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PubMed2006

EGFR and erbB2 mutation status in Japanese lung cancer patients.

Much evidence has accumulated that the epidermal growth factor receptor (EGFR) and its family members are strongly implicated in the development and progression of lung cancers. Somatic mutations of the EGFR gene were found in about 25-40% of Japanese lung cancer patients. More recently, erbB2 mutations are found in about 4% of European-derived lung cancer patients. We have investigated EGFR and erbB2 mutation status in 95 surgically treated nonsmall cell lung cancer (NSCLC) cases from Nagoya City University Hospital. Seventy-five adenocarcinoma cases were included. The presence or absence of EGFR and ernB2 mutations of kinase domains were analyzed by reverse transcription polymerase chain reaction (RT-PCR) amplifications and direct sequences. We have also investigated erbB2 mutation status in 27 surgically treated NSCLC cases followed by treatment with gefitinib from Kinki-chuo Chest Medical Center. EGFR mutations (CTG-->CGG; L858R) were found from 14 of 95 lung cancer patients. We also detected the deletion 1a-type mutations from 9 patients and deletion 4-type mutations from 6 patients in exon 19. In exon 20, 4 mutations including 2 novel mutations were found. Total EGFR mutations were present in 35 patients (36.8%). These mutation statuses were significantly correlated with gender (women 73.3% vs. men 20%, p < 0.0001), smoking status (never smoker 69.4% vs. smoker 16.9%, p < 0.0001), pathologic subtypes (adenocarcinoma 45.1% vs. nonadenocarcinoma 12.5%, p = 0.0089) and differentiation status of the lung cancers (well 51% vs. moderately or poorly 18.4%, p = 0.0021). On the other hand, erbB2 mutation was only found from 1 of 95 patients, at exon 20. This patient was female and a never smoker with adenocarcinoma. This 12 nucleotide insertion mutation (2324-2325 ins ATACGTGATGGC) was located in the exon 20 at kinase domain (775-776 ins YVMA). There was no erbB2 mutation in 27 gefitinib-treated NSCLC patients. In total, we have found only 1 erbB2 mutation from 122 (0.8%) Japanese NSCLC patients. There was a significantly higher erbB2 positive (2+/3+) ratio in EGFR mutant patients (13/25, 52.0%) compared to EGFR wild-type patients (10/62, 16.1%; p = 0.0247). The NSCLC specimen with erbB2 mutation showed 1+ immunoreactivity. The EGFR mutation status might correlate with the clinicopathologic features related to good response to gefitinib, such as gender, smoking history and pathologic subtypes of lung cancers. However, erbB2 mutation is rare from Japanese lung cancer and is of limited value for molecular target therapy.

Aged↗
PubMed2006

Frequent EGFR mutations in brain metastases of lung adenocarcinoma.

Lung adenocarcinomas often metastasize to the brain, and the prognosis of patients with brain metastases is still very poor. The epidermal growth factor receptor (EGFR) gene is mutated in a considerable fraction of primary lung adenocarcinomas, in particular those with drastic response to EGFR tyrosine kinase inhibitors. The present study was designed to elucidate the prevalence of EGFR mutations in brain metastases and the timing of their occurrence during cancer progression. EGFR mutations were detected in 12 of 19 metastatic lung adenocarcinomas to the brain (63%). This frequency was higher than those in previous studies for EGFR mutations at various stages of lung adenocarcinoma in East Asia, including Japan (i.e., 20-55%). In 6 cases with EGFR mutations, the corresponding primary lung tumors were also examined for the mutations, and in all of them, the same types of EGFR mutations were detected also in the primary tumors. In 2 of them, second metastatic brain tumors in addition to the first ones were also available for analysis, and the same types of EGFR mutations were detected in both the first and second ones in both cases. These results indicate that EGFR mutations are present frequently in brain metastases and occur preceding brain metastasis. These findings will be highly informative for treatment of metastatic lung adenocarcinoma to the brain.

Adenocarcinoma↗
PubMed2006

Usefulness of EGFR mutation screening in pleural fluid to predict the clinical outcome of gefitinib treated patients with lung cancer.

