Search Results (194)

Objective: Rat sarcoma (Ras) proteins, Kirsten, Harvey, and Neuroblastoma rat sarcoma viral oncogene homolog (KRAS, HRAS, and NRAS, respectively), are a family of GTPases, which are key regulators of cellular growth, differentiation, and apoptosis through signal transduction pathways modulated by growth factors that have been recognized to be dysregulated in PCOS. This study explores Ras signaling proteins and growth factor-related proteins in polycystic ovary syndrome (PCOS). Methods: In a well-validated PCOS database of 147 PCOS and 97 control women, plasma was batch analyzed using Somascan proteomic analysis for circulating KRas, Ras GTPase-activating protein-1 (RASA1), and 45 growth factor-related proteins. The cohort was subsequently stratified for BMI (body mass index), testosterone, and insulin resistance (HOMA-IR) for subset analysis. Results: Circulating KRas, and RASA1 did not differ between PCOS and control women (p > 0.05). EGF1, EGFR, and EGFRvIII were decreased in PCOS (p = 0.04, p = 0.04 and p < 0.001, respectively). FGF8, FGF9, and FGF17 were increased in PCOS (p = 0.02, p = 0.03 and p = 0.04, respectively), and FGFR1 was decreased in PCOS (p < 0.001). VEGF-D (p < 0.001), IGF1 (p < 0.001), IGF-1sR (p = 0.02), and PDGFRA (p < 0.001) were decreased in PCOS compared to controls. After stratifying for BMI ≤ 29.9 kg/m², EGFR FGF8, FGFR1 VEGF-D, IGF1, and IGF-1sR differed (p < 0.05) though EGF1, EGFRvIII, FGF8, FGFR1, and VEGF-D no longer differed; after subsequently stratifying for HOMA-IR, only FGFR1, VEGF-D, IGF1, and IGF-1sR differed between groups (p < 0.05). Conclusions: Several growth factors that activate Ras differ between women with and without PCOS, and when stratified for BMI and HOMA-IR, only FGFR1, VEGF-D, IGF1, and IGF-1sR differed; these appear to be inherent features of the pathophysiology of PCOS. Full article

B7 homolog 3 (B7-H3) has emerged as a promising target for cancer therapy due to its high expression in various types of cancer cells. It not only regulates the activity of immune cells but also modulates the signal transduction and metabolism of cancer cells. However, the specific interaction partners of B7-H3 still remain unclear, limiting a comprehensive understanding of the precise role of B7-H3 in cancer progression. In this study, we report that B7-H3 can bind to resting Raji cells, stimulated THP-1 cells, and even PC3 prostate cancer cells through its IgV domain alone. Furthermore, to identify the potential interaction partners of B7-H3 on these cells, we adopted an ascorbate peroxidase 2 (APEX2)-based proximity labeling strategy, which revealed about 10 key potential interaction partners. Interestingly, our results suggest that CD45 could be a putative receptor for B7-H3 on Raji cells, while the epidermal growth factor receptor (EGFR) could closely interact with B7-H3 on PC3 cells. Based on further computational structure modeling studies, we show that B7-H3 can bind to the epidermal growth factor (EGF) binding pocket of EGFR—surprisingly, with a stronger affinity than EGF itself. Overall, our study provides an effective approach to identifying B7-H3 interaction partners in both immune and cancer cell lines. Full article

The ultrastructure of submandibular gland (SMG) of Bactrian camels was observed by a transmission electron microscope. Routine HE staining, special staining combined with immunohistochemistry, and immunofluorescence techniques were used to study the histochemical characteristics of the submandibular gland and the localisation and distribution characteristics of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR). HE results showed that the submandibular gland of Bactrian camels was composed of mixed serous and mucinous acini glands. The submandibular striated duct was highly developed and connected with intercalated ducts with larger diameter. Intercalated ducts are shorter and directly connected to acini. In AB-PAS staining, it was observed that the inner wall of striated tube was strongly positive for AB staining. The distribution of the reticular fibres around the follicles and ducts of the submandibular gland is distinct, with collagen fibres distributed mainly in the periphery of the ducts and sparse collagen fibres in the periphery of the acini. Immunohistochemistry and fluorescence show that EGF is strongly positive in striated and intercalated ducts, and EGFR is weakly positive in striated and intercalated ducts. Bactrian camel SMGs secrete more acidic mucins, and EGF and EGFR were mainly secreted and play a role in the pipeline system of SMGs. Full article

