Epidermal Growth Factor Receptor Expression Levels Research Articles

The effects and mechanisms of Gefitinib on airway inflammation and airway remodeling in C57BL/6 mice with asthma

Objective: To investigate the effects of the epidermal growth factor receptor(EGFR) inhibitor Gefitinib on airway inflammation and airway remodelling in asthmatic C57BL/6 mice, and to analyze its possible mechanisms. Methods: Male C57BL/6 mice, aged 6-8 weeks, were randomly assigned into five groups: Group A (control group), Group B (asthma group), Group C (asthma+20 mg/kg gefitinib group), Group D (asthma+40 mg/kg gefitinib group), and Group E (40 mg/kg gefitinib group), with seven mice per group. Mice were sensitized by intraperitoneal injection of a mixture of 0.2 ml solution containing OVA and Al(OH)3 [20 μg OVA+2 mg Al(OH)3 dissolved in 0.2 ml of physiological saline] at Day 0 and 14. Starting from Day 25 to 31, Group B, C, and D were challenged with nebulization of 1% OVA solution (8 ml) to induce asthma, once a day for approximately 40 minutes, with continuous aerosolization for 7 days. Group C and D were given 0.2 ml of Gefitinib dissolved in 0.5% carboxymethylcellulose sodium (CMCNa) by gavage half an hour before challenging, and Group E was simultaneously given with 0.2 ml of Gefitinib dissolved in 0.5% CMCNa only. Group A and B were given an equivalent volume of 0.5% CMCNa by gavage. After 24 h of final challenge, the bronchoalveolar lavage fluid (BALF) was prepared for the determination of total cell count and eosinophil count. The levels of total immune globulin E (IgE) in serum and interleukin (IL)-4, IL-5 and IL-13 in BALF and lung tissue homogenates were measured by ELISA. The mRNA expression levels of IL-4, IL-5, IL-13 in lung were measured. Immunohistochemistry and Western blot experiments were used to detect the expression levels of EGFR in lung tissues. Results: In Group B, the level of total IgE in serum, total cell count, eosinophil count, the levels of IL-4, IL-5, IL-13 in BALF and the phosphorylation of EGFR and its downstream activation in lung were higher than those in Group A (all P<0.05). The levels of total IgE in serum [(261.32±44.38) ng/ml, (194.09±52.39) ng/ml vs (1 023.70±105.51) ng/ml], total cell count [(23.70±4.08)×105/ml, (14.92±4.06)×105/ml vs (35.36±6.30)×105/ml], eosinophil count [(108.00±13.69)×104/ml, (67.00±17.28)×104/ml vs (147.86±20.06)×104/ml], IL-4 [(36.42±4.48) pg/ml, (30.45±8.12) pg/ml vs (58.72±7.17) pg/ml], IL-5 [(16.20±4.62) pg/ml, (13.38±5.14) pg/ml vs (23.46±5.38) pg/ml], IL-13 [(18.45±7.28) pg/ml, (14.33±7.70) pg/ml vs (104.12±24.66) pg/ml] in BALF of Group C and D were lower than those in Group B (all P<0.05). The levels of IL-4, IL-5, and IL-13 as well as their mRNA levels in the lung tissue of Group C and D were lower than those in Group B (all P<0.05). In Group C and D, the positive expression rate of phosphorylated epidermal growth factor receptor (p-EGFR) in lung tissue [(40.53±6.80)%, (23.60±4.42)% vs (70.78±5.36)%], p-EGFR/EGFR (61.68±7.48, 51.13±5.19 vs 105.90±11.66), phosphorylated extracellular regulated protein kinase (p-Erk)/extracellular regulated protein kinase (Erk) (75.28±7.11, 47.54±4.83 vs 98.76±4.71), and phosphorylated protein kinase B (p-Akt)/protein kinase B (Akt) (96.24±5.40, 68.52±2.73 vs 103.30±4.52) was lower than those of Group B (all P<0.05). There was no statistically significant difference in the relevant indicators between Group A and E (all P>0.05). Conclusion: Gefitinib may alleviate airway inflammation and airway remodeling in asthmatic mice by inhibiting EGFR phosphorylation and affecting the activation of downstream Erk and Akt.

β-Elemene Reverses Gefitinib Resistance in NSCLC Cells by Inhibiting lncRNA H19-Mediated Autophagy.

