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Background: The most important factors contributing to multi-drug resistance in oral cancer include overexpression of the EGFR protein and the downstream malignancy regulators that are associated with it. This study investigates the impact of solanine on inflammation, proliferation, and angiogenesis inhibition in multidrug-resistant oral cancer KB-Chr-8-5 cells through inhibition of the EGFR/PI3K/Akt/NF-κB signaling pathway. Methods: Cell viability was assessed using an MTT assay to evaluate cytotoxic effects. Production of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨM), and AO/EtBr staining were analyzed to assess apoptosis and mitochondrial dysfunction. Western blotting was employed to examine protein expression related to angiogenesis, apoptosis, and signaling pathways. Experiments were conducted in triplicate. Results: Solanine treatment at concentrations of 10, 20, and 30 μM significantly increased ROS production, which is indicative of its antioxidant properties. This increase was associated with decreased mitochondrial membrane potential (ΔΨM) with p < 0.05, suggesting mitochondrial dysfunction. Inhibition of EGFR led to reduced activity of PI3K, Akt, and NF-κB, resulting in decreased expression of iNOS, IL-6, Cyclin D1, PCNA, VEGF, Mcl-1, and HIF-1α and increased levels of the apoptotic proteins Bax, caspase-9, and caspase-3. These changes collectively inhibited the growth of multidrug-resistant (MDR) cancer cells. Conclusions: Solanine acts as a potent disruptor of cellular processes by inhibiting the EGFR-mediated PI3K/Akt/NF-κB signaling pathway. These results suggest that solanine holds promise as a potential preventive or therapeutic agent against multidrug-resistant cancers. [ABSTRACT FROM AUTHOR]

The highly lethal brain cancer glioblastoma (GBM) poses a daunting challenge because the blood-brain barrier renders potentially druggable amplified or mutated oncoproteins relatively inaccessible. Here, we identify sphingomyelin phosphodiesterase 1 (SMPD1), an enzyme that regulates the conversion of sphingomyelin to ceramide, as an actionable drug target in GBM. We show that the highly brain-penetrant antidepressant fluoxetine potently inhibits SMPD1 activity, killing GBMs, through inhibition of epidermal growth factor receptor (EGFR) signaling and via activation of lysosomal stress. Combining fluoxetine with temozolomide, a standard of care for GBM, causes massive increases in GBM cell death and complete tumor regression in mice. Incorporation of real-world evidence from electronic medical records from insurance databases reveals significantly increased survival in GBM patients treated with fluoxetine, which was not seen in patients treated with other selective serotonin reuptake inhibitor (SSRI) antidepressants. These results nominate the repurposing of fluoxetine as a potentially safe and promising therapy for patients with GBM and suggest prospective randomized clinical trials.

Apical‐basal polarity is maintained by distinct protein complexes that reside in membrane junctions, and polarity loss in monolayered epithelial cells can lead to formation of multilayers, cell extrusion, and/or malignant overgrowth. Yet, how polarity loss cooperates with intrinsic signals to control directional invasion toward neighboring epithelial cells remains elusive. Using the Drosophila ovarian follicular epithelium as a model, we found that posterior follicle cells with loss of lethal giant larvae (lgl) or Discs large (Dlg) accumulate apically toward germline cells, whereas cells with loss of Bazooka (Baz) or atypical protein kinase C (aPKC) expand toward the basal side of wildtype neighbors. Further studies revealed that these distinct multilayering patterns in the follicular epithelium were determined by epidermal growth factor receptor (EGFR) signaling and its downstream target Pointed, a zinc‐finger transcription factor. Additionally, we identified Rho kinase as a Pointed target that regulates formation of distinct multilayering patterns. These findings provide insight into how cell polarity genes and receptor tyrosine kinase signaling interact to govern epithelial cell organization and directional growth that contribute to epithelial tumor formation. Synopsis: Polarity loss in epithelia can lead to the formation of multilayered tissues and tumorigenesis, yet the molecular pathways involved remain unclear. Here, EGFR signaling is identified to antagonistically regulate cell organization in the Drosophila ovarian follicular epithelium (FE), defining directionality of invasion and cell fate. Loss of polarity genes lgl/Dlg or aPKC/Baz in follicle cells results in apical or basal multilayering patterns, respectively.Depletion of EGFR signaling enhances lgl/Dlg loss‐induced apical growth in the FE.EGFR promotes aPKC/Baz loss‐induced basal growth.EGFR triggers basal growth via transcription factor Pnt‐dependent actomyosin cytoskeletal regulators Rok and Sqh. [ABSTRACT FROM AUTHOR]

