Epidermal Growth Factor Receptor Content Research Articles

Novel EGFR-MAPK kinase and ABC transporter inhibitors for HepG2 resistant to Erlotinib.

Hepatocellular carcinoma (HCC) is the third-leading cause of cancer death in the world, with outlook for most patients having a 5-year survivability of less than 5%. In a previous study from our laboratory, novel estrone inspired analogs act as epidermal growth factor receptor(EGFR) inhibitors in HepG2 cells. This study focuses on the effect of these analogs on an HCC cell line resistance to Erlotinib. Lead compounds MMA132 and MMA102 showed 13 and 20 µM IC50 values, respectively against HepG2-R resistant to Erlotinib. These compounds showed cell cycle arrest of the G2 phase up to 54%, and inhibited cell migration of HepG2-R cells up to 48 h. Western blot analysis revealed that MMA132 reduced total EGFR content after 48 h, while MMA102 inhibited MEK kinase by 84% after 48 h. Western blot analysis also revealed that multidrug resistance protein 2 (MRP2) is overexpressed in HepG2-R, suggesting that ABC transporters play a likely cause in drug resistance. MMA102 showed significant inhibition of both P-glycoprotein (83%) and ABCG2 (53%), two additional ABC transporters. Additionally, MMA102 and MMA132 were used in a combination therapy with MK571(MRP1/2 inhibitor) and produced IC50 values of 18 and 10 µM, respectively, better than either MMA102/132 or MK571 alone. To validate our findings, we conducted molecular dynamic simulations with MMA102 and MMA132 in MEK, P-glycoprotein, MRP1, and MRP2. Results coincided with biological findings in which MMA102 orientation is favored in both MEK and P-glycoprotein pockets, whereas MMA132 likely binds with MRP2, as likely suggested by the combinatorial study.

Novel 3-D cell culture system for in vitro evaluation of anticancer drugs under anchorage-independent conditions.

Anticancer drug discovery efforts have used 2‐D cell‐based assay models, which fail to forecast in vivo efficacy and result in a lower success rate of clinical approval. Recent 3‐D cell culture models are expected to bridge the gap between 2‐D and in vivo models. However, 3‐D cell culture methods that are available for practical anticancer drug screening have not yet been fully attained. In this study, we screened several polymers for their ability to suspend cells or cell spheroids homogeneously in a liquid medium without changing the viscosity behavior, and identified gellan gum (FP001), as the most potent polymer. FP001 promoted cell dispersion in the medium and improved the proliferation of a wide range of cancer cell lines under low attachment conditions by inhibiting the formation of large‐sized spheroids. In addition, cancer cells cultured with FP001‐containing medium were more susceptible to inhibitors of epidermal growth factor (EGF) signaling than those cultured under attachment conditions. We also showed that ligands of the EGF receptor family clearly enhance proliferation of SKOV3 ovarian carcinoma cells under anchorage‐independent conditions with FP001. Consistent with this result, the cells grown with FP001 showed higher EGF receptor content compared with cells cultured under attachment conditions. In conclusion, we developed a novel 3‐D cell culture system that is available for high throughput screening of anticancer agents, and is suitable for evaluation of molecular‐targeted anticancer drugs. Three‐dimensional cell culture using FP001 will be of value in the development of useful technologies for anticancer drug discovery.

GH administration patterns differently regulate epidermal growth factor signaling

