EGF) And Its Receptor Research Articles

Abstract 3211: OQL025, a topical cream for the prevention of epidermal growth factor receptor inhibitor-induced skin rash

Abstract Background: Epidermal growth factor (EGF) and its receptor (EGFR) are expressed by several cell types including epidermal keratinocytes. EGF facilitates skin healing by promoting DNA synthesis and cell proliferation. However, driver mutations in EGFR can cause uncontrolled cell growth and cancers. Thus, EGFR has become a therapeutic target for tumors, especially those with EGFR mutations. EGFR inhibitors (EGFRIs) are widely used to treat various tumor types (e.g., non-small cell lung & colorectal cancers). EGFRIs cause inflammatory skin reactions (e.g., acneiform rash) generally within the first 3 weeks of therapy. We propose a novel topically applied cream (OQL025) containing a prodrug, OQRC184 that releases tofacitinib, a Janus Kinase (JAK) inhibitor, as a prophylaxis for EGFRi-induced skin rash. Methods: OQRC184 is designed to enhance lipophilicity/solubility and when topically applied releases tofacitinib into cutaneous tissue. OQRC184 & tofacitinib potencies were characterized for JAK (JAK1, JAK2, JAK3, TyK2) in a cell-free isolated kinase assay. Pharmacokinetics (PK) were studied in Bama minipigs (n=15) with shaved dorsal skin (~10 mg/cm2) that were randomly assigned to 3 groups of OQRC184 @ 0.15, 0.3, 0.5 mg/cm2. Blood was collected (1 mL in EDTA-K2) at pre-dose & 0.5, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, and 168 h post-dose. Plasma concentrations of OQRC184 and tofacitinib (LLOQ of 0.01 ng/mL range 0.01 to 10 ng/mL) were determined with LC-MS/MS. Prevention of EGFRI-induced rash was studied with topically applied 3% or 6% OQRC184 or vehicle cream (CRM) in an SD rat EGFRI-induced rash model (oral afatinib 30 mg/kg (AF)), QD vs. vehicle (water (VW)). The four groups were VW+CRM; AF+CRM; AF+3%OQRC184; AF+6% OQRC184. Results: OQRC184 had low inhibitory activity compared to tofacitinib against JAK1, JAK2, JAK3, and TyK2 with IC50 ratios of OQRC184 to tofacitinib for JAK1 (3610:1), JAK2 (4435:1), JAK3 (3814:1) and TyK2 (>385:1). After a single topical administration, OQRC184 plasma concentrations were below the LLOQ in 42~52% of samples, and <10-fold of LLOQ in 83%~88% of samples. Tofacitinib plasma concentrations were below the LLOQ in 67~87% of samples and <10-fold of LLOQ in 90%~97% of samples. All rats treated with AF developed a rash by day 5. The incidence of rash grade ≥ 2 was lower in 3% and 6% OQRC184 groups vs. controls. Prophylactic treatment with 6% OQRC184 had almost no rash ≥ grade 2. The mean ±SE rash grade in the 3% and 6% OQRC184 prophylactic treatment groups was significantly lower than in the AF+CRM group (3% (0.53±0.23), 6% (0.30±0.16) vs. AF+CRM (1.03±0.44 (p<0.05)). Conclusion: OQRC184, a prodrug designed for releasing tofacitinib into dermal layers, lacks inhibitory potential for a family of JAK kinases. OQL025 topical cream containing OQRC184 showed minimal systemic exposure for either OQRC184 or tofacitinib and effectively prevented/reduced EGFRI-induced rash in preclinical models. Citation Format: Shilan Liu, Wenqin Zeng, Wenxi Li, Qing You, Robert Claude Tyler, Jie Luo, Hong Tang, Shiyi Zhang. OQL025, a topical cream for the prevention of epidermal growth factor receptor inhibitor-induced skin rash [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3211.

Lack of Association between Epidermal Growth Factor or Its Receptor and Reflux Esophagitis, Barrett's Esophagus, and Esophageal Adenocarcinoma: A Case-Control Study.