Lung cancer is a leading cause of mortality worldwide, especially among Asian patients with non-small cell lung cancer (NSCLC) who have epidermal growth factor receptor (EGFR) mutations. Initially, first-generation EGFR tyrosine kinase inhibitors (TKIs) are commonly administered as the primary treatment option; however, encountering resistance to these medications poses a significant obstacle. Hence, it has become crucial to address initial resistance and ensure continued effectiveness. Recent research has focused on the role of long noncoding RNAs (lncRNAs) in tumor drug resistance, especially lncRNA H19. β-elemene, derived from Curcuma aromatic Salisb., has shown strong anti-tumor effects. However, the relationship between β-elemene, lncRNA H19, and gefitinib resistance in NSCLC is unclear. This study aims to investigate whether β-elemene can enhance the sensitivity of gefitinib-resistant NSCLC cells to gefitinib and to elucidate its mechanism of action. The impact of gefitinib and β-elemene on cell viability was evaluated using the cell counting kit-8 (CCK8) assay. Furthermore, western blotting and qRT-PCR analysis were employed to determine the expression levels of autophagy-related proteins and genes, respectively. The influence on cellular proliferation was gauged through a colony-formation assay, and apoptosis induction was quantified via flow cytometry. Additionally, the tumorigenic potential in vivo was assessed using a xenograft model in nude mice. The expression levels of LC3B, EGFR, and Rab7 proteins were examined through immunofluorescence. Our findings elucidate that the resistance to gefitinib is intricately linked with the dysregulation of autophagy and the overexpression of lncRNA H19. The synergistic administration of β-elemene and gefitinib markedly attenuated the proliferative capacity of resistant cells, expedited apoptotic processes, and inhibited the in vivo proliferation of lung cancer. Notably, β-elemene profoundly diminished the expression of lncRNA H19 and curtailed autophagic activity in resistant cells, thereby bolstering their responsiveness to gefitinib. Moreover, β-elemene disrupted the Rab7-facilitated degradation pathway of EGFR, facilitating its repositioning to the plasma membrane. β-elemene emerges as a promising auxiliary therapeutic for circumventing gefitinib resistance in NSCLC, potentially through the regulation of lncRNA H19-mediated autophagy. The participation of Rab7 in this dynamic unveils novel insights into the resistance mechanisms operative in lung cancer, paving the way for future therapeutic innovations.

Cetuximab-Toxin Conjugate and NPe6 with Light Enhanced Cytotoxic Effects in Head and Neck Squamous Cell Carcinoma In Vitro.

Photodynamic therapy (PDT) is a cancer-targeted treatment that uses a photosensitizer (PS) and irradiation of a specific wavelength to exert cytotoxic effects. To enhance the antitumor effect against head and neck squamous cell carcinoma (HNSCC), we developed a new phototherapy, intelligent targeted antibody phototherapy (iTAP). This treatment uses a combination of immunotoxin (IT) and a PS for PDT and light irradiation. In our prior study, we demonstrated that an immunotoxin (IT) consisting of an anti-ROBO1 antibody conjugated to saporin, when used in combination with the photosensitizer (PS) disulfonated aluminum phthalocyanine (AlPcS2a) and irradiated with light at the appropriate wavelength, resulted in increased cytotoxicity against head and neck squamous cell carcinoma (HNSCC) cells. ROBO1 is a receptor known to be involved in the progression of cancer. In this study, we newly investigate the iTAP targeting epidermal growth factor receptor (EGFR) which is widely used as a therapeutic target for HNSCC. We checked the expression of EGFR in HNSCC cell lines, SAS, HO-1-u-1, Sa3, and HSQ-89. We analyzed the cytotoxicity of saporin-conjugated anti-EGFR antibody (cetuximab) (IT-Cmab), mono-L-aspartyl chlorin e6 (NPe6, talaporfin sodium), and light (664 nm) irradiation (i.e., iTAP) in SAS, HO-1-u-1, Sa3, and HSQ-89 cells. EGFR was expressed highly in Sa3, moderately in HO-1-u-1, SAS, and nearly not in HSQ-89. Cmab alone or IT-Cmab alone did not show cytotoxic effects in Sa3, HO-1-u-1, and HSQ-89 cells, which have moderate or low expression levels of EGFR protein. However, the iTAP method enhanced the cytotoxicity of IT-Cmab by the photodynamic effect in Sa3 and HO-1-u-1 cells, which have moderate levels of EGFR expression. Our study is the first to report on the iTAP method using IT-Cmab and NPe6 for HNSCC. The cytotoxic effects are enhanced in cell lines with moderate levels of EGFR protein expression, but not in nonexpressing cell lines, which is expected to expand the range of therapeutic windows and potentially reduce complications.

Abstract 6956: Role of soluble CD109 in the progression of head and neck squamous cell carcinoma