Current GH administration protocols imply frequent s.c. injections, resulting in suboptimal compliance. Therefore, there is interest in developing delivery systems for sustained release of the hormone. However, GH has different actions depending on its continuous or pulsatile plasma concentration pattern. GH levels and circulating concentration patterns could be involved in the regulation of epidermal growth factor receptor (EGFR) expression in liver. Aberrant expression of this receptor and/or its hyperactivation has been associated with the pathogenesis of different types of carcinoma. Considering that one of the adverse effects associated with GH overexpression and chronic use of GH is the increased incidence of malignancies, the aim of this study was to analyze the effects of GH plasma concentration patterns on EGFR expression and signaling in livers of mice. For this purpose, GH was administered by s.c. daily injections to produce an intermittent plasma pattern or by osmotic pumps to provoke a continuously elevated GH concentration. Intermittent injections of GH induced upregulation of liver EGFR content, augmented the response to EGF, and the induction of proteins involved in promotion of cell proliferation in female mice. In contrast, continuous GH delivery in male mice was associated with diminished EGFR in liver and decreased EGF-induced signaling and expression of early genes. The results indicate that sustained delivery systems that allow continuous GH plasma patterns would be beneficial in terms of treatment safety with regard to the actions of GH on EGFR signaling and its promitogenic activity.

EGF receptor and COX-1/COX-2 enzyme proteins as related to corresponding mRNAs in human per-operative biopsies of colorectal cancer

BackgroundCyclooxygenase (COX) and epidermal growth factor receptor (EGFR) activities promote progression of colorectal cancer. Combined treatment against these targets has not been more effective than single treatments alone. Therefore, our aim was to analyze relationships between COX and EGFR in peroperative colorectal tumor biopsies.MethodTumor and colon mucosa tissue were collected at primary intended curative operations in patients according to well-recognized statistical distributions of tumor stages in colorectal cancer. COX-1, COX-2 and EGFR content in tumor and colon mucosa tissue were quantified by western blot and Q-PCR.ResultsCOX-2 protein appeared as two bands, one at 66 kDa in almost all tumor and mucosa samples and one at 74 kDa in 73% of the tumors and in 23% of the mucosa samples. Tumor COX-2 mRNA was not different from the content in mucosa samples, while COX-2 protein was increased in tumor tissue (p < 0.0003). A correlation between 74 kDa COX-2 protein and COX-2 mRNA occurred in tumor tissue, with significantly increasing COX-2 mRNA across tumor stages. EGFR mRNA content was lower in tumor tissue (p < 0.0001), while EGFR protein was similar in tumor and mucosa samples. COX-2 and EGFR proteins showed a positive correlation in mucosa, while a negative correlation occurred in tumor tissue. Tumor tissue with high COX-2 74 kDa protein lacked EGFR protein.ConclusionOur present results are compatible with the theory that COX-2 and EGFR signalling pathways are inversely related in colorectal cancer tissue. This may explain why combinatorial clinical treatments have been less rewarding.

Tripartite motif protein 23 (TRIM23) regulates degradation of epidermal growth factor receptor (EGFR)

Tripartite motif protein 23 (TRIM23), also known as ADP‐ribosylation factor‐domain protein (ARD) 1, is a multi‐domain 64‐kDa protein that contains ADP‐ribosylation factor, GTPase‐activating protein, and E3 ubiquitin ligase sequences. That EGFR levels were higher in ARD1−/− than in wild type MEFs was known, but the mechanism responsible remained unknown. To explore that question, we used co‐immunoprecipitation (co‐IP) to evaluate possible interactions of ARD1 and EGFR. Endogenous ARD1 and EGFR in HeLa cells were each immunoprecipitated by antibodies against the other, as was cytohesin‐1, the only known activator of the ARD1 ARF domain. Overexpression of ARD1, intended to enhance the interaction between ARD1 and EGFR, decreased the total cell EGFR. We then found that EGFR levels were increased after ARD1 depletion in HeLa cells. It appears that the interaction of ARD1 and EGFR can be important in regulation of EGFR degradation, and thereby the EGFR content of HeLa cells as well as MEFs.