The epidermal growth factor (EGF) and its receptor (EGFR) gene-gene interactions were shown to increase the susceptibility to esophageal cancer. However, the role of the EGF/EGFR pathway in the development of gastroesophageal reflux disease (GERD) and its complications (reflux esophagitis (RE), Barrett's esophagus (BE), and esophageal adenocarcinoma (EAC)) remains unclear. This association study is aimed at investigating functional EGF and EGFR gene polymorphisms, their mRNA expression in esophageal tissues, and EGF plasma levels in relation to RE, BE, and EAC development in the Central European population. 301 patients with RE/BE/EAC (cases) as well as 98 patients with nonerosive reflux disease (NERD) and 8 healthy individuals (controls) were genotyped for +61 A>G EGF (rs4444903) and +142285 G>A EGFR (rs2227983) polymorphisms using the TaqMan quantitative polymerase chain reaction (qPCR). In random subgroups, the EGF and EGFR mRNA expressions were analyzed by reverse transcription qPCR in esophageal tissue with and without endoscopically visible pathological changes; and the EGF plasma levels were determined by enzyme-linked immunosorbent assay. None of the genotyped SNPs nor EGF-EGFR genotype interactions were associated with RE, BE, or EAC development (p > 0.05). Moreover, mRNA expression of neither EGF nor EGFR differed between samples of the esophageal tissue with and without endoscopically visible pathology (p > 0.05) nor between samples from patients with different diagnoses, i.e., RE, BE, or EAC (p > 0.05). Nevertheless, the lower EGF mRNA expression in carriers of combined genotypes AA +61 EGF (rs4444903) and GG +142285 EGFR (rs2227983; p < 0.05) suggests a possible direct/indirect effect of EGF-EGFR gene interactions on EGF gene expression. In conclusion, EGF and EGFR gene variants and their mRNA/protein expression were not associated with RE, BE or EAC development in the Central European population.

Epidermal Growth Factor Is Increased in Conjunctival Malignant Melanoma.

Conjunctival malignant melanoma (CMM) is a rare, but very aggressive tumor with a high metastasis rate. Not much is known about the CMM metastasis mechanisms. So far, epidermal growth factor (EGF) and its receptor (EGF-R) as well as macrophages and matrix metalloproteinase 9 (MMP-9) have been reported to lead to metastasis by epithelial-mesenchymal-transition and tumor migration in different solid tumors. Therefore, we evaluated whether EGF and EGF-R, CD68 and MMP-9 are altered in CMM samples in comparison to conjunctival nevi and healthy conjunctiva. EGF, EGF-R, the macrophage marker CD68 and MMP-9 expression were analyzed in human conjunctival melanoma (CMM, n=16), human conjunctival nevi (n=13) and disease-free human conjunctiva (controls, n=14) by immunohistology. Staining of each sample was evaluated using a standardized score ranging from negative (0) to triple positive (3). The groups were then compared by ANOVA, followed by Tukey's post-hoc test. A statistically significant increase of EGF was seen in CMM samples in comparison to conjunctival nevi (p=0.03). In contrast, no statistically significant differences in EGF-R expression were noted between the three groups. A statistically significant increase of CD68 was only seen in conjunctival nevi compared to controls (p=0.04). MMP-9 expression was similar in all groups. In CMM, the study data demonstrated an up-regulation of EGF in comparison to conjunctival nevi. Hence, EGF might promote proliferation of CMM cells and induce the epithelial-mesenchymal transition. Therefore, our data suggest that an interplay between EGF and CMM might have a critical role in the developing CMM tumors and metastasis.

EGF+61 A>G polymorphism does not predict response to first-generation EGFR tyrosine kinase inhibitors in lung cancer patients.

Epidermal growth factor (EGF) and its receptor (EGFR) play a paramount role in lung carcinogenesis. The polymorphism in the EGF promoter region EGF+61A>G (rs4444903) has been associated with cancer susceptibility, but its role in lung cancer patients treated with tyrosine kinase inhibitors (TKIs) remains unknown. Here, we aimed to evaluate the predictive and prognostic role of EGF+61A>G SNP in lung cancer from Brazilian EGFR‐mutated TKI‐treated patients. Herein, patients carrying EGFR‐sensitizing mutations submitted to TKI treatment (gefitinib/erlotinib) were analyzed (n = 111) for EGF+61A>G genotype by TaqMan genotyping assay. TKI treatment was classified as partial response (PR), stable disease (SD), and disease progression (DP), according to RECIST1.1. Association analysis was assessed by chi‐square and Fisher's test (univariate) and multinomial model (multivariate) and survival analysis by Kaplan‐Meier method and log‐rank test. The EGF+61A>G genotype frequencies observed were: AA = 31.5% (n = 35), AG = 49.6% (n = 55) and GG = 18.9% (n = 21). The allelic frequencies were 56.3% for A, and 43.7% for G and the population was in Hardy‐Weinberg equilibrium (P = 0.94). EGF+61A>G codominant model (AA vs. AG vs. GG) was associated with a response to TKIs (P = 0.046), as well as a recessive model (AA vs. AG + GG; P = 0.023). The multinomial regression showed an association between the codominant model (AG) and recessive model (AG + GG) with SD compared with DP (P = 0.01;OR = 0.08; 95% CI = 0.01–0.60 and P = 0.02;OR = 0.12; 95% CI = 0.20–0.72, respectively). No association between genotypes and progression‐free or overall survival was observed. In conclusion, the EGF+61 polymorphism (AG and AG + GG) was independently associated with stable disease in lung cancer patients although it was not associated with the overall response rate to first‐generation TKIs or patient outcome.

