Study of egfr internalization and degradation in hela cells. The epidermal growth factor receptor egfrher1 and its downstream signaling events are important for regulating cell growth and behavior in many epithelial tumors types. By transfecting hela, mcf7, and 293t cells with cbl sirna or. Epidermal growth factor receptor egfr is a transmembrane protein that is activated by binding of its specific ligands, including epidermal growth factor and transforming growth factor. Quantification of epidermal growth factor receptor expression. Erbb2 has no known direct activating ligand, and may be in an activated state constitutively or become active upon heterodimerization with other family members such as egfr. This positioning allows the receptor to attach bind to other proteins, called ligands. The epidermal growth factor receptor egfr her1 and its downstream signaling events are important for regulating cell growth and behavior in many epithelial tumors types.
Hela cells are stimulated under 3 different conditions by an egf solution to. Egfr and egfrviii expression in primary breast cancer and cell lines james m. The egfr gene provides instructions for making a receptor protein called the epidermal growth factor receptor, which spans the cell membrane so that one end of the protein remains inside the cell and the other end projects from the outer surface of the cell. The prognostic value of egfr overexpression and amplification. Jun 18, 2010 cholangiocarcinoma cc is a malignant neoplasm of the bile ducts or the gallbladder. Understanding the factors, which control erbb2 and egf receptor egfr status in cells is likely to inform future therapeutic approaches directed at these potent oncogenes. The result of qrtpcr showed that egfr mrna level is the same in cells transfected with consi and shp1sirna. Therefore, we analyzed the protein expression and gene copy variation of the epithelial growth factor receptor egfr in chinese esophageal squamous cell carcinoma escc and explored the possible. To examine how shp1 regulates egfr protein expression, quantitative q rtpcr was used to analyze whether egfr mrna expression was affected in mcf10a cells with shp1 knockdown fig. Number of egfr copies in a hela cell human homo sapiens. Sigismund, in progress in molecular biology and translational science, 2016.
Egfr signals were counted from at least 100 cancer cell nuclei, and were divided into six types. Egfr overexpression is involved in multiple tumors and is associated. The results demonstrate that spri is a valuable tool for direct quantification of membrane protein expression level and ligand binding kinetics at single cell resolution. Notch and egfr pathway interaction regulates neural stem.
Summary of egfr erbb, erbb1 expression in human tissue. First, we used a rtpcr assay that can simu ltaneously measure eg frwt and egfrviii mrn a to. Epidermal growth factor receptor expression affects the efficacy of. Oct 27, 2009 biochemical analysis using coimmunoprecipitation and proximitybased quantitative veratag assays demonstrated that high levels of egfr phosphorylation, egfregfr homodimerization, and egfrher2 heterodimerization were present in the trastuzumabresistant cells. The egfr gene provides instructions for making a receptor protein called the epidermal growth factor receptor, which spans the cell membrane so that one end of the protein remains inside. However, current single agent receptor targeting does not achieve a maximal therapeutic effect, and some mutations confer resistance to current available agents. It is also possible that there is another pathway of egfr endocytosis in hela cells that is independent of grb2, analogous to the pathway operating in nih 3t3 cells expressing. Egfr overexpression and activation in high her2, er. This study aimed to detect rbm5 expression in nonsmall cell lung cancer nsclc and to associate rbm5 expression with clinicopathological data from nsclc patients and egfr and kras expression to better understand the potential role of rbm5. However, we have previously shown, using size exclusion.
Epidermal growth factor receptor egfr, also known as erbb1 or her1 is a membrane bound protein that has been associated with a variety of solid tumors and the control of cell survival. Shp1 positively regulates egfr signaling by controlling. Expression of growth factor receptors and targeting of egfr. We have previously shown that an ar egfr autocrine loop is. Egfr has demonstrated expression on vascular endothelial cells, hela cells. Thus, in erlotinibresistant helar30 cells, treatment with rapamycin. In breast cancer, the role of egfr is complex and appears to vary relative to important clinical features including estrogen receptor er status. Complex posttranscriptional regulation of egfreceptor expression by egf and tgf in human prostate cancer cells article pdf available in british journal of cancer 8056.