Multicolor single-molecule tracking (SMT) provides a powerful tool to mechanistically probe molecular interactions in living cells. However, because of the limitations in the optical and chemical properties of currently available fluorophores and the multiprotein labeling strategies, intracellular multicolor SMT remains challenging for general research studies. Here, we introduce a practical method employing a nanopore-electroporation (NanoEP) technique to deliver multiple organic dye-labeled proteins into living cells for imaging. It can be easily expanded to three channels in commercial microscopes or be combined with other in situ labeling methods. Utilizing NanoEP, we demonstrate three-color SMT for both cytosolic and membrane proteins. Specifically, we simultaneously monitored single-molecule events downstream of EGFR signaling pathways in living cells. The results provide detailed resolution of the spatial localization and dynamics of Grb2 and SOS recruitment to activated EGFR along with the resultant Ras activation.

Breast cancer is the second most cancer worldwide in females. The primary factor responsible for tumor recurrence is the presence of breast cancer stem cells (BCSCs), which escape the chemo-radiotherapy. In this study, we have investigated the role of Secretory phospholipase-A2 Group 2A (sPLA 2 -IIA) that is overexpressed in BCSCs of MCF7 and MDA-MB-231 breast cancer cell lines. Further, overexpression of sPLA 2 -IIA revealed an increased EGFR/JNK/c-JUN/c-FOS signaling in BCSCs, while sPLA 2 -IIA knockdown significantly reduced the percentage of BCSCs and decreased signaling in both the cell lines. Importantly, sPLA 2 -IIA knockdown showed differentiation of BCSCs. Strikingly, PET imaging showed a decreased metastatic potential of BCSCs. Our study revealed a novel role of sPLA 2 -IIA in regulating BCSCs, which play a crucial role in regulating the differentiation and metastatic potential of BCSCs. [Display omitted] • SPLA2-IIA knockdown reduces the number of BCSCs. • Loss of sPLA2-IIA decreases the tumorigenic potential of BCSCs in NOD/SCID mice. • SPLA2-IIA knockdown enhances the differentiation potential of BCSCs. • SPLA2-IIA knockdown leads to a decrease in the metastatic potential of BCSCs. • SPLA2-IIA knockdown reduces the EGFR/JNK/c-JUN-c-FOS signaling in BCSCs. [ABSTRACT FROM AUTHOR]