The importance of epidermal growth factor receptor (EGFR) gene mutation has been recognized in nonsmall cell lung cancer (NSCLC), requiring the standardization of mutation screening system including the kind of samples. Here, we examined the EGFR mutation status in 61 pleural fluid samples from NSCLC cases using direct sequencing, nonenriched PCR, mutant-enriched PCR and peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp assay. The mutant-enriched PCR assay detected 16 mutant cases. Among them, the nonenriched PCR assay failed to detect 3 mutant cases. Regarding the discrepancy between mutant-enriched PCR and PNA-LNA PCR clamp assays, 3 cases of exon19-deletions were detected only by mutant-enriched PCR assay and no difference at the L858R mutation. There was no difference in results between direct sequencing and nonenriched PCR assay. We also correlated the EGFR mutation with clinical outcome of gefitinib-treated 29 cases. EGFR mutations were present in 10 cases, revealing 7 partial response and 3 no change (NC). In EGFR wild-type cases, 10 revealed NC and 9 progressive disease. The responders were significantly more frequent among the EGFR mutant cases than among the wild-type (p < 0.0001). Overall survival (p = 0.0092) and progression-free survival (p = 0.018) were significantly longer among the EGFR mutant cases than among the wild-type. In summary, we evaluated the utility of EGFR mutation screening in pleural fluid using 4 assays that showed some discrepancies arising from the designs of the assays. As clinical importance, the EGFR mutation status in pleural fluid can be a biomarker for the favorable outcome of gefitinib-treated NSCLC cases.

Aged↗
PubMed1993

In situ hybridization for epidermal growth factor receptor (EGFR) external domain transcripts in prostatic adenocarcinoma.

We examined prostatic adenocarcinomas from 19 formalin fixed radical prostatectomy specimens for EGFR by in situ hybridization employing a 24 base synthetic biotin-labeled oligonucleotide probe complementary to the 5' end of EGFR mRNA. All slides were examined by light microscopy using a 25x objective. Each field was given three values: 1) Gleason grade (1-5), 2) Nuclear grade [small (< 5.0 mu), intermediate (5-10 mu), large (> 10 mu)], and 3) EGFR staining intensity score (0, absent; 1, weak; 2+, moderate to strong). A total 851 25x fields of prostatic adenocarcinoma were studied. All cancers demonstrated at least some degree of cytoplasmic EGFR message. The EGFR intensity score correlated best with tumor nuclear size. No correlation with Gleason grade was observed. Cytoplasmic staining was also identified in the basal cell layer of benign glands, high grade prostatic intraepithelial neoplasia, stromal nodules, transitional epithelium, periurethral glands, and ganglion cells. Competitive hybridization experiments using an unlabeled EGFR probe showed markedly diminished hybridization signal, while in situ hybridization with a biotin-labeled EGFR sense probe was negative. Immunohistochemistry on 13 of the tumors with 2 monoclonal antibodies against EGFR showed staining in only 1/13 and 10/13 tumors. EGFR expression appears to be most prominent in tumors of high nuclear grade. Further studies will be necessary to explore this growth factor as a prognostic variable in this tumor.

Adenocarcinoma↗
PubMed2001

Comparative genomic sequence analysis and isolation of human and mouse alternative EGFR transcripts encoding truncated receptor isoforms.

This study presents the annotated genomic sequence and exon-intron organization of the human and mouse epidermal growth factor receptor (EGFR) genes located on chromosomes 7p11.2 and 11, respectively. We report that the EGFR gene spans nearly 200 kb and that the full-length 170-kDa EGFR is encoded by 28 exons. In addition, we have identified two human and two mouse alternative EGFR transcripts of 2.4-3.0 kb using both computational and experimental methods. The human 3.0-kb and mouse 2.8-kb EGFR mRNAs are predominantly expressed in placenta and liver, respectively, and both transcripts encode 110-kDa truncated receptor isoforms containing only the extracellular ligand-binding domain. We also have demonstrated that the aberrant 2.8-kb EGFR transcript produced by the human A431 carcinoma cell line is generated by splicing to a recombinant 3'-terminal exon located in EGFR intron 16, which apparently was formed as a result of a chromosomal translocation. Finally, we have shown that the human, mouse, rat, and chicken 1.8- to 3.0-kb alternative EGFR transcripts are generated by distinct splicing mechanisms and that each of these mRNAs contains unique 3' sequences that are not evolutionarily conserved. The presence of truncated receptor isoforms in diverse species suggests that these proteins may have important functional roles in regulating EGFR activity.