Abstract Introduction: About five percent of global cancer deaths are attributed to Head and Neck Squamous Cell Carcinoma (HNSCC), and its annual incidence is predicted to skyrocket by thirty percent by 2030 (according to GLOBOCAN estimates). HNSCC originates from squamous cells in the oral cavity, lips, tongue, larynx, pharynx, and salivary glands. Despite the comparatively easy tumor diagnosis, the number of patients diagnosed at an advanced stage with metastatic spread is substantial. Multiple cell-signaling pathways have been proven to be dysregulated in HNSCC, including the epidermal growth factor receptor (EGFR). Our group has identified CD109, a GPI-anchored membrane-associated protein, as a TGF-β co-receptor that inhibits TGF-β signaling. CD109 expression is upregulated in SCC, and its elevated expression in premalignant lesions is associated with an increased risk of metastatic progression. Recent studies by our team have revealed that CD109 promotes tumor growth in vivo, likely by enhancing EGFR signaling. Interestingly, we have also discovered that membrane CD109 (mCD109) is endogenously released from the cell surface; however, the mechanism of action of the released/soluble form of CD109 (sCD109) remains unknown. In the current study, we examined whether sCD109 opposes the mCD109/EGFR-mediated pro-tumorigenic signaling and HNSCC progression. Research Questions: 1.Does sCD109 regulate EGF-induced EGFR signaling and downstream stem cell marker expression?2.Does sCD109 regulate EGFR expression levels on the HNSCC cell surface?3.Does sCD109 affect the nuclear localization of EGFR?4.Does sCD109 promote EGFR internalization and degradation? Methodology: HNSCC cells and patient-derived primary cells were treated with/without sCD109 recombinant protein (1nM, 5nM, and 10nM) without and with the EGF induction (10ng/ml for 15 mins) and were analyzed by western blotting, immunofluorescence, and immunocytochemistry. The role of sCD109 in regulating membrane EGFR levels was assessed by flow cytometry and surface protein biotinylation assay. Results: Our findings demonstrated that sCD109 inhibited EGF-induced phosphorylation of EGFR (Y1068), STAT3, AKT, and ERK. The sCD109 suppressed the stem cell marker expression (Nanog, OCT4, SOX2). Additionally, sCD109 hindered the nuclear localization of activated EGFR. The sCD109 decreased the membrane EGFR expression levels and activated the phosphorylation of EGFR (Y1045), suggesting a role for sCD109 in promoting EGFR degradation. Conclusion: Our team has previously reported that mCD109 is pro-tumorigenic, and our current findings suggest that sCD109 opposes the pro-tumorigenic effects of mCD109. This study aimed to determine how sCD109 regulates cellular signaling pathways and will provide key insights into the molecular mechanisms underlying the progression of HNSCC, a highly prevalent malignancy. Citation Format: Varsha Reddy Durgempudi, Tenzin Kungyal, Anie Philip. Role of soluble CD109 in the progression of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6956.

Abstract 549: PDZK1 sensitizes TNBC cells to erlotinib via promoting c-Cbl-mediated EGFR degradation and inhibiting EGFR phosphorylation

Abstract Background: Triple-negative breast cancer (TNBC) is lack of effective targeted therapies. Epidermal growth factor receptor (EGFR) is a potential therapeutic target for TNBC. However, TNBC is resistant to EGFR-targeted drugs in clinical trials, and the tolerance mechanism remains unclear. This study aimed to elucidate the possible resistance mechanism for TNBC to EGFR-targeted therapy and how sensitize TNBC cells to erlotinib. Methods: PDZ domain containing 1 (PDZK1) correlated with TNBC development was screened out by bioinformatics analysis and its correlation with resistance to erlotinib was identified by western blotting (WB). Its interaction with EGFR and structural basis were determined by GST-Pull down and coimmunoprecipitation. Its regulation on EGFR signaling activation and expression level were detected by WB in PDZK1 overexpression and knockdown TNBC cells, xenograft tissues and correlation analyses. Correlation was performed by gene set enrichment analysis (GSEA) and immunohistochemistry of tissue microarray followed by Pearson analysis. TNBC-suppressing effects of PDZK1 and its sensitizing effects on erlotinib were investigated using cell viability assay, colony-forming assay, wound healing assay, Matrigel invasion assay and xenograft model in combination with PDZK1 wild type/mutant transfection and rescue experiment. Results: Here we found that PDZK1 was correlated with TNBC development and its level was downregulated in erlotinib-resistant TNBC cells, suggesting that PDZK1 downregulation was related to erlotinib resistance in TNBC. PDZK1 bound with EGFR. Through the interaction, PDZK1 promoted EGFR degradation by enhancing the binding of EGFR with c-Cbl and inhibited EGFR phosphorylation by hindering EGFR dimerization. PDZK1 was specifically downregulated in TNBC tissues and was correlated with poor prognosis of TNBC. Functional assays in vitro and in vivo showed that PDZK1 suppressed TNBC development. Restoring EGFR expression and kinase inhibitor treatment reversed the malignancy caused by PDZK1 overexpression and knockdown, respectively. PDZK1 wild type, but not mutant overexpression enhanced the inhibitory effect of erlotinib on EGFR signaling and TNBC malignancy in vitro and in vivo. Conclusion: PDZK1 is a significant prognostic factor for TNBC and a potential molecular therapeutic target for TNBC to reverse the tolerance of TNBC cells to erlotinib. Citation Format: Junfang Zheng, Yuanzhen Ma, Zhiyu Fang, Yijun Qi. PDZK1 sensitizes TNBC cells to erlotinib via promoting c-Cbl-mediated EGFR degradation and inhibiting EGFR phosphorylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 549.

Near-Infrared Photoimmunotherapy Using a Protein Mimetic for EGFR-Positive Salivary Gland Cancer.

