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PubMed2005

Mutational analysis of EGFR and K-RAS genes in lung adenocarcinomas.

Both epidermal growth factor receptor (EGFR) and RAS gene mutations contribute to the development of non-small cell lung cancer (NSCLC). Because RAS is one of the downstream molecules in the EGFR signal transduction, the association between the somatic mutations of EGFR and RAS may be important in the pathogenesis of NSCLC . However, to date, such data are lacking. In this study, we analyzed the hotspot regions of K-RAS gene (codons 12, 13, 59 and 61) and EGFR gene (exons 18, 19 and 21) in 153 NSCLC tissue samples including 69 adenocarcinomas. Overall, we detected 30 EGFR mutations (19.6%) and 6 K-RAS mutations (3.9%) in the 153 NSCLCs. In the 69 adenocarcinomas, 26 EGFR mutations (37.7%) and six K-RAS mutations (8.7%) were detected. Of note, the 26 tumors with EGFR mutations did not harbor any K-RAS mutations, and the six tumors with K-RAS mutations did not harbor any EGFR mutations. Inverse relationship between K-RAS and EGFR mutations in the lung adenocarcinoma was statistically significant (P=0.046, chi2 test). As regards smoking history, EGFR mutation was significantly associated with never-smoking history, whereas K-RAS mutation was significantly associated with smoking history. Our data suggest that mutations of EGFR and K-RAS genes might separately, but not cooperatively, contribute to lung adenocarcinoma pathogenesis, and that EGFR and K-RAS mutants could separately be anti-neoplastic targets in lung adenocarcinomas.

Adenocarcinoma↗
PubMed2006

EGFR signaling pathway in breast cancers: from traditional signal transduction to direct nuclear translocalization.

Aberrant epidermal growth factor receptor (EGFR) signaling is a major characteristic of many human malignancies including breast cancer. Since the discovery of EGF in 1960's and its receptor in 1980's, our understanding of the EGF/EGFR pathway has been significantly advanced and consequently, EGFR is considered as a major oncogenic factor and an attractive therapeutic target. The well-established traditional function of EGFR is known to transmit extra-cellular mitogenic signals, such as EGF and transforming growth factor-alpha (TGF-alpha), through activating a number of downstream signaling cascades. These include signaling modules that involve phospholipase C-gamma, Ras, and phosphatidylinositol-3 kinase (PI-3K). In cancer cells, the common outcomes following the activation of the EGFR-mediated downstream pathways are altered gene activities, leading to un-controlled tumor proliferation and apoptosis. Interestingly, emerging evidences suggest the existence of a direct mode of the EGFR pathway that is distinct from the traditional transduction pathway. This new mode of EGFR signaling involves cellular transport of EGFR from the cell-surface to the cell nucleus, association of nuclear EGFR complex with gene promoters, and transcriptional regulation of the target genes. Although the nature and pathological consequences of the nuclear EGFR pathway remain elusive, accumulating evidences suggest its association with increased tumor cell proliferation and poor survival rate in breast cancer patients. While several anti-EGFR agents are being tested in breast cancer patients clinically and others under pre-clinical development, a better understanding of the traditional and the nuclear EGFR pathways will facilitate the identification of patients that are likely to respond to these agents as well as future development of more effective anti-EGFR therapeutic interventions.

Breast Neoplasms↗
PubMed2005

Prospective isolation of late development multipotent precursors whose migration is promoted by EGFR.

A simple procedure to isolate neural stem cells would greatly facilitate direct studies of their properties. Here, we exploited the increase in EGF receptor (EGFR) levels, that occurs in late development stem cells or in younger precursors upon exposure to FGF-2, to isolate cells expressing high levels of EGFR (EGFR(high)) from the developing and the adult brain. Independently of age and region of isolation, EGFR(high) cells were highly enriched in multipotent precursors and displayed similar antigenic characteristics, with the exception of GFAP and Lex/SSEA-1 that were mainly expressed in adult EGFR(high) cells. EGFR levels did not correlate with neurogenic potential, indicating that the increase in EGFR expression does not directly affect differentiation. Instead, in the brain, many EGFR(high) precursors showed tangential orientation and, whether isolated from the cortex or striatum, EGFR(high) precursors displayed characteristics of cells originating from the ventral GZ such as expression Dlx and Mash-1 and the ability to generate GABAergic neurons and oligodendrocytes. Moreover, migration of EGFR(high) cells on telencephalic slices required EGFR activity. Thus, the developmentally regulated increase in EGFR levels may affect tangential migration of multipotent precursors. In addition, it can be used as a marker to effectively isolate telencephalic multipotent precursors from embryonic and adult tissue.