Alternative Splicing↗
PubMed2002

Correlation between expression of EGFR and the prognosis of patients with cervical carcinoma.

OBJECTIVE: Overexpression of epidermal growth factor receptor (EGFR) gene has been detected in a large number of human tumors, in most of which the association between overexpression of EGFR and poor prognosis of the disease has been reported. However, the prognostic role of EGFR oncoprotein in cervical carcinoma remains controversial. The current study aimed to determine the prognostic value of EGFR in patients with cervical cancer. METHODS: We measured EGFR oncoprotein with an enzyme-linked immunosorbent assay (ELISA). Results were correlated to clinical data. RESULTS: The presence of measurable levels of EGFR in the tumor was found in all of the explored tumors. The levels varied widely from 31 to 2874 fmol/mg protein with a median at 582 fmol/mg protein, and Q1, Q2, and Q3 quartiles were 156, 562, and 1047, respectively. Overexpression of EGFR was associated with an impaired prognosis with respect to disease-free interval (P = 0.03) and overall survival (P = 0.04). Multivariate analysis revealed that tumor EGFR provided prognostic information with respect to disease-free interval (P = 0.002) and overall survival (P = 0.005) independently of the two established prognosticators, FIGO stage and lesion size. CONCLUSION: Our results are consistent with the concept that EGFR confers prognostic information in addition to that provided by the established clinicopathologic parameters of FIGO stage and lesion size.

Adult↗
PubMed2006

Protocol modifications influence the result of EGF receptor immunodetection by EGFR pharmDx in paraffin-embedded cancer tissues.

EGF receptor (EGFR) became a useful target for several recently introduced therapies of various cancer types including colorectal, lung, head and neck cancers and glioblastoma. The successful clinical application of these novel molecularly targeted therapies requires the expression of their target, EGFR, determined by nucleic acid based or immunohistochemical techniques. However, until now, immunohistochemistry has not become a reliable diagnostic approach for this purpose. The golden standard for the determination of EGFR protein expression in paraffin-embedded cancer tissues is the EGFR pharmDxTM kit. Here we show that the recommended protocol may not be optimal for EGFR immunodetection. Microwave antigen retrieval and extended primary antibody incubation time converted four out of eight EGFR-negative tumors into EGFR-positive in a study of 50 lung adenocarcinoma cases. Accordingly, we recommend retesting cases negative for EGFR with EGFR pharmDxTM using protocol modifications optimizing antigen retrieval and the incubation periods.

Adenocarcinoma↗
PubMed2005

Clinical significance of EGFR amplification and the aberrant EGFRvIII transcript in conventionally treated astrocytic gliomas.

The aim of this study was to evaluate the clinical value of assessing epidermal growth factor receptor (EGFR) amplification and the common 5' rearrangement of EGFR resulting in the EGFRvIII transcript in astrocytic gliomas. Data from 221 tumours were correlated with patient survival. The majority of previous studies evaluated amplification alone and provided contradictory results. Amplification was analysed by a densitometry of Southern blot analysis or quantitative polymerase chain reaction (PCR). EGFR transcripts were examined by reverse transcription PCR and subsequent sequencing. A ribonuclease (RNase) protection assay was carried out on a subgroup to confirm PCR results. Amplification of EGFR was found in 41% (65/160) of glioblastomas (GBs) and 10% (4/41) of anaplastic astrocytomas (AAs). The EGFRvIII rearrangement was identified in 54% (35/65) of GBs and 75% (3/4) of AAs with amplification, as well as in 8% (8/95) of GBs and 5% (2/37) of AAs without amplification (confirmed by RNase protection assay). There were no abnormalities of the EFGR or its transcript in grade II astrocytoma (AII). We found no significant association between EGFR amplification or rearrangement, and age or survival in the 160 GB patients. We noted a tendency towards decreased survival with any EGFR abnormality in the 41 patients with AAs. This was most marked in the five cases with the EGFRvIII transcript (p=0.069), but these were significantly older than those without (p=0.023). No abnormalities of EGFR were identified in AII patients. We conclude that neither EGFR amplification nor the presence of the EGFRvIII transcript predicts patient outcome in conventionally treated GBs. However, in AAs, although uncommon, EGFR aberrations appear to be associated with shorter survival.