Expression of egfr in cancer summary the human protein. First, we used a rtpcr assay that can simu ltaneously measure eg frwt and. Overexpression of igfi receptor in hela cells enhances in. Epidermal growth factor receptor egfrdependent signaling is involved in many. Caski high egfr expression, me180 and dotc2 4510 both. Expression of growth factor receptors and targeting of. Breast cancer resource book order online at atcc org, call 800 638 6597, 703 365 2700, or contact your local distributor 1 worldwide, breast cancer accounts for approximately 23% of all cancer diagnoses in women, and.
The aim of this study was to determine the expression of seven growth factor receptors in cc cell lines and to assess the effect of blocking the egfr receptor in vitro. Expression of egfr in cancer summary the human protein atlas. A431, a549, hela and hek293 cell lines were obtained from the american type culture collection rockville, md. Cell lines a431, bhk21, and sw480 all from german collection of. In the right panel, immunoblot analysis of egfr protein expression in mcf7 cells treated with pma. Enhanced dimerization drives ligandindependent activity of. In their series, the combination of egfr and her2 expression had a negative impact on disease outcome, compared with normal protein levels, which may have. Targeting of growth factor receptors showed therapeutic potential in palliative. In their series, the combination of egfr and her2 expression had a negative impact on disease outcome, compared with normal protein levels, which may have implications in the combined therapy. Activation of egfr on monocytes is required for human. A transcript encoding the epidermal growth factor receptor egfr was found to be among the downregulated transcripts in ad skin. Transgene expression was visualized 48 hours postinfection.
The current study was conducted to determine 1 the exact. Anomalous binding of epidermal growth factor to a431 cells is due to the effect of high receptor densities and a saturable endocytic system. Normal distribution across the dataset is visualized with box plots, shown as median and 25th and 75th percentiles. However, current single agent receptor targeting does not. Additional measurement also revealed that different egfr positive cell lines a431, hela, and a549 show receptor density dependent antiegfr binding kinetics. Recently, molecular therapies targeting epidermal growth factor receptor egfr have been developed for clinical use. Transfected cells expressed either the wildtype egfr. In the right panel, immunoblot analysis of egfr protein. Nevertheless, rna interference experiments provided the first direct evidence of the importance of grb2 and, in fact, any protein in egfr endocytosis. Differential expression of rbm5, egfr and kras mrna and. The egfr expression in these tumor cells was detected by flow cytometry figure s2 in supporting information. In the current study we have examined, in different nsclc cell lines, the combined effect of rna interference. However, egfr expression determined by immunohistochemistry does not predict clinical outcomes of colorectal cancer crc patients treated with cetuximab.
Rna binding motif 5 rbm5 is a tumor suppressor gene that modulates apoptosis through the regulation of alternative splicing of apoptosisrelated genes. The epidermal growth factor receptor egfr is an important chemotherapeutic target for tyrosine kinase inhibitors and antibodies that block the extracellular domain of egfr. Epidermal growth factor receptor egfrmediated positive. Enhanced dimerization drives ligandindependent activity. Betulinic acid ba and curcumin inhibited bladder cancer cell growth and downregulated specificity protein sp transcription factors, and this was accompanied by decreased expression of egfr mrna and protein levels. The endocytosis of egfr and localization of clathrin in hela cells were examined after stimulation by egf at various concentrations i. Expression of epidermal growth factor receptor detected by. Cell deathbased treatment of lung adenocarcinoma cell. Her2 overexpression in human gastric cancer cell lines. Therefore, we evaluated the correlation between egfr levels detected by cetuximab and drug sensitivities of crc cell. Our first job is to listen to and observe what our customers need, and meet those needs with quality products and services. Pdf complex posttranscriptional regulation of egfreceptor. In view of the prominent role in cancer cell biology and alteration in substantial numbers of escc, defining egfr molecular characteristics relevant to patient prognosis is of. Egfr associated expression profiles vary with breast tumor.
Epidermal growth factor receptor endocytic traffic perturbation by. It is a chimeric monoclonal igg1 antibody that binds to egfr ecd, blocks ligand binding and induces receptor internalization and degradation, resulting in downregulation of. Characterization of egfr and erbb2 expression in atopic. Erbb2 is resistant to stimulusinduced degradation and high levels of overexpression can inhibit egf receptor downregulation. C, inhibition of egfr activity decreases the upregulation of rptp.