Animals↗
PubMed2003

Differential responses by pancreatic carcinoma cell lines to prolonged exposure to Erbitux (IMC-C225) anti-EGFR antibody.

BACKGROUND: Pancreatic cancer remains a devastating disease, with 95% of all patients diagnosed with the disease dying within 2 years. The combined therapy using Erbitux, gemcitabine, and radiation caused complete tumor regression using a nude mouse model inoculated with pancreatic MiaPaCa-2 cells but only a delay in tumor growth with BxPC-3. We investigated the effect of prolonged Erbitux treatment to the sensitivity to gemcitabine and/or radiation and the epidermal growth factor receptor (EGFR) signal transduction pathway. METHODS: MiaPaCa-2 and BxPC-3 cells were cultured with or without Erbitux for 6 weeks. Cells were then treated with gemcitabine and/or radiation, harvested 48 h after treatment, and counted. Differences in EGFR expression after exposure to Erbitux were analyzed by FACS. Internalization rates of EGFR induced by Erbitux on these cell lines were determined using 125I-EGF binding assay after removal of Erbitux by acidic wash. Cell lysates were harvested after cells were stimulated with EGF, FGF, or IGF-1 respectively, and EGFR was immunoprecipitated using Erbitux. Samples were separated using SDS-PAGE and transferred to PVDF membrane. The membranes were probed with antibody against human growth factor receptor binding protein (Grb2) to detect the association of this Ras-MAPK upstream adaptor protein to EGFR. Cell lysates were also separated with SDS-PAGE and probed with rabbit anti-human PARP after samples were transferred to PVDF membrane. Expression of BAX and Bcl-(XL) were probed in the cells treated with or without Erbitux. RESULTS: Proliferation assays indicated that prolonged exposure to Erbitux increased the sensitivities of MiaPaCa-2 to gemcitabine and radiation therapy (41 +/- 16% vs 52 +/- 9% for gemcitation, 28 +/- 9 vs 39 +/- 9% for combination; P = 0.015) but not for BxPC-3. FACS analysis showed that the expressed EGFR level decreased by about 42% on MiaPaCa 2 whereas no loss was seen on BxPC-3. Expression of BAX was upregulated on MiaPaCa-2. Poly (ADP-ribose) polymerase cleavage indicated the killing was mediated by apoptosis. Immunoblots showed that Grb2 was co-immunoprecipitated with EGFR after EGF stimulation. Incubation with Erbitux blocked Grb2 binding in MiaPaCa-2 but not BxPC 3. FGF transactivated EGFR down stream Ras-MAPK in the presence or absence of Erbitux. Internalization of EGFR induced by Erbitux did not differ between MiaPaCa-2 and BxPC-3. CONCLUSIONS: 1) Association of Grb2 to EGFR in BxPC-3 induced by EGF in the presence of Erbitux indicates an alternate pathway of Ras-MAPK activation, which may be related with the tumor resistance to treatment; 2) transactivation of EGFR downstream Ras-MAPK pathway by FGF contributes the resistance to treatment; and 3) downregulation of EGFR may increase the response to therapy.

Adaptor Proteins, Signal Transducing↗
PubMed2006

EGFR mutation status in tumour-derived DNA from pleural effusion fluid is a practical basis for predicting the response to gefitinib.

Epidermal growth factor receptor (EGFR) mutations are strong determinants of tumour response to EGFR tyrosine kinase inhibitors in non-small-cell lung cancer (NSCLC). Pleural effusion is a common complication of lung cancer. In this study, we assessed the feasibility of detection of EGFR mutations in samples of pleural effusion fluid. We obtained 43 samples, which was the cell-free supernatant of pleural fluid, from Japanese NSCLC patients, and examined them for EGFR mutations. The epidermal growth factor receptor mutation status was determined by a direct sequencing method (exons 18-21 in EGFR). EGFR mutations were detected in 11 cases (E746_A750del in seven cases, E746_T751del insA in one case, L747_T751del in one case, and L858R in two cases). The EGFR mutations were observed more frequently in women and non-smokers. A comparison between the EGFR mutant status and the response to gefitinib in the 27 patients who received gefitinib revealed that all seven patients with partial response and one of the seven patients with stable disease had an EGFR mutation. No EGFR mutations were detected in the patients with progressive disease. The results suggest that DNA in pleural effusion fluid can be used to detect EGFR mutations and that the EGFR mutation status may be useful as a predictor of the response to gefitinib.