Epidemiological studies suggest an increased risk of colorectal cancer (CRC) aggravation in patients with chronic kidney disease (CKD). Our previous study demonstrated that indoxyl sulfate, a uremic toxin whose concentration increases with CKD progression, exacerbates CRC through activation of the AhR and Akt pathways. Consequently, indoxyl sulfate has been proposed to be a significant link between CKD progression and CRC aggravation. The present study aimed to investigate the roles of c-Myc and β-Catenin, which are hypothesized to be downstream factors of indoxyl sulfate-induced AhR and Akt activation, in CRC cell proliferation and EGF sensitivity in HCT-116 CRC cells. Indoxyl sulfate significantly induced CRC cell proliferation at concentrations exceeding 62.5 µM, a process suppressed by the c-Myc inhibitor 10058-F4. Indoxyl sulfate activated the Akt/β-Catenin/c-Myc pathway as evidenced by the Akt inhibitor MK2206, which decreased both β-Catenin and c-Myc protein levels, and the β-Catenin inhibitor XAV-939, which reduced c-Myc protein levels. The AhR antagonist CH223191 also inhibited c-Myc upregulation, indicating involvement of the AhR/c-Myc pathway. MK2206 partially attenuated the indoxyl sulfate-induced AhR transcriptional activity, suggesting that Akt activation influences the AhR/c-Myc pathway. MK2206, CH223191, and 10058-F4 suppressed the increase in EGFR protein levels induced by indoxyl sulfate, indicating that the Akt/β-Catenin/c-Myc and AhR/c-Myc pathways enhance the sensitivity of HCT-116 CRC cells to EGF. These findings indicate that the elevation of indoxyl sulfate levels in the blood, due to CKD progression, could worsen CRC by promoting the proliferation of CRC cells and enhancing EGF signaling. Therefore, indoxyl sulfate could potentially serve as a therapeutic target for CRC aggravation in patients with CKD. Full article

The Sprouty (SPRY) proteins are evolutionary conserved modulators of receptor tyrosine kinase (RTK) signaling. SPRY2 inhibits fibroblast growth factor (FGF) signaling, whereas it enhances epidermal growth factor (EGF) signaling through inhibition of EGF receptor (EGFR) endocytosis, ubiquitination, and degradation. In this study, we analyzed the effects of SPRY2 on endocytosis and degradation of FGF receptor 1 (FGFR1) using two human glioblastoma (GBM) cell lines with different endogenous SPRY2 levels. SPRY2 overexpression (SPRY2-OE) inhibited clathrin- and caveolae-mediated endocytosis of FGFR1, reduced the number of caveolin-1 vesicles and the uptake of transferrin. Furthermore, FGFR1 protein was decreased by SPRY2-OE, whereas EGFR protein was increased. SPRY2-OE enhanced FGFR1 degradation by increased c-casitas b-lineage lymphoma (c-CBL)-mediated ubiquitination, but it diminished binding of phospholipase Cγ1 (PLCγ1) to FGFR1. Consequently, SPRY2-OE inhibited FGF2-induced activation of PLCγ1, whereas it enhanced EGF-induced PLCγ1 activation. Despite the reduction of FGFR1 protein and the inhibition of FGF signaling, SPRY2-OE increased cell viability, and knockdown of SPRY2 enhanced the sensitivity to cisplatin. These results demonstrate that the inhibitory effect of SPRY2-OE on FGF signaling is at least in part due to the reduction in FGFR1 levels and the decreased binding of PLCγ1 to the receptor. Full article