Breast cancer is the leading cause of cancer deaths for women worldwide. Endocrine therapies represent the cornerstone for hormone-dependent breast cancer treatment. However, in many cases, endocrine resistance is induced with poor prognosis for patients. In the current study, we have developed MCF-7 cell lines resistant to fulvestrant (MCF-7Fulv) and tamoxifen (MCF-7Tam) aiming at investigating mechanisms underlying resistance. Both resistant cell lines exerted lower proliferation capacity in two-dimensional (2-D) cultures but retain estrogen receptor α (ERα) expression and proliferate independent of the presence of estrogens. The established cell lines tend to be more aggressive exhibiting advanced capacity to form colonies, increased expression of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and heterodimerization of ERBB family receptors and activation of EGFR downstream pathways like MEK/ERK1/2 and PI3K/AKT. Tyrosine kinase inhibitors tested against resistant MCF-7Fulv and MCF-7Tam cells showed moderate efficacy to inhibit cell proliferation, except for lapatinib, which concomitantly inhibits both EGFR and HER2 receptors and strongly reduced cell proliferation. Furthermore, increased autophagy was observed in resistant MCF-7Fulv and MCF-7Tam cells as shown by the presence of autophagosomes and increased Beclin-1 levels. The increased autophagy in resistant cells is not associated with increased apoptosis, suggesting a cytoprotective role for autophagy that may favor cells' survival and aggressiveness. Thus, by exploiting those underlying mechanisms, new targets could be established to overcome endocrine resistance. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is one of the most common cancers worldwide. Epidemiological studies indicate that consumption of fruits and vegetables containing procyanidins is associated with lower CRC risk. This study investigated the capacity of two dimeric procyanidins composed of epicatechin gallate (ECG) or epigallocatechin gallate (EGCG) isolated from persimmons, to inhibit CRC cell growth and promote apoptosis, characterizing the underlying mechanisms. ECG and EGCG dimers reduced the growth of five human CRC cell lines in a concentration (10-60 μM)- and time (24-72 h)-dependent manner, with a 72 h-IC50 value in Caco-2 cells of 10 and 30 μM, respectively. ECG and EGCG dimers inhibited Caco-2 cell proliferation by arresting the cell cycle in G2/M phase and by inducing apoptosis via the mitochondrial pathway. In addition, ECG and EGCG dimers inhibited cell migration, invasion, and adhesion, decreasing the activity of matrix metalloproteinases (MMP-2/9). Mechanistically, ECG and EGCG dimers inhibited the activation of lipid raft-associated epidermal growth factor (EGF) receptor (EGFR), without affecting its localization at lipid rafts. In particular, ECG and EGCG dimers reduced EGFR phosphorylation at Tyr1068 residue, prevented EGFR dimerization and activation upon stimulation, and induced EGFR internalization both in the absence and presence of EGF. Furthermore, ECG and EGCG dimers increased EGFR phosphorylation at Tyr1045 residue, providing a docking site for ubiquitin ligase c-Cbl and induced EGFR degradation by the proteasome. Downstream of EGFR, ECG and EGCG dimers inhibited the activation of the MEK/ERK1/2 and PI3K/AKT signaling pathways, downregulating proteins involved in the modulation of cell survival. In conclusion, ECG and EGCG dimers reduced CRC cell growth by inhibiting EGFR activation at multiple steps, including the disruption of lipid rafts integrity and promoting EGFR degradation. These results shed light on a potential molecular mechanism on how procyanidins-rich diets may lower CRC risk.

Abstract Background In insects, an interplay between the activities of distinct hormones, such as juvenile hormone (JH) and 20-hydroxyecdysone (20E), regulates the progression through numerous life history hallmarks. As a crucial endocrine factor, JH is mainly synthesized in the corpora allata (CA) to regulate multiple physiological and developmental processes, including molting, metamorphosis, and reproduction. During the last century, significant progress has been achieved in elucidating the JH signal transduction pathway, while less progress has been made in dissecting the regulatory mechanism of JH biosynthesis. Previous work has shown that receptor tyrosine kinase (RTK) signaling regulates hormone biosynthesis in both insects and mammals. Here, we performed a systematic RNA interference (RNAi) screening to identify RTKs involved in regulating JH biosynthesis in the CA of adult Blattella germanica females. Results We found that the epidermal growth factor receptor (Egfr) is required for promoting JH biosynthesis in the CA of adult females. The Egf ligands Vein and Spitz activate Egfr, followed by Ras/Raf/ERK signaling, and finally activation of the downstream transcription factor Pointed (Pnt). Importantly, Pnt induces the transcriptional expression of two key enzyme-encoding genes in the JH biosynthesis pathway: juvenile hormone acid methyltransferase (JHAMT) and methyl farnesoate epoxidase (CYP15A1). Dual-luciferase reporter assay shows that Pnt is able to activate a promoter region of Jhamt. In addition, electrophoretic mobility shift assay confirms that Pnt directly binds to the − 941~ − 886 nt region of the Jhamt promoter. Conclusions This study reveals the detailed molecular mechanism of Egfr signaling in promoting JH biosynthesis in the German cockroach, shedding light on the intricate regulation of JH biosynthesis during insect development.

