预后差。     

Epidermal growth factor receptor monoclonal antibodies inhibit the growth of lung cancer cell lines.

The ability of monoclonal antibody (MAb 108), an immunoglobulin G (IgG)2a against the epidermal growth factor receptor (EGF-R), to interact with lung cancer cell lines was investigated. 125I-EGF bound with high affinity to non-small-cell lung cancer (NSCLC) cells, and MAb 108 inhibited specific binding of nine NSCLC cell lines in a dose-dependent manner (IC50 = 0.3-3 micrograms per ml). 125I-MAb 108 bound with high affinity (kd = 2 nM) to a single class of sites (Bmax = 70,000 per cell) using NSCLC neuroendocrine cell line NCI-H460. Specific 125I-MAb 108 binding was inhibited with high affinity by MAb 108 but not by a control antibody IgG using large-cell carcinoma cell line NCI-H1299. 125I-MAb 108 binding was not internalized at 37 degrees C using NSCLC neuroendocrine cell line NCI-H460 and adenocarcinoma cell line NCI-H23. Also, 1 microgram per ml of MAb 108 but not of a control IgG inhibited the clonal growth of NCI-H23 and squamous cell carcinoma cell line NCI-H157 in vitro. Also, MAb 108 inhibited xenograft formation of cell lines NCI-H460, NCI-H157, and NCI-H727 in nude mice in vivo. After a palpable tumor had formed using NCI-H460 cells, injection of 100 micrograms of MAb 108 (intraperitoneally three times weekly) inhibited xenograft volume in nude mice by approximately 50%. These data suggest that MAb 108 may interact with EGF receptors on lung cancer cell lines and inhibit NSCLC proliferation.     

Gene delivery by an epidermal growth factor/DNA polyplex to small cell lung cancer cell lines expressing low levels of epidermal growth factor receptor

In the present study, we wanted to determine whether efficient gene delivery using an epidermal growth factor (EGF)/DNA polyplex could be accomplished in small cell lung cancer (SCLC) cell lines expressing low EGF receptor (EGFR) levels. EGFR expression levels and transduction efficiencies with polyplexes were examined in five SCLC cell lines and two controls. EGFR expression was examined by binding assays and demonstrated low EGFR levels ranging from 3.6 to 87.4 fmol/mg protein. The SCLC cell lines exhibited high sensitivity to adenovirus infection, which was an important determinant for transduction efficiency when adenovirus was used as an endosomolytic agent. The transduction efficiencies with EGF/DNA polyplexes ranged from 41% +/- 3.5% to 73% +/- 4.6% in the EGFR-positive SCLC cell lines. In the controls lacking EGFRs, only 5% +/- 1.0% and 8% +/- 1.8% of the cells were transduced. Furthermore, the transduction efficiency could be reduced from 50% +/- 4.9% to 18% +/- 1.1% when excess EGF was added to compete with the EGF/DNA polyplexes. In the present study, receptor-targeted gene delivery to SCLC cell lines has been demonstrated for the first time. Our results indicate that even low receptor expression levels in the target cells are sufficient for efficient and specific in vitro gene delivery with EGF/DNA polyplexes.     

EGFR在非小细胞肺癌中的表达及临床意义

目的：探讨表皮生长因子受体（EGFR）在非小细胞肺癌（NSCLC）中的表达及其与NSCLC发生、发展及预后的关系,指导非小细胞肺癌的靶向治疗。方法：回顾性分析82例NSCLC手术病例临床病理资料及随访资料,用免疫组化（EnVision法）的方法观察EGFR在非小细胞肺癌组织中的表达,对比分析20例非恶性肺组织中EGFR的表达。结果：EGFR在NSCLC中的表达率为53.66%,在非恶性肺组织中EGFR均呈阴性表达;EGFR在NSCLC中的表达与患者的性别、病理类型、TNM分期及淋巴结转移有显著统计学意义（P〈0.05）;EGFR高表达者生存期短。结论：EGFR在NSCLC中高表达;女性患者高于男性,并且与患者的病理类型、TNM分期及淋巴结转移有密切的关系,可作为判断NSCLC患者病情进展及预后的重要指标,还可以指导NSCLC患者的靶向治疗。     