Blockade of epidermal growth factor and its receptor and axial elongation in experimental myopia.

To examine the influence of epidermal growth factor (EGF) and its receptor (EGFR) on axial ocular elongation, we intraocularly injected an EGF antibody and an EGFR antibody into young guinea pigs with lens-induced axial elongation (myopization). Mean axial elongation was reduced in the eyes injected with the EGF/EGFR-antibody compared with the contralateral control eyes injected with PBS (phosphate-buffered solution) (0.43±0.13mm vs 0.53±0.13mm; P<.001). The intereye difference in axial length increased (P=.005) as the doses of the EGF antibody and EGFR antibody increased. As a corollary, the thickness of the retina at the posterior pole was dose-dependently increased in the injected eyes compared to the contralateral control eyes. Immunohistochemical staining for EGF and the relative mRNA expression of EGF and EGFR were the highest in eyes not injected with the EGF antibody or EGFR antibody and decreased (P<.05) as the dose of EGF antibody or EGFR antibody increased. In an in vitro study, EGF had a stimulating effect and the EGF antibody had an inhibitory effect on the proliferation and migration of RPE cells. The findings showed that the intravitreal application of an EGF antibody and EGFR antibody is associated with a dose-dependent reduction in lens-induced axial elongation in young guinea pigs. The EGFR family may play a role in axial elongation of the eye and in the development of myopia.

Cultured Cordyceps sinensis polysaccharides attenuate cyclophosphamide-induced intestinal barrier injury in mice

Abstract Intestine is not only the largest digestive and absorbing organ, but also an important defensive barrier of the body. Maintaining healthy intestinal barrier is a prerequisite for intestinal function. In this study, polysaccharides from the cultured Cordyceps sinensis (CSP) were orally given to cyclophosphamide (Cy)-treated mice to investigate its protective effect on intestinal barrier injury. Results showed that CSP had positive effects on intestinal morphology, including restoring villus length and crypt depth as well as improving quantity of goblet cells and mucins expression. Furthermore, CSP enhanced intestinal alkaline phosphatase (IAP), diamine oxidase (DAO), epidermal growth factor (EGF) and its receptor (EGFR). In addition, occludin, claudin-1 and zonula occludins (ZO)-1 were upregulated and myosin light chain kinase (MLCK), p-MLC were downregulated by CSP. Moreover, CSP promoted p-ERK and inhibited p-JNK, p-p38. These findings suggested that CSP could attenuate Cy-induced intestinal barrier injury which maybe associated with mitogen-activated protein kinases (MAPKs) pathways.

Abstract 2608: EGF signaling activates a TAZ-driven oncogenic program in glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most aggressive and lethal primary brain tumor in humans. Hyperactivation of the signaling pathway driven by the epidermal growth factor (EGF) and its receptor (EGFR) are commonly occurring in GBM. The oncogenic transcriptional program driven by EGF signaling is still incompletely understood in GBM. Here, we found that EGF induces the upregulation and nuclear translocation of TAZ, an oncogenic transcriptional co-activator. TAZ and its paralog gene YAP are commonly known as downstream mediators of the Hippo signaling pathway. TAZ has been found as a driver of mesenchymal transition in GBM cells and promotes GBM radioresistance. Our results support that EGF induces TAZ transcription and the activation of TAZ gene targets, through the signaling axis including EGFR, STAT3 and ERK. EGFRvIII is the most frequently occurring EGFR mutation in GBM causing constitutively active EGFR. We found that EGFRvIII causes EGF-independent activation of TAZ and its downstream oncogenic targets. Next, we used next-generation sequencing to establish a genome-wide map of TAZ gene targets in GBM cells after EGF stimulation. The results suggest that EGF signaling activates a TAZ-driven oncogenic program including autocrine and paracrine factors that promote GBM growth, angiogenesis, stemness and immunoescape. Moreover, we found that TAZ targeting by shRNAs or the FDA-approved drug Verteporfin effectively inhibits the TAZ-driven oncogenic program activated by EGF stimulation or the EGFRvIII mutant. TAZ targeting also inhibits GBM cell growth and xenograft formation. In contrast, enforced TAZ induces the TAZ-driven oncogenic program and promotes GBM cell growth and xenograft formation. In conclusion, our results reveal a novel TAZ-driven oncogenic program that is activated by EGF signaling in GBM. Our discoveries strongly support TAZ as a therapeutic target for GBM and likely other human cancer with hyperactivation of EGF signaling. Our preclinical studies also provide a clinically translatable strategy for GBM therapy based on using the FDA-approved drug Verteporfin. Citation Format: Minling Gao, Yi Fu, Laterra John, Mingyao Ying. EGF signaling activates a TAZ-driven oncogenic program in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2608.