Adult↗
PubMed1990

Epidermal growth factor receptor (EGFr) as a marker for poor prognosis in node-negative breast cancer patients: neu and tamoxifen failure.

Analysis of EGFr and ER was performed on tumour samples from 231 patients with operable breast cancer followed for up to 6 yr after surgery. The median duration of follow-up in patients still alive at the time of analysis was 45 months. Thirty-five percent of patients (82) had tumours greater than 10 fmol/mg 125I-EGF binding (EGFr+) and 47% (109) had cystolic ER concentration greater than 5 fmol/mg (ER+), with a marked inverse relationship between EGFr and ER (P less than 0.00001). EGFr was second only to axillary node status as a prognostic marker for all patients both in terms of relapse-free and overall survival (P less than 0.001, logrank EGFr+ vs EGFr-). For patients with histologically negative axillary nodes EGFr was superior to ER in predicting relapse and survival (P less than 0.01 and P less than 0.005, respectively, compared to P less than 0.1 and P less than 0.1, logrank). In a multivariate (Cox model) analysis only EGFr, out of EGFr, ER, size and grade, was predictive for either relapse-free or overall survival for patients with node-negative disease (P = 0.052 and P = 0.026, respectively). The correlation of neu expression with response to tamoxifen in patients with recurrent disease was assessed immunochemically. Response rate was reduced in the presence of neu from 50 to 17% for ER+ cases and from 26 to 0% for ER- cases.

Biomarkers, Tumor↗
PubMed1997

Prognostic significance of c-erbB-2/neu amplification and epidermal growth factor receptor (EGFR) in primary breast cancer and their relation to estradiol receptor (ER) status.

The aim of this study is to evaluate the prognostic significance of c-erbB-2/neu amplification and epidermal growth factor receptor (EGFR) expression in primary breast cancer (BC) and their prognostic implications when combined with estradiol receptor (ER) status. In this work, 825 BCs were studied. Neu amplification was evaluated by dot-blot and EGFR expression was evaluated by ligand binding assay using I125-EGF. Neu, EGFR, estradiol and progesterone receptors (ER and PR) had a marked influence on disease free survival (DFS) in univariate analysis. In node-negative (NO) cases only neu was associated with short DFS (p = 0.005). However, in node-positive (N+) cases both EGFR (p = 0.005) and neu (p = 0.002) influenced DFS. None of the biological markers were significant predictors for overall survival (OS) in NO/BC. On the contrary, in N+/BC, EGFR + (p = 0.003) was associated with short OS. The EGFR + /neu/phenotype represented a sub-group with an even worse prognosis with respect to DFS (p = 0.0034) as well as EGFR + /ER-tumors (p = 0.005). Moreover, neu + /ER-patients also had a high probability of relapse (p = 0.0000) and death (p = 0.006). C-erbB-2/neu, EGFR, histological grade, pN, pT and ER were subjected to a Cox multivariate regression analysis: neu was the most important parameter in predicting recurrence, and EGFR was a significant predictor for OS.

Adult↗
PubMed1998

Epidermal growth factor receptor (EGFR) is not related to the prognosis of cervical cancer.