Near-infrared photoimmunotherapy (NIR-PIT) is a novel cancer therapy based on a monoclonal antibody (mAb) conjugated to a photosensitizer (IR700Dye). The conjugate can be activated by near-infrared light irradiation, causing necrotic cell death with high selectivity. In this study, we investigated NIR-PIT using a small protein mimetic (6-7 kDa, Affibody) which has more rapid clearance and better tissue penetration than mAbs for epidermal growth factor receptor (EGFR)-positive salivary gland cancer (SGC). The level of EGFR expression was examined in vitro using immunocytochemistry and Western blotting. Cell viability was analyzed using the alamarBlue assay. In vivo, the volume of EGFR-positive tumors treated with NIR-PIT using the EGFR Affibody-IR700Dye conjugate was followed for 43 days. It was found that NIR-PIT using the EGFR Affibody-IR700Dye conjugate induced the selective destruction of EGFR-positive SGC cells and restricted the progression of EGFR-positive tumors. We expect that NIR-PIT using the EGFR Affibody-IR700Dye conjugate can efficiently treat EGFR-positive SGC and preserve normal salivary function.

Quantifying Imaging Agent Binding and Dissociation in 3-D Cancer Spheroid Tissue Culture Using Paired-Agent Principles.

Binding kinetics play an important role in cancer diagnosis and therapeutics. However, current methods of quantifying binding kinetics fail to consider the three-dimensional environment that drugs and imaging agents experience in biological tissue. In response, a methodology to assay agent binding and dissociation in 3-D tissue culture was developed using paired-agent molecular imaging principles. To test the methodology, the uptakes of ABY-029 (an IRDye 800CW-labeled epidermal growth factor receptor (EGFR)-targeted antibody mimetic) and IRDye-700DX carboxylate in 3-D spheroids were measured in four different human cancer cell lines throughout staining and rinsing. A compartment model (optimized for the application) was then fit to the kinetic curves of both imaging agents to estimate binding and dissociation rate constants of the EGFR-targeted ABY-029 agent. A statistically significant correlation was observed between apparent association rate constant (k3) and the receptor concentration experimentally and in simulations (r = 0.99, p < 0.05). A statistically significant difference was found between effective k3 (apparent rate constant of ABY-029 binding to EGFR) values for cell lines with varying levels of EGFR expression (p < 0.05), with no significant difference found between cell lines and controls for other fit parameters. Additionally, a similar binding affinity profile compared to a gold standard method was determined by this model. This low-cost methodology to quantify imaging agent or drug binding affinity in clinically relevant 3-D tumor spheroid models can be used to guide timing of imaging in molecular guided surgery and could have implications in drug development.

The possible regulatory role of miR-514 and miR-642b in cumulus cells on the oocyte maturation in patients with polycystic ovary syndrome

Polycystic ovary syndrome is a common endocrine disorder in reproductive-age women. Accordingly, abnormal microenvironment may negatively influence oocyte developmental competence as a result of the altered expression profile of cumulus cells (CCs), mainly the key players of oocyte maturation, such as epidermal growth factor receptor (EGFR) and prostaglandin E receptor-2 (PTGER2). This study aimed to examine the expression levels of miR-514, miR-642b, and their candidate target genes (EGFR and PTGER2, respectively) in CCs of immature and mature oocytes in patients with PCOS. A total of 40 oocytes at germinal vesicle (GV) and 40 oocytes at metaphase II (MII) stages were retrieved from 30 PCOS women. Quantitative real-time PCR was performed to analyze the expression level of miR-514, miR-642b, EGFR, and PTGER2 in cumulus cells (CCS) of each oocyte. The expression level of miRNAs and their candidate target genes were compared between CCs of GV and MII oocytes. Our study suggests an inverse relationship exists between the expression levels of miR-514 and EGFR, and miR-642b and PTGER2. Furthermore, we observed that CCs of GV oocytes had higher levels of EGFR and PTGER2 mRNA and lower levels of miR-514 and miR-642b expression compared to those of MII oocytes. The present study demonstrated that miR-514 and miR-642b can regulate oocyte development by targeting EGFR and PTGER2, respectively. Therefore, examination of these miRNAs in CCs could be promising parameters to predict oocyte competence in PCOS patients.

Antibody-Conjugated Polymersomes with Encapsulated Indocyanine Green J-Aggregates and High Near-Infrared Absorption for Molecular Photoacoustic Cancer Imaging.

Imaging plays a critical role in all stages of cancer care from early detection to diagnosis, prognosis, and therapy monitoring. Recently, photoacoustic imaging (PAI) has started to emerge into the clinical realm due to its high sensitivity and ability to penetrate tissues up to several centimeters deep. Herein, we encapsulated indocyanine green J (ICGJ) aggregate, one of the only FDA-approved organic exogenous contrast agents that absorbs in the near-infrared range, at high loadings up to ∼40% w/w within biodegradable polymersomes (ICGJ-Ps) composed of poly(lactide-co-glycolide-b-polyethylene glycol) (PLGA-b-PEG). The small Ps hydrodynamic diameter of 80 nm is advantageous for in vivo applications, while directional conjugation with epidermal growth factor receptor (EGFR) targeting cetuximab antibodies renders molecular specificity. Even when exposed to serum, the ∼11 nm-thick membrane of the Ps prevents dissociation of the encapsulated ICGJ for at least 48 h with a high ratio of ICGJ to monomeric ICG absorbances (i.e., I895/I780 ratio) of approximately 5.0 that enables generation of a strong NIR photoacoustic (PA) signal. The PA signal of polymersome-labeled breast cancer cells is proportional to the level of cellular EGFR expression, indicating the feasibility of molecular PAI with antibody-conjugated ICGJ-Ps. Furthermore, the labeled cells were successfully detected with PAI in highly turbid tissue-mimicking phantoms up to a depth of 5 mm with the PA signal proportional to the amount of cells. These data show the potential of molecular PAI with ICGJ-Ps for clinical applications such as tumor margin detection, evaluation of lymph nodes for the presence of micrometastasis, and laparoscopic imaging procedures.