Colorectal cancer (CRC) has become a prevalent and deadly malignancy over the years. Drug resistance remains a major challenge in CRC treatment, significantly affecting patient survival rates. Obesity is a key risk factor for CRC development, and accumulating evidence indicates that increased secretion of adipokines, including Visfatin, under obese conditions contributes to the development of resistance in CRC to various therapeutic methods. Amphiregulin (AREG) is a member of the epidermal growth factor (EGF) family, which activates the EGF receptor (EGFR), influencing multiple tumorigenic characteristics of cancers. Abnormal expression levels of AREG in cancer cells have been associated with resistance to anti-EGFR therapy in patients. However, it remains unclear whether this abnormal expression also impacts CRC resistance to other chemotherapeutic drugs. The aim of this study is to examine whether AREG expression levels could be affected in CRC cells under Visfatin stimulation, thereby initiating the development of resistance to 5-fluororacil (5-FU). Through our results, we found that Visfatin indeed increases AREG expression, reducing the sensitivity of HCT-116 CRC cells to 5-FU cytotoxicity. Moreover, AREG upregulation is regulated by STAT3-CREB transcription factors activated by JNK1/2 and p38 signaling. This study highlights the significant role of AREG upregulation in CRC cells in initiating chemotherapeutic resistance to 5-FU under Visfatin stimulation. These findings provide a deeper understanding of drug resistance development in CRC under obese conditions and offer new insights into the correlation between an abnormal increase in AREG levels and the development of 5-FU-resistance in CRC cells, which should be considered in future clinical applications. Full article

Keratinocytes play an essential role in the inflammatory phase of wound regeneration. In addition to migrating and proliferating for tissue regeneration, they produce a large amount of cytokines that modulate the inflammatory process. Previous studies have shown that subthermal treatment with radiofrequency (RF) currents used in capacitive resistive electric transfer (CRET) therapy promotes the proliferation of HaCat keratinocytes and modulates their cytokine production. Although physical therapies have been shown to have anti-inflammatory effects in a variety of experimental models and in patients, knowledge of the biological basis of these effects is still limited. The aim of this study was to investigate the effect of CRET on keratinocyte proliferation, cytokine production (IL-8, MCP-1, RANTES, IL-6, IL-11), TNF-α secretion, and the expression of MMP9, MMP1, NF-κB, ERK1/2, and EGFR. Human keratinocytes (HaCat) were treated with an intermittent 448 kHz electric current (CRET signal) in subthermal conditions and for different periods of time. Cell proliferation was analyzed by XTT assay, cytokine and TNF-α production by ELISA, NF-κB expression and activation by immunofluorescence, and MMP9, MMP1, ERK1/2, and EGF receptor expression and activation by immunoblot. Compared to a control, CRET increases keratinocyte proliferation, increases the transient release of MCP-1, TNF-α, and IL-6 while decreasing IL-8. In addition, it modifies the expression of MMPs and activates EGFR, NF-κB, and ERK1/2 proteins. Our results indicate that CRET reasonably modifies cytokine production through the EGF receptor and the ERK1/2/NF-κB pathway, ultimately modulating the inflammatory response of human keratinocytes. Full article