miR-134靶向EGFR对非小细胞肺癌增殖的影响

目的 探讨微小RNA-134（miR-134）对非小细胞肺癌（NSCLC）细胞增殖和凋亡的影响及可能的机制。方法 采用实时荧光定量PCR（QPCR）检测miR 134在NSCLC细胞株（A549、H252）和正常胚肺细胞株WI38中的表达情况;将A549和H252细胞分为3组,分别为空白对照组（不转染）、miR-NC组（转染不相关siRNA）和miR-134组（转染miR-134 mimics）。分别于转染后24、48、72、96h收集细胞,采用MTS法检测细胞的增殖情况;转染后96h用流式细胞仪检测细胞的凋亡情况;双荧光素酶报告基因实验检测miR-134与表皮生长因子受体（EGFR）3’UTR的结合情况,QPCR检测过表达miR-134的A549和H252细胞中EGFR的表达情况。结果 与WI38 细胞相比,miR-134在A549细胞中的表达下调85.91％,在H252细胞中下调78.13％（P＜0.05）。MTS检测显示,miR-134能显著降低A549和H252细胞的增殖能力,并呈时间依赖性。流式细胞仪检测显示,与空白对照组比较,miR-134组A549细胞的凋亡比例提高226.31％,H252细胞提高47.85％（P＜0.05）。双荧光素酶报告基因实验显示miR 134能与EGFR3’UTR结合,显著降低荧光值（P＜0.05）。QPCR检测显示,与空白对照组比较,转染miR-134 mimics 后,EGFR在A549细胞中的相对表达量下调57.0％,在H252中下调35.0％,差异均有统计学意义（P＜0.01）。结论 miR-134在NSCLC中低表达,能通过靶向EGFR抑制NSCLC细胞增殖,并诱导凋亡。     

Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines.

Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data demonstrated that the cells bound between 3 and 52 fmol/mg protein with a KD ranging from 0.5 x 10(-10) to 2.7 x 10(-10) M. EGF binding to the receptor was confirmed by affinity-labeling EGF to the EGF receptor. The cross-linked complex had a M(r) of 170,000-180,000. Northern blotting showed the expression of EGF receptor mRNA in all 10 cell lines that were found to be EGF receptor-positive and in one cell line that was found to be EGF receptor-negative in the radioreceptor assay and affinity labeling. Our results provide, for the first time, evidence that a large proportion of a broad panel of small cell lung cancer cell lines express the EGF receptor.     

EGFR在女性非小细胞肺癌中的表达及临床意义

背景与目的：近年来女性肺癌发病率的明显增加引起人们的关注.本研究主要是以女性非小细胞肺癌为研究对象,探讨表皮生长因子受体（epidermal growth fator receptor,EGFR）在女性非小细胞肺癌（non-small cell lung cancer,NSCLC）中的表达及其与女性NSCLC发生、发展及预后的关系,指导女性非小细胞肺癌的靶向治疗.方法：回顾性分析62例女性非小细胞肺癌手术病例临床病理资料及随访资料,用免疫组化的方法检测EGFR在女性非小细胞肺癌组织中的表达,对比分析10例非恶性肺组织中EGFR的表达,用Cox模型进行生存分析.结果：EGFR在62例女性NSCLC中的表达率70.97%,在非恶性肺组织中均呈阴性表达,两者比较差异有显著性（P＜0.05）;EGFR在女性腺癌的表达高于鳞癌（P＜0.05）,尤其在细支气管肺泡癌中的表达更高（P＜0.05）;EGFR表达与女性NSCLC的TNM分期及淋巴结转移呈显著正相关（P＜0.05）;EGFR高表达的女性患者生存期短、预后差（P＜0.05）;Cox比例风险模型分析表明,患者术后生存时间与病理类型、淋巴结是否转移显著相关（P＜0.05）,腺癌、有淋巴结转移是患者独立的不良预后因素.结论：女性NSCLC中EGFR的表达与患者病理类型、TNM分期及淋巴结转移有密切的关系,EGFR在女性NSCLC中高表达,可作为判断女性NSCLC病情进展及预后的重要指标,还对NSCLC女性患者的靶向治疗的选择有一定的指导意义.     