Adenocarcinoma↗
PubMed2007

All-cause and cardiovascular mortality in diabetic subjects increases significantly with reduced estimated glomerular filtration rate (eGFR): 10 years' data from the South Tees Diabetes Mortality study.

AIMS: To investigate the association between estimated glomerular filtration rate (eGFR) and total and cardiovascular mortality in a population-based cohort of diabetic subjects. METHODS: A longitudinal study using a population-based district diabetes register comprising 3288 subjects in South Tees, UK. The eGFR was calculated using the Modification of Diet in Renal Disease (MDRD) study equation. Patients were stratified by baseline eGFR into five stages as per the National Kidney Foundation guidelines: Stage 1, eGFR > 90; Stage 2, eGFR 60-89; Stage 3, eGFR 30-59; Stage 4, eGFR 15-29; and Stage 5, eGFR < 15 ml/min per 1.73 m(2). Main outcome was all-cause and cardiovascular mortality between 1 January 1994 and 31 July 2004. RESULTS: At baseline, mean age (58.4 years) differed between groups. Persons with lower eGFR were older (P < 0.001). Thirty-six percent (n = 1193, males 56%) had died by 10 years (cardiovascular cause in 60%). Median follow-up was 10.5 years amounting to 28 342 person years. Stages 4 and 5 (eGFR <or= 29 ml/min per 1.73 m(2)) were amalgamated for mortality analysis. Total and cardiovascular mortality increased with reduced eGFR. Adjusted hazard ratios (HR) [95% confidence interval (CI)] for all-cause mortality comparing groups 2 and 3, and 4 and 5 combined with group 1 were 1.28 (1.02, 1.60), 2.58 (2.05, 3.25) and 6.42 (4.25, 9.71), respectively. Adjusted HRs (95% CI) for mortality due to circulatory disease comparing groups 2 and 3, and 4 and 5 combined with group 1 were 1.50 (1.10, 2.06), 3.32 (2.41, 4.58) and 7.99 (4.69, 13.62), respectively. CONCLUSIONS: In diabetic subjects, mortality increases significantly with reduced GFR. Low eGFR identifies patients at high risk of cardiovascular mortality who should be targeted for aggressive risk factor modification.

Diabetes Mellitus, Type 2↗
PubMed2004

Predominant expression of mutant EGFR (EGFRvIII) is rare in primary glioblastomas.

EGFR amplification is a frequent genetic alteration in primary (de novo) glioblastomas, and is often associated with structural alterations. Most common is variant III (EGFRvIII), which results from a non-random 801 bp in-frame deletion of exons 2 to 7 of the EGFR gene. We assessed amplification and overexpression of EGFRvIII and wild-type EGFR in 30 glioblastoma biopsies. Immunohistochemically, EGFR overexpression was observed in 20 (67%) of 30 glioblastomas. Eight (27%) cases also showed immunoreactivity to an EGFRvIII antibody. In 6 of these cases, the pattern of EGFR and EGFRvIII overexpression was compared in serial sections: In 4 cases, areas with immunoreactivity to EGFRvIII largely coincided with wild-type EGFR expression. In the other 2 cases, the areas immunoreactive to EGFRvIII were significantly less extensive than EGFR-positive areas. To assess whether EGFRvIII is predominantly amplified in tumors with concurrent wild-type EGFR amplification, we carried our real-time quantitative PCR using 2 sets of primers located in exon 2 and intron 15 of the EGFR gene. A > 5-fold ratio of relative copy numbers between intron 15 (present both in wild-type EGFR and EGFRvIII) and exon 2 (present only in wild-type EGFR, but missing in EGFRvIII) suggested predominant amplification of EGFRvIII in only 3 (10%) of 30 glioblastomas. The observation that intratumoral wild-type EGFR overexpression is often more extensive and that predominant amplification of EGFRvIII is a rare event would limit the effectiveness of therapeutic approaches based on selective targeting of EGFRvIII.

Adult↗
PubMed2004

Wound-induced HB-EGF ectodomain shedding and EGFR activation in corneal epithelial cells.