Introduction: Cancer stem cells (CSC), a major culprit of drug-resistant phenotypes and tumor relapse, represent less than 2 % of the bulk of TNBC cells, making them difficult to isolate, study, and thus, limiting our understanding of the pathogenesis of the disease. Current methods for CSC enrichment, such as 3D spheroid culture, genetic modification, and stem cell conditioning, are time consuming, expensive, and unsuitable for high-throughput assays. One way to address these limitations is to use topographical stimuli to enhance CSC populations in planar culture. Physical cues in the breast tumor microenvironment can influence cell behavior through changes in the mechanical properties of the extracellular matrix (ECM). In this study, we used topographical cues on polystyrene films to investigate their effect on the proteome and stemness of standard TNBC cell lines.Methods: The topographical polystyrene-based array was generated using razor printing and polishing methods. Proteome data were analyzed and enriched bioprocesses were identified using R software. Stemness was assessed measuring CD44, CD24 and ALDH markers using flow cytometry, immunofluorescence, detection assays, and further validated with mammosphere assay. EGF/EGFR expression and activity was evaluated using enzyme-linked immunosorbent assay (ELISA), immunofluorescence and antibody membrane array. A dose-response assay was performed to further investigate the effect of surface topography on the sensitivity of cells to the EGFR inhibitor.Results: Surface roughness enriched the CSC population and modulated epidermal growth factor receptor (EGFR) signaling activity in TNBC cells. Enhanced proliferation of MDA-MB-468 cells in roughness correlated with upregulation of the epidermal growth factor (EGF) ligand, which in turn corresponded with a 3-fold increase in the expression of EGFR and a 42% increase in its phosphorylation compared to standard smooth culture surfaces. The results also demonstrated that phenotypic changes associated with topographical (roughness) stimuli significantly decreased the drug sensitivity to the EGFR inhibitor gefitinib. In addition, the proportion of CD44+/CD24−/ALDH+ was enhanced on surface roughness in both MDA-MB-231 and MDA-MB-468 cell lines. We also demonstrated that YAP/TAZ activation decreased in a roughness-dependent manner, confirming the mechanosensing effect of the topographies on the oncogenic activity of the cells.Discussion: Overall, this study demonstrates the potential of surface roughness as a culture strategy to influence oncogenic activity in TNBC cells and enrich CSC populations in planar cultures. Such a culture strategy may benefit high-throughput screening studies seeking to identify compounds with broader tumor efficacy.

Growth factor binding to EGFR drives conformational changes that promote homodimerization and transphosphorylation, followed by adaptor recruitment, oligomerization, and signaling through Ras. Whether specific receptor conformations and oligomerization states are necessary for efficient activation of Ras is unclear. We therefore evaluated the sufficiency of a phosphorylated EGFR dimer to activate Ras without growth factor by developing a chemical-genetic strategy to crosslink and "trap" full-length EGFR homodimers on cells. Trapped dimers become phosphorylated and recruit adaptor proteins at stoichiometry equivalent to that of EGF-stimulated receptors. Surprisingly, these phosphorylated dimers do not activate Ras, Erk, or Akt. In the absence of EGF, phosphorylated dimers do not further oligomerize or reorganize on cell membranes. These results suggest that a phosphorylated EGFR dimer loaded with core signaling adapters is not sufficient to activate Ras and that EGFR ligands contribute to conformational changes or receptor dynamics necessary for oligomerization and efficient signal propagation through the SOS-Ras-MAPK pathway.

Tks4 is a large scaffold protein in the EGFR signal transduction pathway that is involved in several cellular processes, such as cellular motility, reactive oxygen species-dependent processes, and embryonic development. It is also implicated in a rare developmental disorder, Frank–ter Haar syndrome. Loss of Tks4 resulted in the induction of an EMT-like process, with increased motility and overexpression of EMT markers in colorectal carcinoma cells. In this work, we explored the broader effects of deletion of Tks4 on the gene expression pattern of HCT116 colorectal carcinoma cells by transcriptome sequencing of wild-type and Tks4 knockout (KO) cells. We identified several protein coding genes with altered mRNA levels in the Tks4 KO cell line, as well as a set of long non-coding RNAs, and confirmed these changes with quantitative PCR on a selected set of genes. Our results show a significant perturbation of gene expression upon the deletion of Tks4, suggesting the involvement of different signal transduction pathways over the well-known EGFR signaling. [ABSTRACT FROM AUTHOR]