99mTc-Hydrazinonicotinamide Epidermal Growth Factor–Polyethylene Glycol–Quantum Dot Imaging Allows Quantification of Breast Cancer Epidermal Growth Factor Receptor Expression and Monitors Receptor Downregulation in Response to Cetuximab Therapy

Therapy of cancer, including basallike breast tumors, that targets the epidermal growth factor receptor (EGFR) would greatly benefit from noninvasive methods that can quantitatively monitor receptor status and treatment response. Here, we investigated the potential of a novel technique based on streptavidin cadmium selenide/zinc sulfide quantum dots (Qdots) multiplexed with polyethylene glycol (PEG), epidermal growth factor (EGF), and (99m)Tc-hydrazinonicotinamide. In vitro binding affinity and specificity were evaluated in cultured cells. Biodistribution studies and in vivo imaging were performed in murine breast tumor xenografts of basallike phenotype MDA-MB-468 cells and EGFR-negative cells. (99m)Tc-hydrazinonicotinamide EGF-PEG-Qdot showed specific and high-affinity EGFR targeting on confocal microscopy, immunoblotting, and binding assays. When intravenously injected, MDA-MB-468 tumors were visualized with high contrast by both optical and scintigraphic imaging. Scintigraphic image-based quantification correctly discriminated high-EGFR-expressing MDA-MB-468 tumors from other tumors, and image-based tumor uptake closely correlated to EGFR content. Importantly, serial imaging of MDA-MB-468 tumors responding to cetuximab therapy could detect a significant reduction of tumor uptake that was paralleled by downregulation of EGFR expression. Furthermore, high baseline uptake predicted good response to cetuximab therapy. (99m)Tc-hydrazinonicotinamide EGF-PEG-Qdot provides EGFR-targeted imaging of breast tumors and may allow noninvasive monitoring of EGFR status in living subjects before and after targeted therapies.

GH modulates hepatic epidermal growth factor signaling in the mouse.

Epidermal growth factor (EGF) is a key regulator of cell survival and proliferation involved in the pathogenesis and progression of different types of cancer. The EGF receptor (EGFR) is activated by binding of the specific ligand but also by transactivation triggered by different growth factors including GH. Chronically, elevated GH levels have been associated with the progression of hepatocellular carcinoma. Considering EGF and GH involvement in cell proliferation and their signaling crosstalk, the objective of the present study was to analyze GH modulatory effects on EGF signaling in liver. For this purpose, GH receptor-knockout (GHR-KO) and GH-overexpressing transgenic mice were used. EGFR content was significantly decreased in GHR-KO mice. Consequently, EGF-induced phosphorylation of EGFR, AKT, ERK1/2, STAT3, and STAT5 was significantly decreased in these mice. In contrast, EGFR content as well as its basal tyrosine phosphorylation was increased in transgenic mice overexpressing GH. However, EGF stimulation caused similar levels of EGFR, AKT, and ERK1/2 phosphorylation in normal and transgenic mice, while EGF induction of STAT3 and STAT5 phosphorylation was inhibited in the transgenic mice. Desensitization of the STATs was related to decreased association of these proteins to the EGFR and increased association between STAT5 and the tyrosine phosphatase SH2-containing phosphatase-2. While GHR knockout is associated with diminished expression of the EGFR and a concomitant decrease in EGF signaling, GH overexpression results in EGFR overexpression with different effects depending on the signaling pathway analyzed: AKT and ERK1/2 pathways are induced by EGF, while STAT3 and STAT5 activation is heterologously desensitized.

Correlates and Determinants of Nuclear Epidermal Growth Factor Receptor Content in an Oropharyngeal Cancer Tissue Microarray