We analyzed the prognostic significance of epidermal growth factor receptor (EGFR) in a large prospective series of 90 cervical cancer patients observed for a long follow-up period. EGFR levels ranged from 0 to 52.1 fmol/mg protein, with a median value of 6.0 fmol/mg protein. Patients with an advanced stage of disease expressed lower EGFR levels than those with an early stage of disease (median values were 7.8 fmol/mg protein for patients with stage I-II and 4.2 fmol/mg protein for patients with stage III-IV, P = 0.013). There was no correlation between EGFR expression and other clinicopathological parameters analyzed. No significant relationship was shown between EGFR positivity and overall survival. No significant relationship between EGFR status and disease-free survival was observed. Cox univariate regression analysis using EGFR as a continuous variable showed that EGFR levels are not associated with the risk of disease recurrence after treatment or death (P-value not significant). Our data didn't seem to indicate a prognostic role of EGFR in cervical cancer.

Adult↗
PubMed2005

Gene copy number for epidermal growth factor receptor (EGFR) and clinical response to antiEGFR treatment in colorectal cancer: a cohort study.

BACKGROUND: The antiepidermal growth factor receptor (antiEGFR) monoclonal antibodies cetuximab and panitumumab have good clinical activity in about 10% of patients with metastatic colorectal cancer that is resistant to chemotherapy. The molecular mechanisms underlying clinical response or resistance to these agents are unknown. METHODS: Tumours from 31 patients with metastatic colorectal cancer who had either an objective response (n=10) or stable disease or progressive disease (n=21) after treatment with cetuximab or panitumumab were screened for genetic changes in EGFR or its immediate intracellular effectors. Specifically, we assessed the EGFR copy number and the mutation profile of the EGFR catalytic domain and of selected exons in KRAS, BRAF, and PIK3CA. RESULTS: Eight of nine of patients with objective responses who were assessable by fluorescence in-situ hybridisation (FISH) had an increased EGFR copy number. By contrast, one of 21 non-responders assessable by FISH had an increased EGFR copy number (p<0.0001 for responders vs non-responders, Fisher's exact test). The mutation status of the EGFR catalytic domain and its immediate downstream effectors PIK3CA, KRAS, and BRAF did not correlate with disease response. In colorectal-cancer cell lines, the concentration of cetuximab that completely inhibited proliferation of cells with amplified EGFR copy number did not affect proliferation of cells with unamplified EGFR. INTERPRETATION: We propose that the response to antiEGFR treatment has a genetic basis and suggest that patients might be selected for treatment on the basis of EGFR copy number.

Aged↗
PubMed2002

EGFR is a transducer of the urokinase receptor initiated signal that is required for in vivo growth of a human carcinoma.

Urokinase plasminogen activator receptor (uPAR) activates alpha5beta1 integrin and ERK signaling, inducing in vivo proliferation of HEp3 human carcinoma. Here we demonstrate that EGFR mediates the uPAR/integrin/fibronectin (FN) induced growth pathway. Its activation is ligand-independent and does not require high EGFR, but does require high uPAR expression. Only when uPAR level is constitutively elevated does EGFR become alpha5beta1-associated and activated. Domain 1 of uPAR is crucial for EGFR activation, and FAK links integrin and EGFR signaling. Inhibition of EGFR kinase blocks uPAR induced signal to ERK, implicating EGFR as an important effector of the pathway. Disruption of uPAR or EGFR signaling reduces HEp3 proliferation in vivo. These findings unveil a mechanism whereby uPAR subverts ligand-regulated EGFR signaling, providing cancer cells with proliferative advantage.

Blotting, Northern↗
PubMed2004

Growth and EGFR regulation in breast cancer cells by vitamin D and retinoid compounds.