EGF+61 A>G polymorphism is not associated with lung cancer risk in the Brazilian population.

Epidermal growth factor (EGF) and its receptor (EGFR) play an important role in lung carcinogenesis. A functional single nucleotide polymorphism (SNP) in EGF promoter region (EGF+61 A>G-rs4444903) has been associated with cancer susceptibility. Yet, in lung cancer, the EGF+61 A>G role is unclear. The aim of this study was to evaluate the risk of lung cancer associated with EGF+61 A>G SNP in the Brazilian population. For that, 669 lung cancer patients and 1104 controls were analyzed. EGF+61 A>G genotype was assessed by PCR-RFLP and TaqMan genotyping assay. Both patients and controls were in Hardy-Weinberg equilibrium. As expected, uni- and multivariate analyses showed that tobacco consumption and age were significant risk factors for lung cancer. The genotype frequencies in lung cancer patients were 27.3% of AA, 47.4% of AG and 25.3% of GG, and for controls were 25.3% of AA, 51.6% of AG and 23.1% of GG. The allele frequencies were 51.1% of A and 48.9% of G for both cases and controls. No significant differences for the three genotypes (AA, AG and GG-codominant model) were observed between cases and controls. We then grouped AG and GG (recessive model) genotypes, as well as AA and AG (dominant model), and again, no significant differences were also found. This is the largest study to explore EGF+61 A>G polymorphism association with lung cancer risk and suggests that this SNP is not a risk factor for lung cancer in the Brazilian population.

From the Editor’s desk....: September 2018

From the Editor’s desk....: September 2018

Abstract 1406: EGF+61 A&amp;gt;G polymorphism is not a lung cancer risk: A case-control study in a large Brazilian population

Abstract Lung cancer is a malignancy with high incidence and mortality worldwide, being in Brazil the first most lethal cancer in men and second in women. Epidermal growth factor (EGF) and its receptor (EGFR) play a central role in lung carcinogenesis, once EGF/EGFR interaction activates several intracellular pathways that control cellular growth, proliferation, differentiation, migration and apoptosis. It has been described the association between a single nucleotide polymorphism (SNP) in EGF promoter region (EGF+61 A&amp;gt;G - rs4444903) and cancer susceptibility to distinct tumors. In lung cancer, the results are still scarce and unclear, with different reports showing discrepant results. Therefore, the aim of this study is to evaluate the risk of lung cancer development associated with the EGF+61 A&amp;gt;G SNP in the Brazilian population. For that, 669 lung cancer patients and 1104 controls were analyzed. Following DNA isolation from both cases (FFPE or blood) and controls (blood), the EGF+61 A&amp;gt;G genotype was assessed by PCR-RFLP in FFPE samples, and TaqMan genotyping assay for blood's DNAs. As expected, uni- and multivariate analyses, showed that tobacco consumption (p&amp;lt;0.001; OR=11.47; 95% CI 8.44-15.58 for smokers and p&amp;lt;0.001; OR 3.06; 95% CI 2.28-4.10 for ex-smokers) and age (p&amp;lt;0.001; OR=3.74; 95% CI 1.2.95-4.74 for &amp;gt;65 years) were important risk factors for lung cancer. Both patients and controls were in Hardy-Weinberg equilibrium. The genotype frequencies observed in lung cancer patients were 27.4% of AA, 47.4% of AG and 25.3% of GG, and for controls were 25.3% of AA, 51.6% of AG and 23.1% of GG. The allele frequencies were 51.1% of A and 48.9% of G in patients for both cases and controls. No significant differences for the three genotypes (AA, AG and GG - model 1) were observed between cases and controls (p=0.116; AG genotype: OR=0.80; 95% CI 0.61-1.05 and GG genotype: OR=1.03; 95% CI 0.75-1.42). We further grouped AG and GG and compared with the AA genotype (model 2), as well grouped AA and AG, compared to GG genotype (model 3), and no significant differences were found (model 2 - p=0.3; OR=0.87; 95% CI 0.67-1.12; model 3 - p=0.197; OR= 1.19; 95% CI 0.91-1.55). Moreover, disease-free and overall survivals were calculated considering the 3 models, and no significant differences were observed. In conclusion, the present study suggest that EGF+61 A&amp;gt;G polymorphism is not a risk factor for lung cancer in Brazilian population. Citation Format: Ana C. Laus, Flavia E. de Paula, Marcos A. Lima, Carolina D. Carlos, Izabela N. Gomes, Pedro R. de Marchi, Luciano S. Viana, Cristovam Scapulatempo Neto, Rui M. Reis. EGF+61 A&amp;gt;G polymorphism is not a lung cancer risk: A case-control study in a large Brazilian population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1406.