Silencing of egfr in hela cells was achieved by double transient transfection, using oligofectamine invitrogen, of the following short interfering rna sirna oligo. Nov 25, 2014 study of egfr protein expression and mutation 1. Quantification of epidermal growth factor receptor. We tested egfr expression on different blood leukocyte populations by flow cytometry. Pdf egfr and egfrviii expression in primary breast cancer. Hela cells were either mocktransfected or stably transfected with the wildtype wt or a cs mutant mtcptp expression plasmid, and were infected with ssaav2egfp or scaav2egfp vectors. Grb2 regulates internalization of egf receptors through. In view of the prominent role in cancer cell biology and alteration in substantial numbers of escc, defining egfr molecular characteristics relevant to patient prognosis is of great importance. Drugs that target specificity proteins downregulate epidermal. Jan 25, 2018 however, when autophagy is further elevated by treatment in addition to egfr tkis, it can induce autophagic cell death.
By transfecting hela, mcf7, and 293t cells with cbl sirna or dominantnegative 70zcbl, we found that at high egf doses, cbl is required for egfr endocytosis in mitotic cells, but not in interphase cells. Five cervical cancer cell lines, covering the entire range of egfr mrna expression, were selected for further study. Pdf study of egfr internalization and degradation in hela cells. Knockdown of hip1 expression promotes ligandinduced. Targeting the epidermal growth factor receptor in non. Apr 26, 2012 rna binding motif 5 rbm5 is a tumor suppressor gene that modulates apoptosis through the regulation of alternative splicing of apoptosisrelated genes. Caski high egfr expression, me180 and dotc2 4510 both midrange egfr expression, hela low egfr expression, and c33a negative for egfr expression. Impact of epidermal growth factor receptor egfr cell surface. Egfr and egfrviii expression in primary breast cancer and. The current study was conducted to determine 1 the exact frequency of egfr protein overexpression, 2 the correlation between protein overexpression and egfr amplification, and 3 the correlation between the status of the genetic and clinicopathologic features in nonsmall cell. Targeting the epidermal growth factor receptor in nonsmall. Sep 02, 2015 we stably expressed egfr with an nterminal hemagglutinin tag haegfr in cho cells, which lack endogenous egfr.
We stably expressed egfr with an nterminal hemagglutinin tag haegfr in cho cells, which lack endogenous egfr. In contrast to the wt receptor, expression of egfrl858r or egfr. The epidermal growth factor receptor egfr is a validated therapeutic target in nonsmall cell lung cancer nsclc. However, when autophagy is further elevated by treatment in addition to egfr tkis, it can induce autophagic cell death. The cell lines in the human protein atlas have been analyzed by rnaseq to estimate the transcript abundance of each proteincoding gene. In conclusion, the current study presents novel insights into the impact of. Complex posttranscriptional regulation of egfreceptor. The epidermal growth factor receptor egfr is a member of the erbb family of receptor tyrosine kinases and is upregulated in many cancer cell types, including breast cancer and head and neck squamous cell carcinoma hnscc 36,62. Mar 12, 2020 it is a chimeric monoclonal igg1 antibody that binds to egfr ecd, blocks ligand binding and induces receptor internalization and degradation, resulting in downregulation of surface egfr expression. To examine how shp1 regulates egfr protein expression, quantitative q rtpcr was used to analyze whether egfr mrna expression was affected in mcf10a cells with shp1 knockdown. A pharmacologic approach for our initial studies was a logical first step, as drugs can be added to cells via the medium and quickly perturb trafficking. Quantification of the egfr expression level in cell membranes and the interaction kinetics with drugs are thus important for cancer diagnosis and treatment. Cytoplasmic and membranous expression in several tissues, most abundant in placenta.
Her2 and her3 cooperatively regulate cancer cell growth. Atcc stands ready to support our customers needs during the coronavirus pandemic. Correlating egfr expression with receptorbinding properties. Quantitative analysis reveals how egfr activation and. The rnaseq data was then used to classify all genes according to their cell linespecific expression into one of six different categories, defined based on the total set of all nx values in all analyzed cell. Oct 06, 2015 additional measurement also revealed that different egfr positive cell lines a431, hela, and a549 show receptor density dependent anti egfr binding kinetics. Because her3 does not possess kinase activity and a selective inhibitor of her2 but not of an epidermal growth factor receptor reduced the phosphoher3 level, her3 was suggested to be transphosphorylated by her2. Epidermal growth factor receptors with tyrosine kinase domain. In control hela cells, we detected significant ubiquitylation of egfr after.