Effects of rosmarinic acid and doxorubicine on an ovarian adenocarsinoma cell line (OVCAR3) via the EGFR pathway.

We aimed to reveal the effects of rosmarinic acid (RA), which has come to the forefront with its antitumor and antioxidant properties in many studies recently in the ovarian adenocarcinoma cell line, on the epidermal growth factor receptor (EFGR) signaling pathway in the presence of doxorubicin (DOX). Ovarian adenocarcinoma cell line (OVCAR3) and human skin keratinocyte cell line human skin keratinocyte cell line (HaCaT) were used as control. (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was applied to determine the effect of RA and DOX on the proliferation of OVCAR3 and HaCaT cells. Bcl2 expression and epidermal growth factor receptor (EGFR) and western blot analysis were performed to determine the expression levels of the markers. It was determined that RA (IC50 = 437.6 μM) and DOX (IC50 = 0.08 μM) have the ability to inhibit the proliferation of OVCAR3 cells and induce apoptosis in a 72-hour time and dose-dependent manner. Western blot showed that the expression level of Bcl-2 and EGFR in OVCAR3 cells was down-regulated by RA and DOX. Apoptosis in OVCAR3 cells can potentially be induced by RA via the EGFR pathway, and RA may be a potent agent for cancer therapy.

Immunohistochemical Observation and Correlation of Epidermal Growth Factor Receptor Downstream Signaling Proteins with Plasma Medium-Chain Fatty Acids Levels in Breast Cancer

Abstract Aim: The aim of this study was to correlate the expression of epidermal growth factor receptor (EGFR) downstream signaling molecules with plasma medium-chain fatty acids in breast cancer (BC). Materials and Methods: One hundred and sixteen plasma samples were collected from BC and control subjects. mRNA was extracted and gene expression of mRNA was analyzed by reverse transcriptase-quantitative polymerase chain reaction. Plasma levels of phosphorylated EGFR (p-EGFR), p-Akt, and phosphorylated mammalian target of rapamycin (p-mTOR) were measured using an ELISA kit. Thirty BC tissue sections were used to analyze the expression of EGFR/p-EGFR, Akt/p-Akt, and mammalian target of rapamycin (mTOR)/p-mTOR by immunohistochemistry. Results: The relative mRNA expression levels of EGFR, Akt, and mTOR were high in BC compared to the control subjects, whereas plasma levels of phosphorylated EGFR downstream signaling molecules were upregulated in BC patients, which was statistically significant. The expression of p-EGFR was high in both the cytoplasm and the nuclei, and also, the higher the expression of the downstream molecules in BC tissue sections. Conclusion: In the present investigation, there was a noteworthy inverse relationship between plasma levels of caprylic and lauric acid and the expression of EGFR downstream signaling molecules in BC. These findings suggest that EGFR signaling in BC may be efficiently repressed by increasing the amount of caprylic and lauric acid supplements taken through diet.

Modulation of EGFR Activity by Molecularly Imprinted Polymer Nanoparticles Targeting Intracellular Epitopes.

In recent years, molecularly imprinted polymer nanoparticles (nanoMIPs) have proven to be an attractive alternative to antibodies in diagnostic and therapeutic applications. However, several key questions remain: how suitable are intracellular epitopes as targets for nanoMIP binding? And to what extent can protein function be modulated via targeting specific epitopes? To investigate this, three extracellular and three intracellular epitopes of epidermal growth factor receptor (EGFR) were used as templates for the synthesis of nanoMIPs which were then used to treat cancer cells with different expression levels of EGFR. It was observed that nanoMIPs imprinted with epitopes from the intracellular kinase domain and the extracellular ligand binding domain of EGFR caused cells to form large foci of EGFR sequestered away from the cell surface, caused a reduction in autophosphorylation, and demonstrated effects on cell viability. Collectively, this suggests that intracellular domain-targeting nanoMIPs can be a potential new tool for cancer therapy.