PURPOSE: Epithelial wound healing is, at least in part, mediated in an autocrine fashion by epidermal growth factor (EGF) receptor (EGFR)-ligand interactions. This study sought to identify the endogenous EGFR ligand and the mechanism by which it is generated in response to wounding in cultured porcine corneas and human corneal epithelial cells. METHODS: Epithelial debridement wounds in cultured porcine corneas and scratch wounds in an epithelial monolayer of SV40-immortalized human corneal epithelial (THCE) cells were allowed to heal in the presence of tyrphostin AG1478 (an EGFR inhibitor), GM6001 (a matrix metalloproteinase [MMP] inhibitor), or CRM197 (a diphtheria toxin mutant), with or without HB-EGF. The activation of EGFR and extracellular signal-regulated kinase (ERK) was analyzed by immunoprecipitation using EGFR antibodies and Western blot analysis with phosphotyrosine antibody. Wound induced HB-EGF shedding was assessed by isolation of secreted HB-EGF from wounded THCE cells and by measuring the release of alkaline phosphatase (AP) in THCE stable cell lines expressing HB-EGF-AP. RESULTS: In THCE cells, wound-induced EGFR phosphorylation and ERK activation. In both organ and cell culture models, epithelial wounds were healed in basal media and inhibition of EGFR activation by AG1478 blocked wound closure with or without exogenously added HB-EGF. GM6001 delayed wound closure. Its effects diminished in the presence of exogenous EGF or HB-EGF, suggesting that the MMP inhibitor primarily blocks the release of EGFR ligands. CRM197, a highly specific antagonist of HB-EGF, impaired epithelial wound closure, suggesting that HB-EGF is an endogenous ligand released on epithelial wounding. Consistent with the effects on epithelial migration, all inhibitors as well as HB-EGF function-blocking antibodies retarded wound-induced EGFR phosphorylation in cultured THCE cells. The release of HB-EGF in response to wounding was demonstrated by the fact that heparin-binding proteins isolated from wounded, but not control, THCE-conditioned medium stimulated EGFR and ERK phosphorylation and by the expression of HB-EGF-AP in THCE cells, in which wounding induced the release of AP activity in an MMP-inhibitor-sensitive manner. CONCLUSIONS: HB-EGF released on wounding acts as an autocrine-paracrine EGFR ligand. HB-EGF shedding and EGFR activation represent a critical event during corneal epithelial wound healing, suggesting a possible manipulation of wound healing during the early phases.

Alkaline Phosphatase↗
PubMed2000

[Epidermal growth factor receptor (EGFR) in the prognosis of bladder carcinoma. Experience of 5 years].

METHODS: From November 1992 to November 1993, a prospective study was conducted on 20 controls and 61 patients with bladder carcinoma. EGFR expression was determined by radioimmunoassay and the correlation of the results of histological analysis and the clinical course was analyzed. The follow-up period was from November 1992 to July 1998. The association between qualitative variables and the x2 or Fisher exact test was compared using the hypothesis of the proportional ordinal trend for the ordinal variables, and the quantitative variables were analyzed using Student's t test and/or variance analysis (ANOVA). Survival was analyzed by the Kaplan-Meier method and comparison was performed using the Breslow exact test. The Cox proportional hazards regression model was utilized. The SPSS software for Windows 7.0 was used for the analysis. RESULTS: The EGFR values were higher for patients with bladder carcinoma than in controls (14.48 vs 2.54 fmol/mg of protein). EGFR values were higher in patients with superficial bladder tumor than in those with infiltrating tumors (27.03 fmol/mg vs. 10.05 fmol/mg of protein; p = 0.000). Poorly differentiated tumors showed higher values of EGFR (6.73, 14.48 and 17.07 fmol/mg of protein for grades I, II and III, respectively; p < 0.05). The EGFR values were higher in patients that died from cancer during follow-up (64.8) than in those who died from other causes (47.5) and those who are alive and on follow-up (42). An increase in EGFR values did not carry a risk of death from cancer (p = 0.1269; ns). Analysis of the grade of tumor differentiation showed that for the more aggressive tumor grade, a positive EGFR was a sign of reduced survival. Survival in patients with superficial and infiltrating tumor did not appear to change significantly according to the EGFR value. EGFR determination was not useful in predicting recurrence and increased EGFR values did not correlate with a higher risk of recurrence. CONCLUSIONS: 1) The normal pattern of EGFR could not be established. 2) EGFR was not useful in identifying subgroups at risk of death. 3) Knowledge about these proteins synthesized by oncogenes offers new possibilities in the treatment of cancer.

ErbB Receptors↗
PubMed2026

Spironolactone, early acute eGFR changes, and clinical outcomes in patients with heart failure with preserved ejection fraction: insights from TOPCAT Americas.