Thus, in erlotinibresistant helar30 cells, treatment. Here, we found that contrary to interphase cells, mitotic egfr endocytosis is more reliant on the activation of the e3 ligase cbl. In the lower left panel, qpcr analysis of egfr expression in mcf7 cells treated with pma. Targeting of growth factor receptors showed therapeutic potential in palliative settings for many solid tumors. Biochemical analysis using coimmunoprecipitation and proximitybased quantitative veratag assays demonstrated that high levels of egfr phosphorylation.
All the cells were cultured in a humidified incubator at 37c with 5% co 2 and 70% relative humidity. A her2 expression determined by immunohistochemistry of xenografts in nude mice formed after subcutaneous injection of glm1, glm2, glm4 and ncin87 cells. Three classes of epidermal growth factor receptors on hela cells. Here, we further investigated the expression pattern of two egfr family members egfr and erbb2 in ad skin on a protein level. Hopp n, hagen j, aggeler b and a kalyuzhny 2017 express. Regulation of egfr endocytosis by cbl during mitosis mdpi. Cholangiocarcinoma cc is a malignant neoplasm of the bile ducts or the gallbladder. Levels of expression of endogenous egfr in hela cells and hcc827 cells, related to figure 2. Atcc is the premier global biological materials resource and standards. Protein overexpression and gene amplification of epidermal. Tissue expression of egfr summary the human protein atlas. Regulation of erbb2 receptor status by the proteasomal dub. To investigate egfr signaling using a genomics approach, several breast basal. Our first job is to listen to and observe what our customers need, and meet those needs with quality.
Egfr del19t790m stably expressing baf3 stable cell line forprofit el014fpgvoso. Report egfr and egfrviii expression in primary breast cancer and cell lines james m. As can be seen in figure 2a, whereas mockinfected hela cells. Our previous studies showed that the overexpression of novel. Egfrmediated beclin 1 phosphorylation in autophagy. Regulation of erbb2 receptor status by the proteasomal.
We now show that for hela cells expressing similar numbers of egfr and erbb2, egfr down. Transfected cells expressed either the wildtype egfr egfrwt, egfrl858r, or egfr. Drugs that target specificity proteins downregulate. Her2 and her3 cooperatively regulate cancer cell growth and. Pdf egfr and egfrviii expression in primary breast. Mar 21, 2012 the epidermal growth factor receptor egfr is a validated therapeutic target in nonsmall cell lung cancer nsclc. Shp1 positively regulates egfr signaling by controlling egfr. Epidermal growth factor receptor egfr inhibitors have been studied in head and neck cancer. Immunohistochemical ihc analysis of egfr and erbb2 showed decreased expression of.
We observed that hbecs expressing egfr deletion e746a750, or egfr l858r. Egfr overexpression and activation in high her2, er negative. Her3 was highly phosphorylated in tak285sensitive cells, where tak285 treatment reduced her3 phosphorylation level. The pathologies associated with egfrs have led to novel treatment. Figure 1 compares the levels of egfr phosphorylation on tyr1068. We conclude that egfr overexpression can mediate trastuzumab resistance in both. Rna cancer tissue category is calculated based on mrna expression levels across all 17 cancer tissues and include. The epidermal growth factor receptor egfr and its ligand amphiregulin ar have been shown to be coover expressed in breast cancer. Cells free fulltext regulation of egfr endocytosis by. The egfr trafficking in hela cells has been characterized in detail 21, 23, making these cells an excellent model for our studies. Cetuximab is a chimeric mousehuman monoclonal antibody that targets the human epidermal growth factor receptor egfr. Endosomal accumulation of the activated epidermal growth. Because her3 does not possess kinase activity and a selective. A431, hela and hek293 cells were grown in dulbecco modified eagle medium dmem lonza, walkersville, md usa with 10% fetal bovine serum fbs and 1% penicillin and.