The effect of 1,25-dihydroxyvitamin D(3), analog C (1,25-(OH)(2)-16-en-23-yn-26,27-F(6)-vitamin D(3)), 9-cis retinoic acid, and all-trans retinoic acid on the growth and expression of EGFR in MCF7, T47D, BT474, and BT549 breast cancer cells was examined. Significant growth inhibition was noted in MCF7, T47D, and BT474 cells by 8 days of treatment, while BT549 cells showed none. MCF7, T47D, and BT549 cells treated with 1,25-dihydroxyvitamin D(3) demonstrated a 50% decrease in EGFR mRNA within 2 h which was sustained to 72 h, while BT474 cells demonstrated a 200-500% increase. EGFR protein levels correlated with these mRNA changes in BT474 and BT549 cells. Measurement of mRNA stability in vitamin D treated BT474 cells indicated that there was no change in EGFR mRNA half-life. Transfection of an EGFR promoter containing reporter plasmid demonstrated vitamin D induced changes in reporter gene activity that paralleled the changes observed in EGFR mRNA and protein. Electrophoretic mobility shift assays using a putative vitamin D response element within this region of the EGFR promoter demonstrated specific VDR binding. These results indicate that the vitamin D effect on EGFR expression in breast cancer cells has a transcriptional component likely mediated through a vitamin D responsive promoter sequence. They also suggest that growth inhibition and EGFR down-regulation by vitamin D and retinoids may be related events in some breast cancer cells, but not in all.

Breast Neoplasms↗
PubMed2004

Lack of relationship between EGFR-1 immunohistochemical expression and prognosis in a multicentre clinical trial of 93 patients with advanced primary ovarian epithelial cancer (GINECO group).

Epidermal growth factor receptor 1 (EGFR-1) overexpression is usually described as linked with a worse prognosis in a variety of tumours of epithelial origin. However, its role in ovarian cancer is still controversial. The aim of the present study was to analyse the prognostic impact of EGFR-1 in a retrospective series of 93 stage III-IV primary ovarian epithelial tumours. All patients, enrolled in a multicentre GINECO prospective clinical trial, were treated with the same platinum-based combination chemotherapy, and were followed up with a median of 69 months. Epidermal growth factor receptor 1 plasma membrane expression, assessed by immunohistochemistry on paraffin-embedded tissues, was correlated with clinical parameters as well as immunohistochemical expression results of HER-2 (c-erbB-2), BAX, BCL-2, p53 and anti-Ki-67, previously studied in the same series of patients. Positive immunostaining for EGFR-1 was seen in 31 of the 93 analysed cases (33%). No correlation was found between EGFR-1 expression and clinical parameters. No correlation was found between EGFR-1 expression and other biological markers, except for HER-2, which was limit for significance. Indeed, among the EGFR-1-negative cases, 10.3% expressed HER-2, whereas the HER-2-expressing tumours accounted for 27.6% of EGFR-1-positive cases (P=0.06). Epidermal growth factor receptor 1 overexpression had no prognostic impact on both overall and progression-free survival through univariate and multivariate analyses. The potential effect of EGFR-1 and HER-2 co-expression on targeted therapy against EGFR-1 and/or HER-2 molecules has to be further analysed.

Adult↗
PubMed2005

Luteinising hormone-releasing hormone analogue reverses the cell adhesion profile of EGFR overexpressing DU-145 human prostate carcinoma subline.

Cetrorelix, a luteinising hormone-releasing hormone (LHRH) analogue, has been shown to limit growth of the human androgen-independent prostate cell line DU-145, although other inhibitory actions may also be affected. Both growth and invasion of DU-145 cells are linked to autocrine epidermal growth factor receptor (EGFR) signalling. Invasiveness requires not only cells to migrate to conduits, but also reduced adhesiveness between tumour cells to enable separation from the tumour mass. Thus, we investigated whether Cetrorelix alters the DU-145 cell-cell adhesion and if this occurs via altered EGFR signalling. Pharmacologic levels of Cetrorelix limited the invasiveness of a highly invasive DU-145 subline overexpressing full-length EGFR (DU-145 WT). Extended exposure of the cells to Cetrorelix resulted in increased levels of the cell-cell adhesion complex molecules E-cadherin, alpha- and beta-catenin, and p120. Puromycin blocked the increases in E-cadherin and beta-catenin levels, suggesting that de novo protein synthesis is required. The Cetrorelix effect appears to occur via transmodulation of EGFR by a protein kinase C (PKC)-dependent mechanism, as there were no changes in DU-145 cells expressing EGFR engineered to negate the PKC transattenuation site (DU-145 A654); downregulation of EGFR signalling produced a similar upregulation in adhesion complex proteins, further suggesting a role for autocrine signalling. Cetrorelix increased the cell-cell adhesiveness of DU-145 WT cells to an extent similar to that seen when autocrine EGFR signalling is blocked; as expected, DU-145 A654 cell-cell adhesion also was unaffected by Cetrorelix. The increased adhesiveness is expected as the adhesion complex molecules moved to the cells' periphery. These data offer direct insight into the possible crosstalk pathways between the LHRH and EGFR receptor signalling. The ability of Cetrorelix to downregulate EGFR signalling and subsequently reverse the antiadhesiveness found in metastatic prostate cancer highlights a novel potential target for therapeutic strategies.