We have previously reported nuclear localization of epidermal growth factor receptor (EGFR) protein in oropharyngeal cancer tissue. Nuclear EGFR levels were inversely correlated with survival and response to radiotherapy. Here, we sought to identify the determinants and correlates of nuclear EGFR content. We analyzed an oropharyngeal cancer tissue microarray for the expression of the key molecules of the EGFR signaling cascade using an automated image analysis technique (AQUA) scored on a scale of 0 to 255, which permits protein quantitation and subcellular localization. Patients with oropharyngeal squamous cell cancer treated with radiotherapy or surgery and radiotherapy were eligible. Data were analyzed using Spearman correlations and multiple linear regression with robust SEs. Of the 95 tumors included in this study, 72 (75%) had sufficient tissue for analysis of nuclear EGFR. Nuclear EGFR levels were associated with membranous/cytoplasmic EGFR levels (rho = 0.82, P < 0.001), nuclear extracellular signal-regulated kinase-2 (rho = 0.30, P = 0.01), and nuclear proliferating cell nuclear antigen (PCNA; rho = 0.36, P = 0.003). Nuclear phosphorylated-Akt, cyclin D1, phosphatase and tensin homolog (mutated in multiple cancers 1) (PTEN), p53, and proliferation marker Ki-67 levels did not correlate with nuclear EGFR level. In multivariable analysis, only PCNA retained its significant association (P = 0.01). These results are consistent with preclinical data showing that EGFR may function as a tyrosine kinase in the nucleus, phosphorylating and stabilizing PCNA. The nuclear activity of EGFR may constitute a novel therapeutic target.

Differential impact of Cetuximab, Pertuzumab and Trastuzumab on BT474 and SK‐BR‐3 breast cancer cell proliferation

The potential of epidermal growth factor receptor (EGFR)- and Her2-targeted antibodies Cetuximab, Pertuzumab and Trastuzumab, used in combination to inhibit cell proliferation of breast cancer cells in vitro, has not been extensively investigated. It is anticipated that there would be differences between specific erbB receptor co-expression profiles that would affect tumour cell growth. We have examined the effects of Cetuximab, Pertuzumab and Trastuzumab, applied separately or in combination, on cell proliferation of BT474 and SK-BR-3 breast cancer cell lines. Cell cycle progression of BT474 and SK-BR-3 cells was statically and dynamically assessed using flow cytometry. In order to discover a potential influence of differential EGFR co-expression on sensitivity to antibody treatment, EGFR was down-regulated by siRNA in SK-BR-3. An annexinV/propidium iodide assay was used to identify potential induction of apoptosis. Treatment with Pertuzumab and Trastuzumab, both targeted to Her2, resulted in a reduced fraction of proliferating cells, prolongation of G(1) phase and a great increase in quiescent BT474 cells. Cetuximab had no additional contribution to the effect of either Pertuzumab or Trastuzumab when administered simultaneously. Treatment with the antibodies did not induce an appreciable amount of apoptosis in either BT474 or SK-BR-3 cells. In contrast to SK-BR-3, the BT474 cell line appears to be more sensitive to antibody treatment due to low EGFR content besides Her2 overexpression. The extent of decelerated or blocked cell proliferation after antibody treatment that is targeted to EGFR and to Her2 depends both on EGFR and Her2 co-expression and on antibody combination used in the treatment setting. Cetuximab did not enhance any inhibitory effect of Trastuzumab or Pertuzumab, most probably due to the dominant overexpression of Her2. Cell susceptibility to Trastuzumab/Pertuzumab, both targeted to Her2, was defined by the ratio of EGFR/Her2 co-expression.

Correlates and determinants of nuclear epidermal growth factor receptor (EGFR) content in an oropharyngeal cancer tissue microarray

6022 Background: We have previously reported nuclear localization of EGFR protein in oropharyngeal cancer tissue. Nuclear EGFR levels were inversely correlated with survival and response to radiotherapy. Here, we sought to identify determinants and correlates of nuclear EGFR content. Methods: We analyzed an oropharyngeal cancer tissue microarray for expression of key molecules of EGFR signaling cascade utilizing an automated image analysis technique (AQUA) scored on a scale of 0–255 which permits protein quantitation and subcellular localization. Patients with OSCC treated with radiotherapy (RT) or surgery and RT were eligible. Data were analyzed using Pearson correlations and multiple linear regression with robust standard errors. Results: Of the 95 tumors included in this study, 72 (75%) had sufficient tissue for analysis of nuclear EGFR. Nuclear EGFR levels were associated with membranous/cytoplasmic EGFR levels (rho=0.85, p&lt;0.01), nuclear ERK2 (rho=0.34, p=0.01), tumor cyclin D1 (rho=−0.25, p=0.06) and the proliferation markers Ki-67 (rho=0.24,p=0.06) and PCNA (rho=0.38, p&lt;0.01). Nuclear phosphorylated-Akt, PTEN, p53 levels did not correlate with nuclear EGFR level. In multivariable analysis, only PCNA retained its significant association (p=0.01). Conclusions: Our results are consistent with the preclinical model of translocation of membranous EGFR to the nucleus acting as a transcriptional factor and fostering cell proliferation. Nuclear EGFR may constitute a mechanism of resistance to current EGFR-targeted therapies. No significant financial relationships to disclose.