Abstract A78: EGF+61 A&amp;gt;G polymorphism is not associated with lung cancer risk in Brazilian population

Abstract Lung cancer is a malignancy with high incidence and mortality, being in Brazil the second most common cancer in men and the fourth in women. Epidermal growth factor (EGF) and its receptor (EGFR) play a central role in lung carcinogenesis, once EGF/EGFR interaction activates several intracellular pathways that control cellular growth, proliferation, differentiation, migration, and apoptosis. The association has been described between a single nucleotide polymorphism (SNP) in EGF promoter region (EGF+61 A&amp;gt;G – rs4444903) and cancer susceptibility to distinct tumors, including melanoma, gliomas, gastric cancer, hepatocellular carcinoma, and others. In lung cancer, the results are still scarce and unclear, with different reports showing discrepant results. Therefore, the aim of this study is to evaluate the risk of lung cancer development associated with the EGF+61 A&amp;gt;G SNP in a Brazilian population. For that, 437 lung cancer patients and 1104 controls were included in this study. Following DNA isolation from both tumor (FFPE) and controls (blood), the EGF+61 A&amp;gt;G genotype was assessed by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), and TaqMan Genotyping Assay, respectively. Statistical analysis included chi-square and Mann-Whitney tests, and logistic regression model to estimate Odds Ratios. As expected, uni- and multivariate analyses showed that tobacco consumption (p&amp;lt;0.001; OR=4.6; 95% CI 3.53-6.2 for smokers and p&amp;lt;0.001; OR 1.9; 95% CI 1.48-2.56 for ex-smokers) and age (p&amp;lt;0.001; OR=1.03; 95% CI 1.02-1.04 per year) were important risk factors for lung cancer. The genotype frequencies observed in lung cancer patients were 27.9% of AA, 45.1% of AG, and 20% of GG, and for controls were 25.3% of AA, 51.6% of AG, and 23.1% of GG. The allele frequencies were 51% of A and 49% of G in patients, and 50% of each allele in controls. Hardy-Weinberg equilibrium was estimated among patients and controls and no deviation was found in control group. However, patient's group revealed a p=0.039. No significant differences for the three genotypes (AA, AG, and GG) were observed between cases and controls (p=0.064; AG genotype: OR=0.79; 95% CI 0.605-1.033 and GG genotype: OR=1.05; 95% CI 0.78-1.43). We further grouped AG and GG genotypes and compared with the AA genotype, and also did not found significant differences (p=0.3; OR=0.87; 95% CI 0.681-1.120). Moreover, overall survival was calculated for patients considering AG/GG versus AA genotypes, and no significant differences were observed (p=0.21). In conclusion, the present study suggests that EGF+61 A&amp;gt;G polymorphism is not a risk factor for lung cancer in the Brazilian population. Citation Format: Ana Carolina Laus, Flavia Escremim de Paula, Marcos Alves de Lima, Carolina Dias Carlos, Izabela Natalia Faria Gomes, Pedro Rafael Martins de Marchi, Luciano de Souza Viana, Rui Manuel Vieira Reis. EGF+61 A&amp;gt;G polymorphism is not associated with lung cancer risk in Brazilian population [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A78.

Bioactive factors in milk across lactation: Maternal effects and influence on infant growth in rhesus macaques (Macaca mulatta).

Among mammals, numerous bioactive factors in milk vary across mothers and influence offspring outcomes. This emerging area of research has primarily investigated such dynamics within rodent biomedical models, domesticated dairy breeds, and among humans in clinical contexts. Less understood are signaling factors in the milk of non-human primates. Here, we report on multiple bioactive components in rhesus macaque (Macaca mulatta) milk and their associations with maternal and infant characteristics. Milk samples were collected from 59 macaques at multiple time points across lactation in conjunction with maternal and infant morphometrics and life-history animal records. Milk was assayed for adiponectin (APN), epidermal growth factor (EGF) and its receptor (EGF-R), and transforming growth factor beta 2 (TGF-β2 ). Regression models were constructed to assess the contributions of maternal factors on variation in milk bioactives, and on the relationship of this variation to infant body mass and growth. Maternal body mass, parity, social rank, and infant sex were all predictive of concentrations of milk bioactives. Primiparous mothers produced milk with higher adiponectin, but lower EGF, than multiparous mothers. Heavier mothers produced milk with lower EGF and EGF-R, but higher TGF-β2 . Mothers of daughters produced milk with higher TGF-β2 . Mid-ranking mothers produced milk with higher mean EGF and adiponectin concentrations than low-ranking mothers. Milk EGF and EGF-R were positively associated with infant body mass and growth rate. Importantly, these signaling bioactives (APN, EGF, EGF-R, and TGF-β2 ) were significantly correlated with nutritional values of milk. The effects of milk signals remained after controlling for the available energy in milk revealing the added physiological role of non-nutritive milk bioactives in the developing infant. Integrating analyses of energetic and other bioactive components of milk yields an important perspective for interpreting the magnitude, sources, and consequences of inter-individual variation in milk synthesis. Am. J. Primatol. 78:838-850, 2016. © 2016 Wiley Periodicals, Inc.