AIMS: Early acute changes in estimated glomerular filtration rate (eGFR) have been well described with renin-angiotensin system inhibitors and sodium-glucose cotransporter-2 inhibitors, but less is known about the frequency, prognostic relevance, and implications of these changes after mineralocorticoid receptor antagonist (MRA) initiation in patients with heart failure with preserved ejection fraction (HFpEF). METHODS: We performed a post-hoc analysis of 1648 patients enrolled in the TOPCAT trial (Americas regional subgroup), defining an early eGFR dip as a &#x2265;15% decrease in eGFR between baseline and week 4. Landmark analyses assessed the association of eGFR changes, treatment, and the primary composite endpoint (cardiovascular death, HF hospitalization, or aborted cardiac arrest). RESULTS: Within 4 weeks of treatment initiation, 431 (26%) patients experienced acute eGFR decrease with a higher proportion of patients assigned to spironolactone [269 (33%)] compared with placebo [162 (20%)] (odds ratio 1.97; 95% confidence interval 1.58-2.47). An acute eGFR decrease was independently associated with higher risk of subsequent cardiovascular outcomes, irrespective of treatment arm. However, treatment with spironolactone appeared beneficial in reducing the primary cardiovascular outcome irrespective of the presence [hazard ratio 0.75 (0.53-1.08)] or absence [0.80 (0.64-1.00)] of early eGFR decrease (Pinteraction = .81). At any given magnitude of eGFR decline, risk of the primary endpoint was consistently lower with spironolactone compared with placebo (Pinteraction = .64). CONCLUSIONS: Early acute eGFR changes were common and adversely prognostic in patients with HFpEF. Spironolactone treatment was beneficial in improving cardiovascular outcomes, despite a modest increase in the likelihood of acute eGFR decrease. An acute eGFR decrease early after MRA initiation should not automatically prompt treatment discontinuation. TRIAL REGISTRATION: ClinicalTrials.gov NCT00094302.

Humans↗
PubMed1999

Human mammary epithelial cells rapidly exchange empty EGFR between surface and intracellular pools.

Binding of ligand to the epidermal growth factor receptor (EGFR) initiates a series of processes including activation of the intrinsic EGFR tyrosine kinase, receptor autophosphorylation, and the assembly of active signaling complexes at the plasma membrane. Concomitantly, receptor trafficking is initiated, and the receptor is ultimately delivered to the lysosome, where it is degraded. Virtually all studies on EGFR trafficking have used fibroblasts and transformed cells. Because EGFR exerts a potent effect on the physiology of epithelial cells, we examined the regulation of EGFR activity and trafficking in nontransformed human mammary epithelial cells (HMEC). We found that HMEC that displayed a luminal phenotype were largely unresponsive to EGF and maintained a majority of their EGFR at the cell surface. In contrast, HMEC with a basal phenotype were highly responsive to EGF and, at steady state in the absence of exogenous ligand, distributed empty EGFR into intracellular pools. Maintenance of the intracellular pools was a direct consequence of specific and rapid endocytosis of the empty EGFR. The trafficking pattern was EGFR specific, used coated pits, and did not require receptor tyrosine kinase activity. Such an mechanism redistributes EGFR signaling potential among different membrane domains and into vesicles with unique biochemical microenviroments. In addition, our data show that EGFR endocytosis can be regulated in the absence of ligand binding and receptor activation in a cell-type-specific manner.

Breast↗
PubMed2004

Synergistic interaction between the EGFR tyrosine kinase inhibitor gefitinib ("Iressa") and the DNA topoisomerase I inhibitor CPT-11 (irinotecan) in human colorectal cancer cells.