Evaluation of radiolabeled ML04, a putative irreversible inhibitor of epidermal growth factor receptor, as a bioprobe for PET imaging of EGFR-overexpressing tumors

Overexpression of epidermal growth factor receptor (EGFR) has been implicated in tumor development and malignancy. Evaluating the degree of EGFR expression in tumors could aid in identifying patients for EGFR-targeted therapies and in monitoring treatment. Nevertheless, no currently available assay can reliably quantify receptor content in tumors. Radiolabeled inhibitors of EGFR-TK could be developed as bioprobes for positron emission tomography imaging. Such imaging agents would not only provide a noninvasive quantitative measurement of EGFR content in tumors but also serve as radionuclide carriers for targeted radiotherapy. The potency, reversibility, selectivity and specific binding characteristics of ML04, an alleged irreversible inhibitor of EGFR, were established in vitro. The distribution of the F-18-labeled compound and the extent of EGFR-specific tumor uptake were evaluated in tumor-bearing mice. ML04 demonstrated potent, irreversible and selective inhibition of EGFR, combined with specific binding to the receptor in intact cells. In vivo distribution of the radiolabeled compound revealed tumor/blood and tumor/muscle activity uptake ratios of about 7 and 5, respectively, 3 h following administration of a radiotracer. Nevertheless, only minor EGFR-specific uptake of the compound was detected in these studies, using either EGFR-negative tumors or blocking studies as controls. To improve the in vivo performance of ML04, administration via prolonged intravenous infusion is proposed. Detailed pharmacokinetic characterization of this bioprobe could assist in the development of a kinetic model that would afford accurate measurement of EGFR content in tumors.

Molecular determination of epidermal growth factor receptor in normal and neoplastic colorectal mucosa

The epidermal growth factor receptor (EGFr) is considered a major target for treatment of colorectal cancer (CRC). We found a mean EGFr content significantly lower but more activated in colonic neoplastic tissue than in paired normal mucosa. Phosphorylated (pY1068) EGFr detection in CRC may be a better tool than EGFr detection to select patients for targeted therapies.

Prognostic role of epidermal growth factor receptor in localized breast cancer: 15 Years of follow-up

Background: The expression of epidermal growth factor receptor (EGF-R) in breast cancer (BC) is generally considered as an unfavorable event during tumor progression. Its predictive role has been fairly well defined: EGF-R expression predicts tamoxifen unresponsiveness. EGF-R role in autocrine growth regulation was confirmed. However, reported results on its prognostic role in BC patients were conflicting. The prognostic role of EGF-R after 15 years of follow-up is analyzed in a group of 70 localized BC patients, presented at diagnosis in clinical stages I-III. Methods: BC patients newly diagnosed from December 1990 until March 1991, treated in accordance to the National Protocol, were selected for EGF-R analysis. Steroid receptors and EGF-R were determined at diagnosis in same frozen tissue samples, using biochemical methods. Except 6 patients who were lost from follow-up in the interval shorter than 60 months, the remaining patients were followed-up from 60-188 months. The total number of events was 32 relapses (46%), and 27 deaths (38.5%). Results: EGF-R expression was found in 28/70 patients (40%), and the EGF-R content higher than 26 fmol - in 15/70 patients (21%). Neither the expression, nor the high content of EGF-R showed any influence on disease-free or overall survival. Levels of EGF-R were similar in relapsing and relapse-free patients. Nodal status had the strongest infuence on prognosis. Conclusion: Our results suggest that the controversial findings, regarding the EGF-R prognostic role, might be the consequence of a genuine weak influence of EGF-R expression on disease outcome. .