Chemopreventive effect of leflunomide against Ehrlich's solid tumor grown in mice: Effect on EGF and EGFR expression and tumor proliferation.

i) The current study aimed to examine the effect of leflunomide on tumoral expression of epidermal growth factor and its receptor (EGFR) in Ehrlich's ascites carcinoma (EAC) grown in mice. ii) Mice were injected subcutaneously with EAC cells and allocated into four groups; Group i: EAC control group. Groups ii-iv: mice treated with leflunomide (3, 10 or 30mg/kg/day, p.o.), respectively. Pharmacologic treatments were initiated at day 8 and continued for 14days. iii) Treatment with leflunomide evoked antitumor properties as indicated by reduction in tumor mass, histopathological score, number of intratumoral PCNA immunopositive nuclei. Leflunomide (3, 10 or 30mg/kg) exerted an anti-inflammatory effect as indicated by the reduction in serum tumor necrosis factor-α. Furthermore, leflunomide demonstrated anti-angiogenic activity which was expressed as a decline in serum vascular endothelial growth factor and down-regulation of intratumoral EGF protein and mRNA expression as well as EGFR expression in addition to suppression of immunostaining for the endothelial marker, CD31. iv) Taken together, the present results demonstrated that leflunomide possessed anti-angiogenic and anti-proliferative activity against EAC solid tumors that might be correlated to down regulation of EGF and EGFR. Further, the current data indicated that leflunomide may have utility in the management of human cancer.

Asporin participates in gastric cancer cell growth and migration by influencing EGF receptor signaling.

Asporin (ASPN), a novel member of the small leucine-rich proteoglycan (SLRP) family, serves as a key component of the tumor stroma and has been reported to be abnormally expressed in certain types of tumors. Specifically, the proteoglycan was proven to activate the coordinated invasion of scirrhous gastric cancer and cancer-associated fibroblasts. However, the role of ASPN in cancer cell growth and metastasis has not yet been addressed. In the present study, we aimed to evaluate the tumoricidal benefits of ASPN on tumorigenesis and progression of gastric cancer. Firstly, it was demonstrated that ASPN was overexpressed in gastric carcinoma tissues when compared to the corresponding non‑cancerous tissues, and it had varied levels of expression in gastric cancer epithelial cell lines. Additionally, we assessed the effects of transient siRNA‑mediated ASPN knockdown on gastric cancer cells. ASPN silencing inhibited proliferation and suppressed the migration of immortalized neoplastic epithelial cells. Furthermore, at the molecular level, we found that downregulation of ASPN blocked the anti-apoptotic molecule Bcl-2, increased the expression of pro-apoptotic molecule Bad, reduced the expression of migration-related proteins CD44 and matrix metalloproteinase (MMP)-2, and abrogated the activation of the phosphorylation status of ERK and epidermal growth factor (EGF) and its receptor (EGFR). Collectively, our findings indicate that ASPN is upregulated and plays an oncogenic role in gastric cancer progression and metastasis by influencing the EGFR signaling pathway.

Is salivary epidermal growth factor a biomarker for oral leukoplakia? A preliminary study

The aim of this study was to compare the salivary epidermal growth factor (EGF) levels between patients with oral leukoplakia (OL) and clinically healthy individuals, to evaluate the association between salivary and tissular EGF, and to correlate EGF with clinicopathologic data, including the presence of dysplasia. Salivary EGF levels were measured in 32 patients and 32 controls. The tissue expressions of EGF and its receptor (EGFR) were immunohistochemically evaluated. Salivary EGF levels were similar in patients with OL compared with controls. There was no association between the salivary levels and immunohistochemical expression of EGF. An absence of EGF detection by immunohistochemistry was associated with development of multiple lesions. Dysplastic lesions showed a tendency toward presenting higher salivary EGF levels. Currently, it is not possible to indicate salivary EGF as a biomarker for OL. Further studies are needed to elucidate the role of EGF in oral carcinogenesis. A follow-up study is necessary to evaluate the changes in EGF values following the surgical excision of OL.

EGF and EGFR genetic polymorphisms predict prognosis in locally advanced pharyngolaryngeal squamous cell carcinoma patients receiving postoperative concurrent chemoradiotherapy.