Cell Adhesion↗
PubMed2006

Distinctive activation patterns in constitutively active and gefitinib-sensitive EGFR mutants.

Mutations in the kinase domain of epidermal growth factor receptor (EGFR) are associated with clinical responsiveness to gefitinib in patients with non-small-cell lung cancers (NSCLC). Recently, we have identified many novel EGFR mutations in NSCLC tissues. In this study, we found that gefitinib could suppress the tyrosine phosphorylation of most EGFR mutants better than the wild-type receptor. However, gefitinib had quite variable growth-suppressive effects on different EGFR mutant-expressing cells. All tested EGFR mutants have high basal phosphorylation at multiple tyrosine residues. Upon EGF stimulation, the mutated EGFRs did not have apparently stronger phosphorylation at tyrosines 845, 992, 1,068, and 1,173 than the wild-type receptor. However, stronger phosphorylation at tyrosine 1,045 was observed in the S768I, L861Q, E709G, and G719S mutants. The E746-A750 deletion mutant was less responsive to EGF than the wild-type and other mutant receptors. The S768I, L861Q, E709G, and G719S mutants were refractory to EGF-induced ubiquitination and had more sustained tyrosine phosphorylation. E709G and G719S also lacked EGF-induced receptor downregulation. Our results indicate that, in addition to sensitivity to gefitinib, EGFR mutations also caused various changes in EGFR's regulatory mechanisms, which may contribute to the constitutive activation of EGFR mutants and oncogenesis in NSCLC.

Animals↗
PubMed2005

EGFR kinase possesses a broad specificity for ErbB phosphorylation sites, and ligand increases catalytic-centre activity without affecting substrate binding affinity.

We previously found that EGF (epidermal growth factor) increases the EGFR (EGF receptor) kinase-binding affinity towards the major tyrosine phosphorylation sites in downstream adaptor proteins such as Gab1 (Grb2-associated binding protein 1) and Shc [Src homology 2 (SH2) domain and collagen containing protein], but not that towards EGFR autophosphorylation sites [Fan, Wong, Deb and Johnson (2004) J. Biol. Chem. 279 , 38143-38150]. EGFR activation can also result in transphosphorylation of tyrosine resides in the C-terminal region of the related receptors ErbB2, ErbB3 and ErbB4 in heterodimers which are formed upon ligand stimulation. In the present study, we investigated the specificity of EGFR kinase by comparing the steady state kinetic parameters for peptides derived from all four ErbBs in the absence or presence of EGF. Our results demonstrated that (i) EGFR kinase can efficiently phosphorylate a broad range of diverse peptide sequences representing ErbB sites; (ii) certain ErbB2, ErbB3 and ErbB4 sites had higher specificity constants than any EGFR sequence and (iii) EGF stimulation consistently increases the k(cat) approx. 5-fold, but does not significantly alter the K(m) for any ErbB peptides. Furthermore, peptides containing lysine at position -2 or -3 N-terminal to the target tyrosine were found to be poor EGFR kinase substrates, and substitution of these lysines with glutamine decreased the K(m) and increased the k(cat) for these substrates. We conclude that EGFR kinase-mediated ErbB transphosphorylations are mostly controlled at the level of oligomerization, and not by a preference of the EGFR kinase for phosphorylation sites in any particular ErbB. The results also demonstrated that, unlike phosphorylation sites in select downstream targets, EGF does not regulate the recognition of phosphorylation sites in the C-terminal region of any of the ErbBs.

Amino Acid Sequence↗