Expression and prognostic value of EGFR in invasive breast cancer

Epidermal growth factor receptor (EGFR) is a membrane receptor expressed in a variety of solid human cancers and directly related with poor prognosis. The objective of this work was to evaluate the EGFR content in breast carcinomas, its possible relationship with different clinical-pathological parameters, and its potential prognostic significance and predictive value. EGFR levels were examined by radioligand binding assays in 846 patients with invasive breast cancer. The median follow-up period was 50 months. There was a wide variability of EGFR levels among the studied tumors (0.01-403 fmol/mg protein). Statistical analysis showed that EGFR levels were significantly higher in younger patients (p=0.0001). EGFR were also notably higher in ER-negative or PgR-negative tumors than in ER-positive (p=0.0001) or PgR-positive tumors (p=0.001). In addition, the presence of high intratumoral EGFR levels (cut-off: 6 fmol/mg protein) was associated with both shorter relapse-free survival (p=0.04) and overall survival (p=0.01) in the group of patients as a whole, as well as with overall survival in the subgroup of patients without any type of systemic adjuvant treatment (p=0.02). However, EGFR levels did not achieve significance as independent prognostic factor in the multivariate analysis. There is a wide variability of intratumoral EGFR levels in breast carcinomas, and these protein levels correlated positively with a poor prognosis in the t univariate analysis. However, further studies are necessary in order to assess the possible clinical value of EGFR in combination with other essential components of the EGFR family network.

Decorin Evokes Protracted Internalization and Degradation of the Epidermal Growth Factor Receptor via Caveolar Endocytosis

Decorin inhibits the epidermal growth factor receptor (EGFR) by down-regulating its tyrosine kinase activity, thereby blocking the growth of a variety of transformed cells and tumor xenografts. In this study we provide evidence that decorin directly binds to the EGFR causing its dimerization, internalization, and ultimately its degradation. Using various pharmacological agents to disrupt clathrin-dependent and -independent endocytosis, we demonstrate that decorin evokes a protracted internalization of the EGFR primarily via caveolar-mediated endocytosis. In contrast to EGF, decorin targets the EGFR to caveolae, but not to early or recycling endosomes. Ultimately, however, both EGF- and decorin-induced pathways converge into late endosomes/lysosomes for final degradation. Thus, we have discovered a novel biological mechanism for decorin that could explain its anti-proliferative and anti-oncogenic mode of action.

A critical role for enhanced TGF-α and EGFR expression in the initiation of parathyroid hyperplasia in experimental kidney disease

The parathyroid hyperplasia secondary to kidney disease is associated with enhanced expression of the growth promoter transforming growth factor-alpha (TGF-alpha). TGF-alpha stimulates growth through activation of its receptor, the epidermal growth factor receptor (EGFR), normally expressed in the parathyroid glands. Because enhanced coexpression of TGF-alpha and EGFR causes aggressive cellular growth, these studies utilized highly specific inhibitors of EGFR tyrosine kinase, a step mandatory for TGF-alpha-induced EGFR activation, to assess the contribution of growth signals from enhanced expression of TGF-alpha exclusively or both TGF-alpha and EGFR to the rapid parathyroid growth induced by kidney disease and exacerbated by high-phosphorus (P) and low-calcium (Ca) diets in rats. The enhancement in parathyroid gland weight and proliferating activity (proliferating cell nuclear antigen/Ki67) induced by kidney disease and aggravated by either high P or low Ca intake, within the first week after 5/6 nephrectomy, in rats, coincided with simultaneous increases (2- to 3-fold) in TGF-alpha and EGFR content. Conversely, prevention of the increases in both TGF-alpha and EGFR paralleled the efficacy of either P restriction or high-Ca intake in ameliorating uremia-induced parathyroid hyperplasia. More importantly, suppression of TGF-alpha/EGFR signaling, through prophylactic administration of potent and highly selective inhibitors of ligand-induced EGFR activation, completely prevented both high-P- and low-Ca-induced parathyroid hyperplasia as well as TGF-alpha self-upregulation. Thus enhanced parathyroid TGF-alpha/EGFR expression, self-upregulation, and growth signals occur early in kidney disease, are aggravated by low-Ca and high-P intake, and constitute the main pathogenic mechanism of the severity of parathyroid hyperplasia.