The synthetic peptide of lumican C-terminal 13 amino acids with the cysteine replaced by an alanine, hereafter referred to as lumikine (LumC13_C-A: YEALRVANEVTLN), binds to TGFβ type I receptor/activin-like kinase5 (TBR1/ALK5) in the activated TGFβ receptor complex to promote corneal epithelial wound healing. The present study aimed to identify the minimum essential amino acid epitope necessary to exert the effects of lumikine via ALK5 and to determine the role of the Y (tyrosine) residue for promoting corneal epithelium wound healing. This study also aimed to determine the signaling pathway(s) triggered by lumican–ALK5 binding. For such, adult Lum knockout (Lum^−/−) mice (~8–12 weeks old) were subjected to corneal epithelium debridement using an Agerbrush^®. The injured eyes were treated with 10 µL eye drops containing 0.3 µM synthetic peptides designed based on the C-terminal region of lumican for 5–6 h. To unveil the downstream signaling pathways involved, inhibitors of the Alk5 and EGFR signaling pathways were co-administered or not. Corneas isolated from the experimental mice were subjected to whole-mount staining and imaged under a ZEISS Observer to determine the distance of epithelium migration. The expression of EGFR ligands was determined following a scratch assay with HTCE (human telomerase-immortalized cornea epithelial cells) in the presence or not of lumikine. Results indicated that shorter LumC-terminal peptides containing EVTLN and substitution of Y with F in lumikine abolishes its capability to promote epithelium migration indicating that Y and EVTLN are essential but insufficient for Lum activity. Lumikine activity is blocked by inhibitors of Alk5, EGFR, and MAPK signaling pathways, while EGF activity is only suppressed by EGFR and MAPK inhibitors. qRT-PCR of scratched HTCE cells cultures treated with lumikine showed upregulated expression of several EGFR ligands including epiregulin (EREG). Treatment with anti-EREG antibodies abolished the effects of lumikine in corneal epithelium debridement healing. The observations suggest that Lum/lumikine binds Alk5 and promotes the noncanonical Smad-independent TGFβ/TBRs signaling pathways during the healing of corneal epithelium debridement. Full article

Cancer cells can release EGF-like peptides, acquiring the capacity of autocrine stimulation via EGFR-mediated signaling. One of these peptides (HBEGF) was found to be released from a membrane-bound precursor protein and is critically implicated in the proliferative potential of cancer cells. We observed that the increased lactate levels characterizing neoplastic tissues can induce the release of uPA, a protease promoting HBEGF shedding. This effect led to EGFR activation and increased ERK1/2 phosphorylation. Since EGFR-mediated signaling potentiates glycolytic metabolism, this phenomenon can induce a self-sustaining deleterious loop, favoring tumor growth. A well characterized HBEGF inhibitor is CRM197, a single-site variant of diphtheria toxin. We observed that, when administered individually, CRM197 did not trigger evident antineoplastic effects. However, its association with a uPA inhibitor caused dampening of EGFR-mediated signaling and apoptosis induction. Overall, our study highlights that the increased glycolytic metabolism and lactate production can foster the activated state of EGFR receptor and suggests that the inhibition of EGFR-mediated signaling can be attempted by means of CRM197 administered with an appropriate protease inhibitor. This attempt could help in overcoming the problem of the acquired resistance to the conventionally used EGFR inhibitors. Full article

EGFR-targeted therapies are efficacious, but toxicity is common and can be severe. Urokinase type plasminogen activator receptor (uPAR)-targeted drugs are only emerging, so neither their efficacy nor toxicity is fully established. Recombinant eBAT was created by combining cytokines EGF and uPA on the same single-chain molecule with truncated Pseudomonas toxin. Its purpose was to simultaneously target tumors and their vasculature in the tumor microenvironment. In prior studies on mice and dogs, the drug proved efficacious. Here, we report the safety of eBAT in normal wildtype, uPAR knockout, and immunoreplete and immunodeficient tumor-bearing mice, as well as in dogs with spontaneous sarcoma that more closely mirror human cancer onset. In immunocompetent mice, tumor-bearing mice, uPAR knockout mice, and mice receiving species-optimized eBAT, toxicities were mild and self-limiting. Likewise, in dogs with life-threatening sarcoma given dosages found to be biologically active, eBAT was well tolerated. In mice receiving higher doses, eBAT was associated with dose-dependent evidence of liver injury, including portal biliary hyperplasia, oval cell proliferation, lymphoplasmacytic inflammation, periportal hepatocellular microvesicular change, hemorrhage, necrosis, and apoptosis. The results support continuing the clinical development of eBAT as a therapeutic agent for individuals with sarcoma and other cancers. Full article