Epidermal growth factor receptor [EGFR (HER1, erbB1)] is a receptor with associated tyrosine kinase activity, and is expressed in colorectal cancers and many other solid tumors. We examined the effect of the selective EGFR tyrosine kinase inhibitor (EGFR-TKI) gefitinib ("Iressa") in combination with the DNA topoisomerase I inhibitor CPT-11 (irinotecan) on human colorectal cancer cells. EGFR mRNA and protein expression were detected by RT-PCR and immunoblotting in all 7 colorectal cancer cell lines studied. Gefitinib inhibited the cell growth of the cancer cell lines in vitro with an IC(50) range of 1.2-160 microM by 3,(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Lovo cells exhibited the highest level of protein and autophosphorylation of EGFR and were the most sensitive to gefitinib. The combination of gefitinib and CPT-11 induced supra-additive inhibitory effects in COLO320DM, WiDR and Lovo cells, assessed by an in vitro MTT assay. Administration of gefitinib and CPT-11 had a supra-additive inhibitory effect on WiDR cells and tumor shrinkage was observed in Lovo cell xenografts established in nude mice, whereas no additive effect of combination therapy was observed in COLO320DM cells. To elucidate the mechanisms of synergistic effects, the effect of CPT-11-exposure on phosphorylation of EGFR was examined by immunoprecipitation. CPT-11 increased phosphorylation of EGFR in Lovo and WiDR cells in time- and dose-dependent manners. This EGFR activation was completely inhibited by 5 microM gefitinib and gefitinib-induced apoptosis was enhanced by combination with CPT-11, measured by PARP activation although no PARP activation was induced by 5 microM CPT-11 alone. These results suggested that these modification of EGFR by CPT-11, in Lovo cells, is a possible mechanism for the synergistic effect of CPT-11 and gefitinib. These findings imply that the EGFR-TKI gefitinib and CPT-11 will be effective against colorectal tumor cells that express high levels of EGFR, and support clinical evaluation of gefitinib in combination with CPT-11, in the treatment of colorectal cancers.

Animals↗
PubMed2005

Epidermal growth factor receptor (EGFR) downstream molecules as response predictive markers for gefitinib (Iressa, ZD1839) in chemotherapy-resistant non-small cell lung cancer.

Gefitinib has shown meaningful antitumor activity with tolerable toxicity in chemotherapy-refractory NSCLC in previous studies. Moreover, EGFR expression failed to show a correlation with response. In an attempt to identify predictive markers of response, we have investigated the tumoral expression of key signaling molecules of EGFR (EGFR, p-EGFR, p-Akt, p-Erk, p-STAT3) by immunohistochemistry and analyzed their correlations with response. Of 65 patients who received gefitinib (250 mg/day) for chemotherapy-refractory NSCLC, there were 14 partial responses (21.5%), 21 stable diseases (32.3%) and 21 progressive diseases (32.3%). Median durations of overall survival and time to progression were 6.7 months and 2.8 months, respectively. Immunohistochemistry was performed in 34 patients with evaluable tissue specimens. EGFR was overexpressed (2+ or 3+) in 32.4% and p-EGFR was positive in 26.5%. The expressions of p-Akt, p-Erk and p-STAT3 were positive (1+ or 2+) in 50%, 38.2% and 79.4%, respectively. The EGFR expression was not correlated with p-EGFR or the downstream molecules. EGFR or p-EGFR status did not correlate with response. Positive expression of p-Erk was significantly associated with poor response (38.1% in -, 14.3% in 1+, 0% in 2+; p = 0.046). Furthermore, tumors with positive p-Akt and negative p-Erk nuclear expression exhibited the best response (60%), whereas there was no response in the opposite [p-Akt (-), p-Erk (+)] cases. Intense nuclear staining of p-Akt (2+) was associated with prolonged TTP (HR 0.25, 95% confidence interval [CI] 0.08-0.79, p = 0.018) and OS (HR 0.16, 95% CI 0.04-0.62, p = 0.008). These results support the assumption that gefitinib responsiveness might be predicted by activated EGFR downstream molecules such as p-Akt and p-Erk.

Adult↗
PubMed2006

EGFR protein overexpression and gene amplification in squamous cell carcinomas of the esophagus.

Overexpression of epidermal growth factor receptor (EGFR) is observed in many cancers, sometimes accompanied by gene amplification. Recently, several clinical therapies targeting EGFR were developed, but the eligibility criteria for these therapies is not fully established. To develop such eligibility criteria for esophageal squamous cell carcinoma (ESCC), we sought to clarify: (i) the exact frequency of EGFR overexpression, (ii) the relationship between protein overexpression and gene amplification, (iii) the relationship between gene amplification and specific gene mutations and (iv) the correlation between the status of EGFR and clinical or pathological features. Immunohistochemistry revealed that EGFR protein is overexpressed in 53 (50%) of the 106 ESCC examined. Fluorescence in situ hybridization (FISH) indicated clear EGFR gene amplification in 15 of the 53 tumors, somewhat higher EGFR copy in 32 cases, and no increase in 6 cases. Gene amplification was significantly associated with high level overexpression. Direct sequencing of exons 19 and 21 of EGFR revealed no mutations in 15 tumors exhibiting gene amplification, and no mutations in 25 tumors not exhibiting gene amplification. Overexpression of EGFR was significantly correlated with depth of invasion of the tumor. In conclusion, anti-EGFR therapies may be appropriate for patients with ESCC. We assume that combined analyses by immunohistochemistry/FISH would clarify aberrations in protein and gene function, and could help to identify those patients who may benefit from anti-EGFR therapy.