Lack of response to epidermal growth factor in adult neural progenitor cells

Neural progenitor/stem cells reside in the subventricular zone and dentate gyrus of the adult rat brain, which proliferate in response to several growth factors. Here we show that the neurospheres generated from the adult dentate gyrus and subventricular zone proliferate in vitro in response to fibroblast growth factor-2, but not epidermal growth factor. Likewise, extracellular signal-regulated kinase phosphorylation was stimulated by fibroblast growth factor-2, but not epidermal growth factor. Immunoblotting demonstrated negligible epidermal growth factor receptor content in these neurospheres, indicating that neural progenitor cells isolated from the adult rat brain are not responsive to epidermal growth factor in vitro, because of the lack of epidermal growth factor receptors and/or their signaling. However, the lack of response by epidermal growth factor in our study could be because of the culture conditions, and may not reflect the physiological condition.

Prognostic significance of EGF receptor expression in early glottic cancer

Objective: A positive relationship between epidermal growth factor receptor (EGFR) expression and radioresistance has been shown both in vitro and in vivo. In a group of 31 patients with early glottic cancer treated with definitive radiotherapy, the relationship of EGFR expression with patient and tumor related parameters were analyzed and the prognostic effect of EGFR expression on local control (LC) was assessed. Material and method: Between 1991 and 2001, 114 patients with early glottic (Tis-T2N0M0) squamous cell carcinoma were treated with radiotherapy at our institution. Among these, 31 patients whose pretreatment pathology specimens were available for immunohistochemical analysis formed the study population. Median age was 64 (46–77). Anterior commissure involvement was evident in 12 (38.7%) patients. Distribution according to T stage was as follows: Tis 6 (19.3%), T1 22 (71%), and T2 3 (9.7%). Histopathological grades of the 25 T1-2 tumors were 10/25 (40%) grade 1, 9/25 (36%) grade 2 and 6/25 (24%) grade 3. Our radiotherapy regimen was 66–70 Gy in 33–35 fractions over 6.5–7 weeks. The median follow-up period was 45 months (range, 5–116). Following immunohistochemical staining, quantitative immunohistochemistry (IHC) was performed by image analysis software and stained tumoral area percentage (STAP) was identified. The cut-off value was ≤5% versus >5%. The relationship of EGFR expression with patient (age) and tumor related (T stage, histopathological grade, and anterior commissure involvement) parameters was evaluated using chi-square test. Prognostic significance of EGFR expression, age, T stage, histopathological grade, and anterior commissure involvement on LC was assessed using log-rank test. Results: No difference was found in EGFR content distribution in relation to age, T stage, histopathological grade, and anterior commissure involvement. In the univariate analysis including age (≤60 versus >60), T stage (Tis and T1 versus T2), histopathological grade (grade 1 and 2 versus grade 3), anterior commissure involvement (present versus absent), and EGFR expression (high versus low), only T stage and EGFR expression were found to be significant prognostic factors affecting LC ( P = 0.0006 and P = 0.03, respectively). Conclusion: The results of this series support that EGFR expression is an unfavorable prognostic factor in early glottic carcinomas. For this reason EGFR IHC may be considered for selecting patients for more aggressive therapies (radiotherapy with different fractionation schemes or surgery) or enrollment into trials targeting EGFR signaling pathways.