Background: In insects, an interplay between the activities of distinct hormones, such as juvenile hormone (JH) and 20-hydroxyecdysone (20E), regulates the progression through numerous life history hallmarks. As a crucial endocrine factor, JH is mainly synthesized in the corpora allata (CA) to regulate multiple physiological and developmental processes, including molting, metamorphosis, and reproduction. During the last century, significant progress has been achieved in elucidating the JH signal transduction pathway, while less progress has been made in dissecting the regulatory mechanism of JH biosynthesis. Previous work has shown that receptor tyrosine kinase (RTK) signaling regulates hormone biosynthesis in both insects and mammals. Here, we performed a systematic RNA interference (RNAi) screening to identify RTKs involved in regulating JH biosynthesis in the CA of adult Blattella germanica females. Results: We found that the epidermal growth factor receptor (Egfr) is required for promoting JH biosynthesis in the CA of adult females. The Egf ligands Vein and Spitz activate Egfr, followed by Ras/Raf/ERK signaling, and finally activation of the downstream transcription factor Pointed (Pnt). Importantly, Pnt induces the transcriptional expression of two key enzyme-encoding genes in the JH biosynthesis pathway: juvenile hormone acid methyltransferase (JHAMT) and methyl farnesoate epoxidase (CYP15A1). Dual-luciferase reporter assay shows that Pnt is able to activate a promoter region of Jhamt. In addition, electrophoretic mobility shift assay confirms that Pnt directly binds to the − 941~ − 886 nt region of the Jhamt promoter. Conclusions: This study reveals the detailed molecular mechanism of Egfr signaling in promoting JH biosynthesis in the German cockroach, shedding light on the intricate regulation of JH biosynthesis during insect development. [ABSTRACT FROM AUTHOR]

Oleanolic acid (OA) and its derivatives show potent anticancer function. Pancreatic cancer (PC) is the fourth core motive of cancer‐related deaths worldwide. Epidermal growth factor receptor (EGFR) has been implicated in PC and has been validated as a therapeutic target. Our study demonstrated that K73‐03, an OA derivative, was identified as a potent inhibitor of EGFR by using reverse pharmacophore screening and molecular dynamics simulation assays. Moreover, Western blot analysis showed that K73‐03 markedly suppressed the levels of phosphorylated‐EGFR (p‐EGFR) and phosphorylated‐Akt (p‐Akt). The inhibitory effect of K73‐03 on PC cells was assessed in vitro and in vivo. Mechanistically, K73‐03 effectively inhibited the cell proliferation of PC cells, and induced apoptosis and autophagy of ASPC‐1 cells in a dose‐dependent manner. Additionally, pretreatment with chloroquine, an autophagy inhibitor, significantly inhibited K73‐03‐induced autophagy and enhanced K73‐03‐induced apoptotic cell death. K73‐03 also strongly repressed ASPC‐1 cells xenograft growth in vivo. Thus, all these findings provided new clues about OA analog K73‐03 as an effective anticancer agent targeted EGFR against ASPC‐1 cells, it is worth further evaluation in the future. [ABSTRACT FROM AUTHOR]

Pemphigus is a severe blistering disease caused by autoantibodies primarily against the desmosomal cadherins desmoglein (DSG)1 and DSG3, which impair desmosome integrity. Especially for the acute phase, additional treatment options allowing to reduce corticosteroids would fulfill an unmet medical need. In this study, we provide evidence that EGFR inhibition by erlotinib ameliorates pemphigus vulgaris IgG-induced acantholysis in intact human epidermis. Pemphigus vulgaris IgG caused phosphorylation of EGFR (Y845) and Rous sarcoma-related kinase in human epidermis. In line with this, a phosphotyrosine kinome analysis revealed a robust response associated with EGFR and Rous sarcoma-related kinase family kinase signaling in response to pemphigus vulgaris IgG but not to pemphigus foliaceus autoantibodies. Erlotinib inhibited pemphigus vulgaris IgG-induced epidermal blistering and EGFR phosphorylation, loss of desmosomes, as well as ultrastructural alterations of desmosome size, plaque symmetry, and keratin filament insertion and restored the desmosome midline considered as hallmark of mature desmosomes. Erlotinib enhanced both single-molecule DSG3-binding frequency and strength and delayed DSG3 fluorescence recovery, supporting that EGFR inhibition increases DSG3 availability and cytoskeletal anchorage. Our data indicate that EGFR is a promising target for pemphigus therapy owing to its link to several signaling pathways known to be involved in pemphigus pathogenesis., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract Background Bladder cancer (BLCA) is one of the most common malignancies worldwide. One of the main reasons for the unsatisfactory management of BLCA is the complex molecular biological mechanism. Annexin A1 (ANXA1), a Ca2+-regulated phospholipid-binding protein, has been demonstrated to be implicated in the progression and prognosis of many cancers. However, the expression pattern, biological function and mechanism of ANXA1 in BLCA remain unclear. Methods The clinical relevance of ANXA1 in BLCA was investigated by bioinformatics analysis based on TCGA and GEO datasets. Immunohistochemical (IHC) analysis was performed to detect the expression of ANXA1 in BLCA tissues, and the relationships between ANXA1 and clinical parameters were analyzed. In vitro and in vivo experiments were conducted to study the biological functions of ANXA1 in BLCA. Finally, the potential mechanism of ANXA1 in BLCA was explored by bioinformatics analysis and verified by in vitro and in vivo experiments. Results Bioinformatics and IHC analyses indicated that a high expression level of ANXA1 was strongly associated with the progression and poor prognosis of patients with BLCA. Functional studies demonstrated that ANXA1 silencing inhibited the proliferation, migration, invasion and epithelial–mesenchymal transition (EMT) of BLCA cells in vitro, and suppressed the growth of xenografted bladder tumors in vivo. Mechanistically, loss of ANXA1 decreased the expression and phosphorylation level of EGFR and the activation of downstream signaling pathways. In addition, knockdown of ANXA1 accelerated ubiquitination and degradation of P-EGFR to downregulate the activation of EGFR signaling. Conclusions These findings indicate that ANXA1 is a reliable clinical predictor for the prognosis of BLCA and promotes proliferation and migration by activating EGFR signaling in BLCA. Therefore, ANXA1 may be a promising biomarker for the prognosis of patients with BLCA, thus shedding light on precise and personalized therapy for BLCA in the future.

Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease. The lack of targeted therapies and poor patient outcome have fostered efforts to discover new molecular targets to treat patients with TNBC. Here, we showed that baculoviral IAP repeat containing 6 (BIRC6) is overexpressed and positively correlated with epidermal growth factor (EGF) receptor (EGFR) in TNBC cells and tissues and that BIRC6 overexpression is associated with poor patient survival. Mechanistic studies revealed that BIRC6 stability is increased by EGF-JNK signaling, which prevents ubiquitination and degradation of BIRC6 mediated by the E3 ubiquitin ligase HECTD1. BIRC6 in turn decreases SMAC expression by inducing the ubiquitin-proteasome pathway, thereby antagonizing apoptosis and promoting the proliferation, colony formation, tumorsphere formation, and tumor growth capacity of TNBC cells. Therapeutically, the PEGylated cationic lipid nanoparticle (pCLN)-assisted delivery of BIRC6 small interfering RNA (siRNA) efficiently silences BIRC6 expression in TNBC cells, thus suppressing TNBC cell growth in vitro and in vivo, and its antitumor activity is significantly superior to that of the EGFR inhibitor gefitinib. Our findings identify an important regulatory mechanism of BIRC6 overexpression and provide a potential therapeutic option for treating TNBC.

Simple Summary: Resistance to standard therapies impose a huge challenge on the treatment for glioblastoma multiforme (GBM), which is often considered as a cell intrinsic property of either GBM or, more significantly, of GBM stem-like cells. Tumor-associated macrophages and microglia (TAMs) take up the majority of the immune population in the tumor microenvironment of GBM and potentially participating in modulating therapy responses. However, little is known about the mechanisms underlying the effect of TAMs on temozolomide (TMZ) induced chemoresistance. Members of the metzincin superfamily such as Matrix Metalloproteases (MMPs) and A Disintegrin and Metalloprotease (ADAM) proteases are important participants in the process of intercellular communications in the tumor microenvironment. Herein, we revealed a novel concept of an intra-tumoral ADAM8 mediated malignant positive feedback loop constituted by the intimate interaction of tumor associated macrophages (TAMs) and GBM cells under TMZ treatment. These findings provide a convincing example and further support the notion that the tumor microenvironment, in addition to GBM cells and GBM stem-like cells, should be considered as an essential modulator of therapy in GBM. In conclusion, our study provides a rational basis for TAM sparing ADAM8-targeting in GBM to optimize standard chemotherapy. Standard chemotherapy of Glioblastoma multiforme (GBM) using temozolomide (TMZ) frequently fails due to acquired chemoresistance. Tumor-associated macrophages and microglia (TAMs) as major immune cell population in the tumor microenvironment are potential modulators of TMZ response. However; little is known about how TAMs participate in TMZ induced chemoresistance. Members of the metzincin superfamily such as Matrix Metalloproteases (MMPs) and A Disintegrin and Metalloprotease (ADAM) proteases are important mediators of cellular communication in the tumor microenvironment. A qPCR screening was performed to identify potential targets within the ADAM and MMP family members in GBM cells. In co-culture with macrophages ADAM8 was the only signature gene up-regulated in GBM cells induced by macrophages under TMZ treatment. The relationship between ADAM8 expression and TAM infiltration in GBM was determined in a patient cohort by qPCR; IF; and IHC staining and TCGA data analysis. Moreover; RNA-seq was carried out to identify the potential targets regulated by ADAM8. CCL2 expression levels were determined by qPCR; Western blot; IF; and ELISA. Utilizing qPCR; IF; and IHC staining; we observed a positive relationship between ADAM8 expression and TAMs infiltration level in GBM patient tissues. Furthermore; ADAM8 induced TAMs recruitment in vitro and in vivo. Mechanistically; we revealed that ADAM8 activated HB-EGF/EGFR signaling and subsequently up-regulated production of CCL2 in GBM cells in the presence of TMZ treatment; promoting TAMs recruitment; which further induced ADAM8 expression in GBM cells to mediate TMZ chemoresistance. Thus; we revealed an ADAM8 dependent positive feedback loop between TAMs and GBM cells under TMZ treatment which involves CCL2 and EGFR signaling to cause TMZ resistance in GBM. [ABSTRACT FROM AUTHOR]