Adult↗
PubMed2005

Up-regulated expression of ADAM17 in human colon carcinoma: co-expression with EGFR in neoplastic and endothelial cells.

The ADAM17 metalloproteinase (a disintegrin and metalloprotease 17) controls epidermal growth factor receptor (EGFR) activation through regulated shedding of EGFR ligands. With the advent of new therapeutic options targeting EGFR signalling in colon carcinoma, it was decided to determine ADAM17 status in relation to clinico-pathological parameters and EGFR status. To this end, a series of 39 colon carcinomas were analysed. Immunohistochemistry and immunofluorescence were used to localize ADAM17, EGFR, and the activated forms of EGFR. The activated form of ADAM17 was assessed in primary cancers and colon cell lines by immunoblotting. ADAM17 and EGFR mRNA levels were assessed by quantitative RT-PCR. Chromogenic in situ hybridization (CISH) was used to quantify the HER1 gene. ADAM17 was strongly expressed in all tumours, by both neoplastic and endothelial cells. It was expressed both as a pro- and as an active form in tumours and colonic cancer cell lines. ADAM17 mRNA was up-regulated in 90% of colon carcinomas relative to the paired normal mucosa, whatever the tumour grade or stage. When present, activated EGFR was co-expressed with ADAM17 by colon carcinomas, although at a variable level among tumour cells, and by endothelial cells. EGFR mRNA was overexpressed in 77% of colon carcinomas compared with the paired normal mucosa. One case showed high-level amplification of HER1. In conclusion, this study is the first demonstration that ADAM17 is overexpressed in human primary colon carcinoma, whatever the tumour stage and differentiation and whatever the level of EGFR expression. Its co-expression with EGFR, in both neoplastic and endothelial cells, suggests a role for ADAM17 in tumour growth and angiogenesis.

ADAM Proteins↗
PubMed2002

Differential responses of EGFR-/AGT-expressing cells to the "combi-triazene" SMA41.

PURPOSE: Previous studies have demonstrated enhanced potency associated with the binary [DNA/epidermal growth factor receptor (EGFR)] targeting properties of SMA41 (a chimeric 3-(alkyl)-1,2,3-triazene linked to a 4-anilinoquinazoline backbone) in the A431 (epidermal carcinoma of the vulva) cell line. We now report on the dependence of its antiproliferative effects (e.g. DNA damage, cell survival) on the EGFR and the DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) contents of 12 solid tumor cell lines, two of which, NIH3T3 and NIH3T3 HER14 (engineered to overexpress EGFR), were isogenic. METHODS: Receptor type specificity was determined using ELISA for competitive binding, as well as growth factor-stimulation assays. DNA damage was studied using single-cell microelectrophoresis (comet) assays, and levels of EGFR were determined by Western blotting. The effects of SMA41 on the cell cycle of NIH3T3 cells were investigated using univariate flow cytometry. RESULTS: Studies of receptor type specificity showed that SMA41: (a) preferentially inhibited the kinase activity of EGFR over those of Src, insulin receptor and protein kinase C (PKC, a serine/threonine kinase), (b) induced stronger inhibition of growth stimulated with EGF than of growth stimulated with platelet-derived growth factor (PDGF) or fetal bovine serum (FBS). Despite the EGFR specificity of SMA41, there was an absence of a linear correlation between the EGFR status of our solid tumor cell lines and levels of DNA damage induced by the alkylating component. Similarly, EGFR levels did not correlate with IC(50) values. The antiproliferative activities of SMA41 correlated more with the AGT status of these cells and paralleled those of the clinical triazene temozolomide (TEM). However, throughout the panel, tumor cell sensitivity to SMA41 was consistently stronger than to its closest analogue TEM. Experiments performed with the isogenic cells showed that SMA41 was capable of inducing twofold higher levels of DNA damage in the EGFR transfectant and delayed cell entry to G(2)/M in both cell types. When the cells were starved and growth-stimulated with FBS (conditions under which both cell types were growth-stimulated), in contrast to TEM, SMA41 and its hydrolytic metabolite SMA52 exhibited approximately nine- and threefold stronger inhibition of growth of the EGFR transfectant. CONCLUSIONS: These results suggest that, in addition to its ability to induce DNA damage and cell cycle perturbations, SMA41 is capable of selectively targeting the cells with a growth advantage conferred by EGFR transfection. When compared with the monoalkyltriazene prodrug TEM, its potency may be further enhanced by its ability to hydrolyze to another signal transduction inhibitor (SMA52) plus a DNA alkylating agent that may further contribute to chemosensitivity. Thus, our new "combi-targeting" strategy may well represent a tandem approach to selectively blocking receptor tyrosine kinase-mediated growth signaling while inducing significant levels of cytotoxic DNA lesions in refractory tumors.