Aberrant N-glycosylation has been associated with progression of the pediatric cancer neuroblastoma (NB) but remains understudied. Here we investigated oligomannose N-glycans in NB by genetic editing of MGAT1 in a human NB cell line, BE(2)-C, called BE(2)-C(MGAT1^−/−). Lectin binding studies confirmed that BE(2)-C(MGAT1^−/−) had decreased complex and increased oligomannose N-glycans. The relevance of 2D and 3D cell cultures was demonstrated for cell morphology, cell proliferation, and cell invasion, thereby highlighting the necessity for 3D cell culture in investigating cancerous properties. Western blotting revealed that oligomannosylated EGFR had increased autophosphorylation. Proliferation was decreased in BE(2)-C(MGAT1^−/−) using 2D and 3D cultures, but both cell lines had similar proliferation rates using 3D cultures without serum. Upon EGF treatment, BE(2)-C(MGAT1^−/−), but not BE(2)-C, showed increased proliferation, and furthermore, the mutant proliferated much faster than BE(2)-C under 3D conditions. Cell spheroid invasiveness was greatly increased in BE(2)-C(MGAT1^−/−) compared with BE(2)-C. Moreover, invasiveness was reduced in both cell lines with either EGF or RhoA activator treatment, regardless of the N-glycan population. Thus, this study further extends our earlier findings that oligomannose N-glycans enhance NB cell invasiveness, and that EGF stimulation of oligomannosylated EGFR greatly enhances cell proliferation rates, underlining the role of oligomannose N-glycans in the promotion of NB. Full article

Background: EGFR has been suggested to contribute to COPD development and progression. Excessive ligand activation of the receptor leads to epithelial hyperproliferation and increased production of mucus, together with alterations in the primary cilia. The present study was designed to evaluate the safety and effect of depleting EGF in moderate-to-severe COPD patients, with an EGF-based vaccine. Patients and methods: A phase I trial was conducted in subjects with moderate or severe COPD. The anti-EGF vaccine schedule consisted of 4 biweekly doses followed by 4 monthly boosters. The primary endpoint was the evaluation of the safety and immunogenicity of the vaccine, together with the change in FEV1 and physical function at week 24. Results: Twenty-six patients with moderate or severe COPD were included in the trial. The vaccine was well tolerated and no serious related adverse events were reported. Ninety percent of the individuals developed a protective antibody response. The specific anti-EGF antibodies had high avidity and were able to inhibit EGFR phosphorylation. At the end of vaccination, serum EGF became undetectable. At week 24, there was a clinically significant improvement in lung function, with a mean change in trough FEV1 of 106 mL. Patients also increased their physical functioning. Conclusions: The EGF-based vaccine was immunogenic and provoked an EGF exhaustion in patients with moderate-to-severe COPD. Depleting EGF might result in a meaningful increase in FEV1, with good tolerability. The current results provide new avenues to treat chronic inflammatory lung diseases associated with EGFR aberrant signaling. Full article

In tumor cells, interleukin-6 (IL-6) signaling can lead to activation of the epidermal growth factor receptor (EGFR), which prolongs Stat3 activation. In the present experiments, we tested the hypothesis that IL-6 signaling activates EGFR signaling in peripheral and spinal nociception and examined whether EGFR localization and activation coincide with pain-related behaviors in arthritis. In vivo in anesthetized rats, spinal application of the EGFR receptor blocker gefitinib reduced the responses of spinal cord neurons to noxious joint stimulation, but only after spinal pretreatment with IL-6 and soluble IL-6 receptor. Using Western blots, we found that IL-6-induced Stat3 activation was reduced by gefitinib in microglial cells of the BV2 cell line, but not in cultured DRG neurons. Immunohistochemistry showed EGFR localization in most DRG neurons from normal rats, but significant downregulation in the acute and most painful arthritis phase. In the spinal cord of mice, EGFR was highly activated mainly in the chronic phase of inflammation, with localization in neurons. These data suggest that spinal IL-6 signaling may activate spinal EGFR signaling. Downregulation of EGFR in DRG neurons in acute arthritis may limit nociception, but pronounced delayed activation of EGFR in the spinal cord may be involved in chronic inflammatory pain. Full article