BackgroundEpidermal growth factor (EGF) and its receptor (EGFR) are part of an important signaling pathway that is involved in the pathogenesis of squamous cell carcinoma of the head and neck (SCCHN). We hypothesized that EGF/EGFR genetic polymorphisms might have a prognostic impact on disease-free survival and overall survival (OS) in locally advanced SCCHN.Materials and methodsThe patient group included a consecutive cohort of 180 patients with locally advanced SCCHN who underwent postoperative concurrent chemoradiotherapy between 2002 and 2010. DNA from formalin-fixed, paraffin-embedded tumor tissues was genotyped for the single nucleotide polymorphism (SNP) of EGF A61G A>G, EGFR R521K G>A and G-216T. The log-rank test was applied to evaluate the impact of SNPs on the outcomes. Survival was estimated using the Kaplan–Meier statistical method.ResultsWe demonstrated that EGF/EGFR SNPs might predict prognosis in patients with primary pharyngolaryngeal tumors, but not in those with oral cavity tumors. In pharyngolaryngeal tumor subgroup, EGF61 G/G genotype led to worse 5 year OS rate when compared to G/A or A/A genotypes (13.3% versus 34.3% versus 50.0%, P=0.017). The 5 year OS of patients with EGFR R521K G/G (11.1%) and G/A (15.9%) were lower than the A/A (62.5%) genotype (P=0.054). Patients carrying one or two unfavorable alleles had worse 5 year OS than those without unfavorable allele (not available versus 20% versus 71.4%, P=0.002). Multivariate analysis revealed that the highest risk of death was associated with the coexistence of two unfavorable genotypes (hazard ratio 25.7, 95% confidence interval =3.4–193.4; P=0.002).ConclusionIn this study, we were able to demonstrate that the EGF A61G and EGFR R521K genetic polymorphisms might be important prognostic factors in patients with locally advanced primary pharyngolaryngeal squamous cell carcinoma who underwent postoperative concurrent chemoradiotherapy.

Abstract 281: Hsp27 is an essential effector of EGF-induced epithelial to mesenchymal transition in prostate cancer.

Abstract Introduction: Prostate cancer (PCa) is the most common cancer and the second leading cause cancer death in North American men. An important determinant for metastasis is epithelial to mesenchymal transition (EMT) which is known to be correlated to castrate resistant prostate cancer (CRCP). Previous report demonstrated the overexpression of Hsp27 and its phosphorylated form in poor diagnosis prostate cancer. In addition, both epidermal growth factor (EGF) and its receptor (EGFR) are up–regulated in PCa during progression to CRPC. However, the role of EGF and EGFR in PCa progression remains unclear. Our objective is to establish that Hsp27 is a central “node” for several signaling pathways activated by EGF and known to modulate EMT. Methods: To assess the role of Hsp27 in EMT, gain and loss of Hsp27 function has been conducted in LNCaP cells with or without EGF stimulation. EMT markers expression has been evaluated at the protein (WB) and mRNA level (qRT–PCR). Furthermore, invasion, migration assays in addition with MMP–9 activity have been done to evaluate the migratory capacity of those cells. Also, to better understand the molecular mechanism involved in EMT induction, transactivation and Chip assays have been performed. The in vitro results have been confirmed with an in vivo study using an Hsp27 antisense oligonucleotide (OGX-427) in a murine model. Results: We found that Heat shock protein 27 (Hsp27) is up–regulated with CRPC progression and drives EMT in PCa cells by decreasing epithelial cell markers, up–regulating mesenchymal markers and enhancing cell migration and MMP–9 activity. Hsp27 knockdown induces sequestration of beta–catenin in the cytoplasm by increasing its binding to GSK–3beta and abrogating EGF induced beta–catenin nuclear translocation. Additionally, by decreasing the nuclear translocation of beta-catenin, Hsp27 knockdown decreases the binding of beta–catenin to slug promoter thereby lessening its transcriptional activity. As Slug is known to be a critical key in EMT induction, we confirmed the role of EGF and Hsp27 in EMT induction and our capacity to abrogate the EGF-induced EMT effect by Hsp27 knockdown. Finally, targeting Hsp27 using antisense oligonucleotide OGX-427 in vivo, significantly reduced tumor cell metastasis in a PCa murine model. Conclusion: This study shows that EGF through Hsp27 induces modifications in gene expression and morphology toward a mesenchymal phenotype. The mechanism involving Hsp27, β-catenin, nuclear β-catenin/TCF and EGF signaling cascades described in the present study contribute to the comprehension of mechanisms driving metastasis in aggressive castration-resistant prostate cancer. This study reveals that Hsp27 is a crucial effector of EGF–dependent and independent EMT and suggests that targeting Hsp27 holds promises for a new strategy in metastatic PCa. Citation Format: Thomas D. Cordonnier, Jennifer L. Bishop, Masaki Shiota, Ario Takeuchi, Ka Mun Nip, Martin Gleave, Amina Zoubeidi. Hsp27 is an essential effector of EGF-induced epithelial to mesenchymal transition in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 281. doi:10.1158/1538-7445.AM2013-281