3T3 Cells↗
PubMed2005

Helicobacter pylori-stimulated interleukin-8 (IL-8) promotes cell proliferation through transactivation of epidermal growth factor receptor (EGFR) by disintegrin and metalloproteinase (ADAM) activation.

Helicobacter pylori infection increases the risk of hyperplastic polyps and gastric cancer, but the mechanisms remain to be elucidated. H. pylori was recently shown to transactivate epidermal growth factor receptor (EGFR) through metalloprotease stimulation. The present study was designed to investigate the effect of interleukin-8 (IL-8) induced by H. pylori infection on EGFR transactivation and epithelial cell growth. H. pylori Sydney strain 1 (SS1) having wild-type cag(+)A was used. Phospho-EGFR assay was performed by immunoprecipitation using anti-human EGFR and anti-phosphotyrosine antibodies. DNA synthesis was evaluated by [3H]thymidine uptake using the human gastric cancer cell line, KATO III. H. pylori induced EGFR phosphorylation, and a disintegrin and metalloproteinase (ADAM) inhibitor, KB-R7785, completely suppressed EGFR phosphorylation. IL-8 also induced EGFR phosphorylation, while anti-IL-8 and anti-IL-8 receptor (CXCR1) neutralizing antibodies suppressed EGFR phosphorylation. [(3)H]Thymidine uptake analysis demonstrated that H. pylori increased DNA synthesis in gastric epithelial cells, and tyrosine kinase inhibitor, MEK inhibitor, and ADAM inhibitor suppressed the DNA synthesis induced by H. pylori. H. pylori-stimulated IL-8 accelerates processing of EGFR ligands through ADAM activation, and cleaved EGFR ligands bind and stimulate EGFR in paracrine and autocrine manners to induce cell proliferation. This may be one of the mechanisms of hyperplastic polyp and gastric cancer development in H. pylori-infected gastric mucosa.

ADAM Proteins↗
PubMed2006

Combining siRNAs at two different sites in the EGFR to suppress its expression, induce apoptosis, and enhance 5-fluorouracil sensitivity of colon cancer cells.

BACKGROUND: The epidermal growth factor receptor (EGFR) has played an important role in the growth and apoptosis of colon cancer. The RNA interference (RNAi) technique can suppress gene expression, but the effects of double combining sites RNAi targeting EGFR have not been well understood. METHODS: pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA-2 expressive vectors were transfected to the LoVo cells. Five groups were selected for the study: Group 1, the control cells; group 2, the negative control plasmid vector HK; group 3, pU6-EGFR-shRNA-1; group 4, pU6-EGFR-shRNA-2; group 5, pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA-2, half for each. The mRNA and protein expression were assessed by Real Time quantitative PCR and Western blot. Apoptosis was determined via flow cytometry. IC(50) and the inhibition ratio of 5-fluorouracil (5-FU) were carried out by CCK-8. RESULTS: In groups 3, 4, and 5, the mRNA expression was decreased by (80.22 +/- 3.42)%, (81.30 +/- 2.83)%, and (90.58 +/- 2.76)%, respectively, and the protein expression was decreased by (74.11 +/- 4.02)%, (73.39 +/- 2.30)%, and (90.39 +/- 3.34)%, respectively. Meanwhile, the cell apoptosis increased by (10.43 +/- 0.49)%, (10.13 +/- 0.39)%, and (14.17 +/- 0.53)%, respectively. The IC(50) of 5-FU and cell inhibition ratio analysis demonstrated that there were significant differences between the following three: group 5, groups 3 and 4, and groups 1 and 2. CONCLUSIONS: Both pU6-EGFR-shRNA-1 and pU6-EGFR-shRNA-2 are capable of suppressing EGFR expression of the LoVo cell and can promote apoptosis and increase the cell toxicity of 5-FU. The double combining sites RNAi technique is significantly better than a single site.

Antimetabolites, Antineoplastic↗