Although SARS-CoV-2 induces mucin hypersecretion in the respiratory tract, hyposalivation/xerostomia has been reported by COVID-19 patients. We evaluate the submandibular gland (SMGs) pathogenesis in SARS-CoV-2-infected K18-hACE2 mice, focusing on the impact of infection on the mucin production and structural integrity of acini, ductal system, myoepithelial cells (MECs) and telocytes. The spike protein, the nucleocapsid protein, hACE2, actin, EGF, TNF-α and IL-1β were detected by immunofluorescence, and the Egfr and Muc5b expression was evaluated. In the infected animals, significant acinar hypertrophy was observed in contrast to ductal atrophy. Nucleocapsid proteins and/or viral particles were detected in the SMG cells, mainly in the nuclear membrane-derived vesicles, confirming the nuclear role in the viral formation. The acinar cells showed intense TNF-α and IL-1β immunoexpression, and the EGF-EGFR signaling increased, together with Muc5b upregulation. This finding explains mucin hypersecretion and acinar hypertrophy, which compress the ducts. Dying MECs and actin reduction were also observed, indicating failure of contraction and acinar support, favoring acinar hypertrophy. Viral assembly was found in the dying telocytes, pointing to these intercommunicating cells as viral transmitters in SMGs. Therefore, EGF-EGFR-induced mucin hypersecretion was triggered by SARS-CoV-2 in acinar cells, likely mediated by cytokines. The damage to telocytes and MECs may have favored the acinar hypertrophy, leading to ductal obstruction, explaining xerostomia in COVID-19 patients. Thus, acinar cells, telocytes and MECs may be viral targets, which favor replication and cell-to-cell viral transmission in the SMG, corroborating the high viral load in saliva of infected individuals. Full article

Pertuzumab (Perjeta^®), a humanized antibody binding to the dimerization arm of HER2 (Human epidermal growth factor receptor-2), has failed as a monotherapy agent in HER2 overexpressing malignancies. Since the molecular interaction of HER2 with ligand-bound EGFR (epidermal growth factor receptor) has been implied in mitogenic signaling and malignant proliferation, we hypothesized that this interaction, rather than HER2 expression and oligomerization alone, could be a potential molecular target and predictor of the efficacy of pertuzumab treatment. Therefore, we investigated static and dynamic interactions between HER2 and EGFR molecules upon EGF stimulus in the presence and absence of pertuzumab in HER2+ EGFR+ SK-BR-3 breast tumor cells using Förster resonance energy transfer (FRET) microscopy and fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS). The consequential activation of signaling and changes in cell proliferation were measured by Western blotting and MTT assay. The autocorrelation functions of HER2 diffusion were best fitted by a three-component model corrected for triplet formation, and among these components the slowly diffusing membrane component revealed aggregation induced by EGFR ligand binding, as evidenced by photon-counting histograms and co-diffusing fractions. This aggregation has efficiently been prevented by pertuzumab treatment, which also inhibited the post-stimulus interaction of EGFR and HER2, as monitored by changes in FRET efficiency. Overall, the data demonstrated that pertuzumab, by hindering post-stimulus interaction between EGFR and HER2, inhibits EGFR-evoked HER2 aggregation and phosphorylation and leads to a dose-dependent decrease in cell proliferation, particularly when higher amounts of EGF are present. Consequently, we propose that EGFR expression on HER2-positive tumors could be taken into consideration as a potential biomarker when predicting the outcome of pertuzumab treatment. Full article