Overexpressed EGFR and mutant K-Ras play vital roles in therapeutic resistance in colorectal cancer patients. To search for an effective therapeutic protocol is an urgent task. A secondary metabolite in the sponge Hippospongia sp., Heteronemin, has been shown to induce anti-proliferation in several types of cancers. A thyroxine-deaminated analogue, tetrac, binds to integrin αvβ3 to induce anti-proliferation in different cancers. Heteronemin- and in combination with tetrac-induced antiproliferative effects were evaluated. Tetrac enhanced heteronemin-induced anti-proliferation in HT-29 cells (KRAS WT CRC) and HCT-116 cells (KRAS MT CRC). Heteronemin and tetrac arrested cell cycle in different phases. Combined treatment increased the cell accumulation in sub-G1 and S phases. The combined treatment also induced the inactivation of EGFR signaling and downregulated the phosphorylated ERK1/2 protein in both cell lines. Heteronemin and the combination showed the downregulation of the phosphorylated and total PI3K protein in HT-29 cells (KRAS WT CRC). Results by NanoString technology and RT-qPCR revealed that heteronemin and combined treatment suppressed the expression of EGFR and downstream genes in HCT-116 cells (KRAS MT CRC). Heteronemin or combined treatment downregulated genes associated with cancer progression and decreased cell motility. Heteronemin or the combined treatment suppressed PD-L1 expression in both cancer cell lines. However, only tetrac and the combined treatment inhibited PD-L1 protein accumulation in HT-29 cells (KRAS WT CRC) and HCT-116 cells (KRAS MT CRC), respectively. In summary, heteronemin induced anti-proliferation in colorectal cancer cells by blocking the EGFR-dependent signal transduction pathway. The combined treatment further enhanced the anti-proliferative effect via PD-L1 suppression. It can be an alternative strategy to suppress mutant KRAS resistance for anti-EGFR therapy. [ABSTRACT FROM AUTHOR]

Abstract Background Epidermal growth factor receptor (EGFR) and its signaling pathways play a vital role in pathogenesis of lung cancer. By disturbing EGFR signaling, mutations of EGFR may lead to progression of cancer or the emergence of resistance to EGFR-targeted drugs. Results We investigated the correlation between EGFR mutations and EGFR-receptor tyrosine kinase (RTK) crosstalk in the signaling network, in order to uncover the drug resistance mechanism induced by EGFR mutations. For several EGFR wild type (WT) or mutated proteins, we measured the EGFR-RTK interactions using several computational methods based on molecular dynamics (MD) simulations, including geometrical characterization of the interfaces and conventional estimation of free energy of binding. Geometrical properties, namely the matching rate of atomic solid angles in the interfaces and center-of-mass distances between interacting atoms, were extracted relying on Alpha Shape modeling. For a couple of RTK partners (c-Met, ErbB2 and IGF-1R), results have shown a looser EGFR-RTK crosstalk for the drug-sensitive EGFR mutant while a tighter crosstalk for the drug-resistant mutant. It guarantees the genotype-determined EGFR-RTK crosstalk, and further proposes a potential drug resistance mechanism by amplified EGFR-RTK crosstalk induced by EGFR mutations. Conclusions This study will lead to a deeper understanding of EGFR mutation-induced drug resistance mechanisms and promote the design of innovative drugs.