The Effect of Ginger Extract on the Expression of Epidermal Growth Factor Receptor in MDA-mb231 and HT-29 Cell Lines

Background: Cancer is a neoplastic disease that continues to be a global challenge, with a reported prevalence increasing annually. Epidermal growth factor receptor (EGFR) is overexpressed in various epithelial tumors such as breast and colon cancer, and for this reason, it is used for cancer diagnosis and treatment. Because of the high mortality rate associated with cancer and the side effects of chemotherapy and radiotherapy, patients need replaced strategies for therapy. Ginger has biological effects, including antioxidant and anticancer activities. Objectives: This study aims to evaluate ginger extract's effect on the expression of EGFR in MDA-mb231 and HT-29 cell lines. Methods: Fresh ginger rhizomes were purchased from a local food market, washed, grated, and then ginger extract was prepared using ethanol. MDA-mb231 and HT-29 cell lines were treated with different concentrations of ginger (5, 10, and 20 µgr/mL) extract in RPMI-1640 plus 10 % FCS for 18 h. The gene expression of EGFR was measured by real-time PCR. Results: The level of EGFR expression in MDA-mb231 and HT-29 cell lines after treatment with different concentrations of ginger extract was not changed significantly (P &gt; 0.05). Conclusions: The current data indicate that the ginger extract does not have a significant dose-dependent effect, in the concentration range of 5 to 20 µgr/mL, on the expression of EGFR in these cancer cell lines. It is suggested to repeat the experiments with higher concentrations of ginger and in a time-dependent manner.

Urinary exosome proteins PAK6 and EGFR as noninvasive diagnostic biomarkers of diabetic nephropathy

ObjectiveThe actin cytoskeleton plays an essential role in maintaining podocyte functions. However, whether the urinary exosome proteins related to the regulation of the actin cytoskeleton are changed in diabetic nephropathy (DN) is still unknown. This study was to investigate the possibility that related proteins can be applied as diagnostic biomarkers for DN.MethodsUrinary exosomes were obtained from 144 participants (Discovery phase: n = 72; Validation phase: n = 72) by size exclusion chromatography methods. Proteomic analysis of urinary exosome by LC-MS/MS. Western blot and ELISA were applied to validate the selected urinary exosome proteins. The clinical value of selected urinary exosome proteins was evaluated using correlation and receiver operating characteristic curve analyses.ResultsFifteen urinary proteins related to the regulation of the actin cytoskeleton were identified in urinary exosomes. Three upregulated proteins were selected, including Serine/threonine-protein kinase PAK6 (PAK6), Epidermal growth factor receptor (EGFR), and SHC-transforming protein 1(SHC1). The expression level of PAK6 and EGFR was negatively correlated with estimated glomerular filtration rate and positively correlated with serum creatinine levels. For diagnosing DN in the discovery phase: the area under curve (AUC) of PAK6 was 0.903, EGFR was 0.842, and the combination of two proteins was 0.912. These better performances were also observed in the validation phase (For PAK6: AUC = 0.829; For EGFR: AUC = 0.797; For PAK6 + EGFR: AUC = 0.897).ConclusionsUrinary exosome proteins PAK6 and EGFR may be promising and noninvasive biomarkers for diagnosing DN.

Hydroxysafflor Yellow A Promotes HaCaT Cell Proliferation and Migration by Regulating HBEGF/EGFR and PI3K/AKT Pathways and Circ_0084443.

To investigate the effect and possible mechanism of hydroxysafflor yellow A (HSYA) on human immortalized keratinocyte cell proliferation and migration. HaCaT cells were treated with HSYA. Cell proliferation was detected by the cell counting kit-8 assay, and cell migration was measured using wound healing assay and Transwell migration assay. The mRNA and protein expression levels of heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF), EGF receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), mammalian target of rapamycin (mTOR), and hypoxia-inducible factor-1α (HIF-1α) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. Circ_0084443-overexpressing HaCaT cells and empty plasmid HaCaT cells were constructed using the lentiviral stable transfection and treated with HSYA. The expression of circ_0084443 was detected by qRT-PCR. HSYA (800 µmol/L) significantly promoted HaCaT cell proliferation and migration (P<0.05 or P<0.01). It also increased the mRNA and protein expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and increased the phosphorylation levels of PI3K and AKT (P<0.05 or P<0.01). Furthermore, HSYA promoted HaCaT cell proliferation and migration via the HBEGF/EGFR and PI3K/AKT/mTOR signaling pathways (P<0.01). Circ_0084443 attenuated the mRNA expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α (P<0.05). HSYA inhibited the circ_0084443 expression, further antagonized the inhibition of circ_0084443 on HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and promoted the proliferation of circ_0084443-overexpressing HaCaT cells (P<0.05 or P<0.01). However, HSYA could not influence the inhibitory effect of circ_0084443 on HaCaT cell migration (P>0.05). HSYA played an accelerative role in HaCaT cell proliferation and migration, which may be attributable to activating HBEGF/EGFR and PI3K/AKT signaling pathways, and had a particular inhibitory effect on the keratinocyte negative regulator circ_0084443.

Effects of Arctigenin in Proliferation, Migration, and Invasion of Nasopharyngeal Carcinoma 5-8F Cells.