Epidermal growth factor modulates colon epithelial innate immune signaling

Epidermal growth factor (EGF) and its receptor (EGFR) enhance colon epithelial healing through cell migration, proliferation and survival. Since barrier disruption induces epithelial innate immune activation by luminal microbial products, such as lipopolysaccharide (LPS) acting through toll‐like receptor 4 (TLR4), we sought to determine if EGF modulates this response as an additional mechanism to limit inflammatory responses and optimize healing. We hypothesized that EGF inhibits epithelial responses to TLR activation.MethodsMouse colon epithelial cells (YAMC) were used to determine the effect of EGF on TLR expression (qRT‐PCR) and function (NFκB luciferase reporter, CXCL2 mRNA induction, and chemokine secretion by ELISA or multiplex bead array)ResultsEGF suppressed TLR4 and 7 mRNA in a sustained manner without affecting other TLRs. This was specific to epithelial EGFR, and was not observed in macrophages, or in response to other growth factors or cytokines. EGF treatment inhibited colon epithelial NFκB activity in response to LPS and triacylated lipopeptide (TLR1/2 agonist). EGF also reduced LPS‐induced CXCL2 mRNA and secretion of CXCL2, GCSF, LIF, IL‐6 and GM‐CSF. The EGF inhibition of LPS‐induced NFκB activity required the adaptor protein YMER and was abolished by YMER siRNA.DiscussionThese results uncover a novel role for EGF to modulate TLR signaling in colon epithelial cells at the level of expression and function. These effects appeared specific to epithelial cells and required a cytosolic EGFR substrate, YMER. Thus, EGFR modulates epithelial responses to microbial cues and may provide an immunomodulatory signal for the colon epithelium. Since TLR4 transactivates EGFR, this may be a feedback mechanism whereby EGFR activation limits TLR responses. Support by NIH and CCFA.

Resolving the EGF-EGFR interaction characteristics through a multiple-temperature, multiple-inhibitor, real-time interaction analysis approach

Overexpression and aberrant activity of the epidermal growth factor (EGF) have been observed in various cancer types, rendering it an important target in oncology research. The interaction between EGF and its receptor (EGFR), as well as subsequent internalization, is complex and may be affected by various factors including tyrosine kinase inhibitors (TKIs). By combining real-time binding curves produced in LigandTracer® with internalization assays conducted at different temperatures and with different TKIs, the processes of ligand binding, internalization and excretion was visualized. SKOV3 cells had a slower excretion rate compared to A431 and U343 cells, and the tested TKIs (gefitinib, lapatinib, AG1478 and erlotinib) reduced the degree of internalization. The kinetic analysis of the binding curves further demonstrated TKI-dependent balances of EGFR monomer and dimer populations, where lapatinib promoted the monomeric form, while the other TKIs induced dimers. The dimer levels were found to be associated with the apparent affinity of the EGF-EGFR interaction, with EGF binding stronger to EGFR dimers compared to monomers. This study analyzed how real-time molecular interaction analysis may be utilized in combination with perturbations in order to understand the kinetics of a ligand-receptor interaction, as well as some of its associated intracellular processes. Our multiple-temperature and -inhibitor assay setup renders it possible to follow the EGFR monomer, dimer and internalized populations in a detailed manner, allowing for a new perspective of the EGFR biology.

Enterocyte expression of epidermal growth factor receptor is not required for intestinal adaptation in response to massive small bowel resection

PurposeIntestinal adaptation after massive small bowel resection (SBR) permits improved absorption of enteral nutrition despite significant loss of bowel length. Epidermal growth factor (EGF) and its receptor (EGFR) have previously been established to play major roles in the pathogenesis of adaptation. This study tested the hypothesis that EGFR signaling within the epithelial cell compartment (enterocytes) is required for intestinal adaptation. MethodsWe developed a tamoxifen-inducible Villin-Cre/LoxP recombinant system for enterocyte-directed EGFR deletion using EGFR-floxed mice. Epidermal growth factor receptor–null mice and wild-type littermates underwent either 50% proximal SBR or sham operation. Ileal tissue was harvested on postoperative day 7. To assess for adaptation, villus height and crypt depth as well as rates of crypt cell proliferation and apoptosis were measured. ResultsAdaptation after SBR occurred normally, as demonstrated by significant increases in villus height, crypt depth, and crypt proliferative and apoptotic index in both the wild-type and EGFR-null mice. ConclusionEnterocyte EGFR expression is not required for the adaptation response to massive SBR. This novel finding suggests that enterocyte proliferation during adaptation is regulated by EGFR signaling in cells other than enterocytes, perhaps within the mesenchymal cell compartment of the bowel wall via factor(s) that are presently unknown.