Role of the microtubule cytoskeleton in traffic of EGF through the lacrimal acinar cell endomembrane network

We have previously documented a novel biphasic traffic pattern for epidermal growth factor (EGF) in the acinar epithelial cell of the lacrimal gland. Different from the typical paradigm observed in many other cell types, EGF initially accumulates in the acinar basal–lateral recycling endosome, then is re-directed to the prelysosomes and lysosomes and degraded. While the cellular content of intact EGF decreases by 40% between 20 and 120 m of continuous incubation at 37°C, the EGF receptor (EGFR) content decreases only modestly [J. Cell Physiol. 199 (2004) 108]. The purpose of the present study was to investigate the role of the microtubule cytoskeleton in this traffic. Primary cultured rabbit lacrimocytes were incubated with [ 125I]-EGF, lysed, and analyzed by subcellular fractionation on sorbitol density gradients. Nocodazole treatment appeared to slightly decrease the initial uptake rate but to have no significant effect on the total amount of [ 125I] accumulation. However, it enhanced accumulation of [ 125I]-EGF and EGFR in the basal–lateral recycling endosome, and it enhanced accumulation of prepro- and pro- cathepsin B in fractions containing late endosomes and prelysosomes. Nocodazole permitted the time-dependent release of [ 125I]-EGF from the recycling endosome, but it partially inhibited [ 125I]-EGF degradation and decreased accumulation of [ 125I]-labeled degradation products in the lysosome. The microtubule-based molecular motors, cytoplasmic dynein and kinesin, were localized in compartments containing the late endosomes, prelysosomes, and lysosomes, consistent with the suggestion that microtubule-based molecular motors play important roles in traffic within the lysosomal pathway. Confocal fluorescence microscopy imaging of FITC-EGF substantiated the effects observed in biochemical studies by demonstating that nocodazole increased accumulation in a peripheral compartment and decreased traffic to a perinuclear compartment. These data suggest that initial accumulation in the basal–lateral recycling endosome and subsequent release from the recycling endosome to the late endosomes and prelysosome are not microtubule-dependent. On the other hand, microtubule-based motors are more critical for traffic from the prelysosome to the lysosome.

Allelic length of a CA dinucleotide repeat in the egfr gene correlates with the frequency of amplifications of this sequence—first results of an inter‐ethnic breast cancer study

Overexpression of the epidermal growth factor receptor (EGFR) is a common finding in invasive breast cancer and represents a potential target for new treatment options. However, little is known about the parameters that might indicate a potential clinical response for these anti-EGFR-based therapies. In order to gain further insights into the interplay between the length of a CA-SSR I repeat in intron 1 of egfr, copy numbers of this untranslated regulatory sequence, and protein expression, the present study investigated breast cancers from Germans and Japanese patients by microsatellite analysis, quantitative 5' nuclease assay by egfr enzyme-linked immunosorbent assay (ELISA), and comparative genomic hybridization (CGH). Japanese breast cancer patients displayed significantly longer alleles for the CA-SSR I repeat (p < 0.001), associated with significantly lower EGFR expression (mean 65 versus 36 fmol/mg membrane protein). Allelic imbalance (restricted to CA-SSR I) was observed in 55% of the informative Japanese breast cancers compared with only 34% of the German breast cancer reference group. Using a quantitative 5' nuclease assay for egfr, a significantly higher percentage of Japanese breast cancer patients revealed amplifications of the CA-SSR I repeat (p < 0.01). Japanese patients with these amplifications were characterized by a significantly higher EGFR content compared with the German breast cancer patients (p < 0.05). These data show, on the one hand, that the correlation of EGFR overexpression and an inherited CA repeat polymorphism within intron 1 of egfr is a general finding in breast cancer, as has been shown previously. On the other hand, the data demonstrate clearly for the first time an interaction between the length of a polymorphism in intron 1 of egfr as an inherited genetic factor and the frequency of egfr amplification, as an acquired genetic factor, both factors contributing to EGFR overexpression in breast cancer. This new knowledge about mechanisms of regulation of EGFR expression might serve as an additional basis for evaluating anti-EGFR-based therapies.