Nasopharyngeal carcinoma (NPC) is a malignant tumor of the nasopharynx. Here, we aimed to understand better the molecular basis for arctigenin (ARG)'s ability to promote NPC 5- 8F cell invasion. We tested the effects of several doses of ARG on 5-8F cells that had been cultured in vitro. We estimated the metabolic activity of cells by The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium assay. We examined the influence on cell invasion, and migration using Transwell Evaluation. Real-time polymerase chain reaction analysis was used to determine the relative amounts of epidermal growth factor receptor (EGFR), Janus kinase 2 (JAK2) , and transcriptional activator 3 (STAT 3) mRNA expression. Using western blotting, we looked at the level of phosphorylation of specific proteins like EGFR, phosphorylated EGFR, JAK2, and STAT 3. Our findings revealed that ARG inhibited NPC 5-8F cell development in a dose-and time-dependent manner. The invasiveness and mobility of 5-8F cells were significantly suppressed when ARG was overexpressed in a tumor development model. Expression levels of EGFR, JAK2, and STAT 3 mRNA were considerably low in the experimental group. As a consequence of being treated with ARG, lower levels of EGFR, p-EGFR, p-JAK2, and p-STAT3 expression were observed. These results suggest that ARG may prevent NPC 5-8F cells from proliferating, migrating, and invading other tissues. There are a few potential molecular pathways, two of which are the inhibition of EGFR phosphorylation and the reduction of levels of phospho-JAK2 and phospho-STAT3.

Leveraging innate immunity with AFM24, a novel CD16A and epidermal growth factor receptor (EGFR) bispecific innate cell engager: Interim results for the non-small cell lung cancer (NSCLC) cohort.

2533 Background: Patients with EGFR mutant NSCLC who progress on or after small molecule EGFR inhibitors have only limited treatment options. AFM24 is a tetravalent, bispecific innate cell engager that binds EGFR on solid tumor cells and CD16A on NK cells to redirect and enhance antitumor antibody-dependent cellular cytotoxicity, thus acting independently of the EGFR downstream signaling pathway and EGFR expression levels. The Phase 1 dose escalation phase of the presented study (NCT04259450) established the recommended Phase 2 dose of AFM24 monotherapy at 480 mg based on tolerability, receptor occupancy, and pharmacokinetics (PK). Correlative science data revealed an activation and enrichment of immune cells in the tumor microenvironment. Methods: The Phase 2a expansion phase is evaluating 480 mg AFM24 in patients with EGFR mutant NSCLC, relapsed or refractory to ≥1 prior lines of therapy; EGFR-positivity is defined as positive staining for EGFR in ≥1% of tumor cells per local immunohistochemistry assay. The study follows Simon’s two-stage design. The primary endpoint is overall response rate per local RECIST v1.1; secondary endpoints include safety, PK, and immunogenicity. Efficacy per iRECIST is an exploratory endpoint. AFM24 is administered intravenously once weekly in a 28-day cycle. Tumor assessments are performed at initial screening, cycles 2, 4, 6, 8, and every 3 cycles thereafter. Results: As of December 2022, 14 patients (median [range] age 55 [37–82]; 12 male; 11 Asian, 3 White) were enrolled; the median number of prior therapies was 2.5 (1–12). Median treatment duration with AFM24 was 6.7 weeks (1.0–26.1). Best objective response in 10 evaluable patients was confirmed partial response in 1 patient (change from baseline –45%); 4 patients exhibited stable disease, 1 was confirmed. Tumor shrinkage occurred in 2 patients with stable disease (–6%, –18%). The tumor control rate was 50%. Treatment-related adverse events (TRAEs) were reported in 13 patients; infusion related reactions (IRRs, 12), dermatitis acneiform, neutrophil count decreased, decreased appetite, and myalgia (2 each) were most common. Grade ≥3 TRAEs occurred in 4 patients and included decreased neutrophil count (2), lymphopenia, and IRR (1 each). Grade 5 pneumonitis was observed in 1 patient with disease progression and multiple comorbidities; relation to AFM24 could not be ruled out. As of January 2023, enrollment is ongoing; results of the interim analysis will be presented. Conclusions: AFM24 demonstrated clinical activity and acceptable safety in heavily pretreated patients with EGFR mutant NSCLC. The novel mechanism of action of AFM24 could add to the therapeutic options for patients with EGFR expressing tumors and is under clinical evaluation as a single agent and in combination with atezolizumab or autologous NK cells. Clinical trial information: NCT04259450 .

Investigating the novel CD16A and epidermal growth factor receptor (EGFR) bispecific innate cell engager, AFM24, to leverage the innate immune system: Interim results from the colorectal cancer (CRC) cohort.