Interleukin-8 stimulates cell proliferation in non-small cell lung cancer through epidermal growth factor receptor transactivation

Interleukin-8 (IL-8; CXCL8) is a cytokine of the CXC chemokine family that is involved in neutrophil recruitment and activation. In addition, IL-8 has been implicated in a wide variety of other processes, including angiogenesis and metastasis in lung cancer. Lung adenocarcinoma and muco-epidermoid carcinoma cells produce substantial amounts of IL-8, and express both CXCR1 and CXCR2 IL-8 receptors. We hypothesized that IL-8 stimulates proliferation of non-small cell lung cancer cells, involving transactivation of the epidermal growth factor receptor (EGFR). The EGFR plays a central role in regulating cell proliferation and it has been therefore implicated in lung cancer. Both EGFR ligands and transactivation of the receptor may lead to downstream signalling events, including mitogen-activated protein kinase (MAPK) activation. Transactivation of the EGFR has been shown to occur in response to ligands of various G-protein coupled receptors (GPCRs) and involves metalloproteinase-mediated release of membrane bound EGFR ligands. The aim of the present study was to investigate the effect of IL-8 on proliferation of lung adenocarcinoma and muco-epidermoid carcinoma cells, and to explore the mechanisms leading to this proliferation in two different non-small cell lung cancer cell lines (A549 and NCI-H292). In both NSCLC cell lines, we observed that IL-8 stimulates epithelial cell proliferation in a dose-dependent manner. The ability of IL-8 to increase cell proliferation was blocked both by an inhibitor of EGFR tyrosine kinase, by a specific anti-EGFR blocking antibody and by a panmetalloproteinase inhibitor. Similar results were obtained using the GPCR inhibitor pertussis toxin. Inhibition of the MAPK p42/44 (ERK1/2) also blocked the mitogenic effect of IL-8, while a p38 MAPK inhibitor did not affect IL-8-induced cell proliferation. These results suggest that IL-8 increases cell proliferation in NSCLC cell lines via transactivation of the EGFR and that this mechanism involves metalloproteinase activity.     

Tumor epidermal growth factor receptor genotype and depression in stage IV non-small cell lung cancer

Introduction.

Depression appears to be associated with worse survival from cancer, but underlying mechanisms for this association are unknown. In the present study, we explored the degree to which tumor genotype may be associated with depression in patients with non-small cell lung cancer (NSCLC). We examined differences in depression severity and rates of positive screens for major depressive disorder among newly diagnosed patients with stage IV NSCLC and known epidermal growth factor receptor (EGFR) genotype.

Methods.

Newly diagnosed patients (n = 53) with metastatic NSCLC attending an initial thoracic oncology consultation completed self-report questionnaires regarding demographics, smoking behavior, and depression before meeting with their oncologist. Biopsy samples were subsequently genotyped, including screening for EGFR mutations. We conducted a retrospective chart review to obtain clinical data, including tumor stage, performance status, and EGFR genotype.

Results.

Twelve patients (22.6%) tested positive for EGFR mutation. No EGFR mutation–positive cases met the screening criteria for major depressive disorder, in comparison with 29.3% of patients with wild-type EGFR (p = .03). Mutations of EGFR were also associated with lower depression severity than with wild-type EGFR, independent of gender, performance status, and smoking history (p < .05). This finding persisted for both the cognitive–affective and somatic domains of depression symptoms.

Conclusions.

EGFR mutations were associated with lower depression severity and lower rates of probable major depressive disorder in patients with metastatic NSCLC, based on mood screening performed before results of genotyping were known. Findings support further work to explore the directionality of the associations and potential biological pathways to depression.     

Radioimmunodetection of human glioma xenografts by monoclonal antibody to epidermal growth factor receptor

Murine IgG2a monoclonal antibody (MAb) 425 specifically detects epidermal growth factor receptor, which is expressed on human gliomas and tumors of other tissue origin but rarely on normal brain tissues, and not at all on bone marrow and peripheral blood cells. 131I-labeled F(ab')2 fragments of this MAb injected into nude mice grafted with U-87 MG glioma cells preferentially localized in tumor tissue compared to normal mouse tissues, as determined by differential tissue counting of radioactivity. The mean tumor-to-tissue ratios of radioactivity ranged between 8.2 (blood) and 55.8 (muscle) at 2 days after the injection of 15 muCi of 131I-425 F(ab')2/mouse. Radiolabeled fragments of an anti-hepatitis virus IgG2a MAb did not localize in tumors. The localization index derived from the ratios of specific antibody to indifferent antibody in tumor tissue relative to blood was 9.94 at 2 days following the MAb injection. The labeled MAb did not localize in a xenograft of colorectal cancer tumor, which does not express the epidermal growth factor receptor. Tumors could be located by whole-body gamma-scintigraphy without background subtraction following the injection of 100 muCi of radiolabeled MAb 425 F(ab')2 fragments. The data suggest that MAb 425 is a likely candidate for clinical diagnostic and radioimmunotherapy trials.