e14508 Background: AFM24 is a tetravalent, bispecific innate cell engager that binds EGFR on solid tumors and CD16A on natural killer (NK) cells. AFM24 redirects NK cells to EGFR-expressing (EGFR+) solid tumors, enhancing antibody-dependent cellular cytotoxicity. AFM24 induced lysis of EGFR+ tumor cell lines in vitro independent of EGFR expression level and mutation status. The dose escalation phase (Phase 1) of the presented study (NCT04259450) previously established the recommended Phase 2 dose at 480 mg based on tolerability, receptor occupancy, and pharmacokinetics. Correlative science data revealed an activation and enrichment of immune cells in the tumor microenvironment. Here we present preliminary data from the Phase 2a CRC expansion cohort. Methods: The Phase 2a expansion phase is evaluating 480 mg AFM24 in patients with histologically or cytologically confirmed microsatellite stable, RAS wild type EGFR+ CRC. Patients have relapsed or refractory disease to ≥2 prior lines of therapy including an EGFR targeted therapy. EGFR+ is defined as positive staining for EGFR in ≥1% of tumor cells per local immunohistochemistry assay. The study follows Simon’s two-stage design. The primary endpoint is the overall response rate per RECIST v1.1 by investigator assessment; secondary endpoints include safety and tolerability; efficacy per iRECIST is exploratory. AFM24 is administered intravenously once weekly in a 28-day cycle. Tumor assessments are performed at screening, cycles 2, 4, 6, 8, and every 3 cycles thereafter. Results: As of December 2022, 16 patients with Stage 4 CRC were enrolled and treated with 480 mg AFM24 in the study. The median (range) age was 66 years (51–77); 11 males and 5 females; 9 Asian, 7 White). The median number of prior therapies was 4 (2–7) and the median treatment duration was 5.1 weeks (1.0–22.0). Treatment-related adverse events (TRAEs) were reported in 14/16 patients; infusion-related reactions (IRRs, 12), pruritus (3), lymphopenia (3), and anemia (2) were the most common. AFM24 TRAEs of Grade ≥3 occurred in 6 patients and included lymphopenia (3), decreased neutrophil count (1), increased blood bilirubin (1) and IRRs (1). To date, no objective responses were observed. Seven patients remain on treatment including 1 patient with confirmed stable disease ongoing for 5 months exhibiting a tumor reduction of –17%. Final interim results will be presented. Conclusions: The novel mechanism of action of AFM24 may provide an alternative treatment approach for patients with EGFR+ solid tumors. In this study, AFM24 monotherapy was adequately tolerated in a heavily pretreated population of patients with EGFR+ CRC. AFM24 is also being investigated in combination with atezolizumab and autologous NK cells in various EGFR+ solid tumors. Clinical trial information: NCT04259450 .

Tumor suppressor miR-218 directly targets epidermal growth factor receptor (EGFR) expression in triple-negative breast cancer, sensitizing cells to irradiation

PurposeMicroRNA-218 (miR-218) is a key regulator of numerous processes relevant to tumor progression. In the present study, we aimed to characterize the relationship between miR-218 and the Epidermal Growth Factor Receptor (EGFR) as well as to understand downstream effects in triple-negative breast cancer (TNBC).MethodsWe assessed miR-218 and EGFR expression in cell lines and publicly available primary breast cancer gene expression data. We then overexpressed miR-218 in two TNBC cell lines and investigated effects on EGFR and downstream mitogen-activated protein (MAP) kinase signaling. Luciferase reporter assay was used to characterize a direct binding interaction between miR-218 and EGFR mRNA. Digital holographic microscopy helped investigate cell migration and dry mass after miR-218 overexpression. Cell division and invasion were assessed microscopically, while radiation response after miR-218 overexpression alone or combined with additional EGFR knockdown was investigated via clonogenic assays.ResultsWe found an inverse correlation between EGFR expression and miR-218 levels in cell lines and primary breast cancer tissues. MiR-218 overexpression resulted in a downregulation of EGFR via direct binding of the mRNA. Activation of EGFR and downstream p44/42 MAPK signaling were reduced after pre-miR-218 transfection. Cell proliferation, motility and invasiveness were inhibited whereas cell death and mitotic catastrophe were upregulated in miR-218 overexpressing cells compared to controls. MiR-218 overexpressing and EGFR siRNA-treated cells were sensitized to irradiation, more than miR-218 overexpressing cells alone.ConclusionThis study characterizes the antagonistic relationship between miR-218 and EGFR. It also demonstrates downstream functional effects of miR-218 overexpression, leading to anti-tumorigenic cellular changes.

Quantitative comparison of EGFR expression levels of optically trapped individual cells using a capacitance biosensor

Cellular endocytosis is an essential phenomenon which induces cellular reactions, such as waste removal, nutrient absorption, and drug delivery, in the process of cell growth, division, and proliferation. To observe capacitance responses upon endocytosis on a single-cell scale, this study combined an optical tweezer that can optically place a single cell on a desired location with a capacitance sensor and a cell incubation chamber. Single HeLa cancer cell was captured and moved to a desired location through optical trapping, and the single-cell capacitance change generated during the epidermal growth factor (EGF) molecule endocytosis was measured in real time. It was found that single HeLa cells showed a larger increase in capacitance values compared to that of the single NIH3T3 cells when exposed to varying EGF concentrations. In addition, the capacitance change was in proportion to the cell's EGF receptor (EGFR) level when cells of different levels of EGFR expression were tested. An equation derived from these results was able to estimate the EGFR expression level of a blind-tested cell. The biosensor developed in this research can not only quickly move a single cell to a desired location in a non-invasive manner but also distinguish specific responses between cancer and normal cells by continuous measurement of real-time interactions of a single cell in culture to the external ligands.