Abstract Loss-of-function mutations in the human oligophrenin-1 (OPHN1) gene cause intellectual disability, a prevailing neurodevelopmental condition. However, the role OPHN1 plays during neuronal development is not well understood. We investigated the role of the Drosophila OPHN1 ortholog Graf in the development of the mushroom body (MB), a key brain structure for learning and memory in insects. We show that loss of Graf causes abnormal crossing of the MB β lobe over the brain midline during metamorphosis. This defect in Graf mutants is rescued by MB-specific expression of Graf and OPHN1. Furthermore, MB α/β neuron-specific RNA interference experiments and mosaic analyses indicate that Graf acts via a cell-autonomous mechanism. Consistent with the negative regulation of epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) signaling by Graf, activation of this pathway is required for the β-lobe midline-crossing phenotype of Graf mutants. Finally, Graf mutants have impaired olfactory long-term memory. Our findings reveal a role for Graf in MB axon development and suggest potential neurodevelopmental functions of human OPHN1.

While classic cadherin-actin connections in adherens junctions (AJs) have ancient origins, intermediate filament (IF) linkages with desmosomal cadherins arose in vertebrate organisms. In this mini-review, we discuss how overlaying the IF-desmosome network onto the existing cadherin-actin network provided new opportunities to coordinate tissue mechanics with the positioning and function of chemical signaling mediators in the ErbB family of receptor tyrosine kinases. We focus in particular on the complex multi-layered outer covering of the skin, the epidermis, which serves essential barrier and stress sensing/responding functions in terrestrial vertebrates. We will review emerging data showing that desmosome-IF connections, AJ-actin interactions, ErbB family members, and membrane tension are all polarized across the multiple layers of the regenerating epidermis. Importantly, their integration generates differentiation-specific roles in each layer of the epidermis that dictate the form and function of the tissue. In the basal layer, the onset of the differentiation-specific desmosomal cadherin desmoglein 1 (Dsg1) dials down EGFR signaling while working with classic cadherins to remodel cortical actin cytoskeleton and decrease membrane tension to promote cell delamination. In the upper layers, Dsg1 and E-cadherin cooperate to maintain high tension and tune EGFR and ErbB2 activity to create the essential tight junction barrier. Our final outlook discusses the emerging appreciation that the desmosome-IF scaffold not only creates the architecture required for skin’s physical barrier but also creates an immune barrier that keeps inflammation in check.

The mechanisms controlling the dynamics of expansion of adherens junctions are significantly less understood than those controlling their static properties. Here, we report that for suspended cell aggregates, the time to form a new junction between two cells speeds up with the number of junctions that the cells are already engaged in. Upon junction formation, the activation of epidermal growth factor receptor (EGFR) distally affects the actin turnover dynamics of the free cortex of the cells. The 'primed' actin cortex results in a faster expansion of the subsequent new junctions. In such aggregates, we show that this mechanism results in a cooperative acceleration of the junction expansion dynamics (kinetype) but does not alter the cell contractility, and hence the final junction size (phenotype). [ABSTRACT FROM AUTHOR]

Chemotherapy is still the main therapeutic strategy for gastric cancer (GC). However, most patients eventually acquire multidrug resistance (MDR). Hyperactivation of the EGFR signaling pathway contributes to MDR by promoting cancer cell proliferation and inhibiting apoptosis. We previously identified the secreted protein CGA as a novel ligand of EGFR and revealed a CGA/EGFR/GATA2 positive feedback circuit that confers MDR in GC. Herein, we outline a microRNA-based treatment approach for MDR reversal that targets both CGA and GATA2. We observed increased expression of CGA and GATA2 and increased activation of EGFR in GC samples. Bioinformatic analysis revealed that miR-107 could simultaneously target CGA and GATA2, and the low expression of miR-107 was correlated with poor prognosis in GC patients. The direct interactions between miR-107 and CGA or GATA2 were validated by luciferase reporter assays and Western blot analysis. Overexpression of miR-107 in MDR GC cells increased their susceptibility to chemotherapeutic agents, including fluorouracil, adriamycin, and vincristine, in vitro. Notably, intratumor injection of the miR-107 prodrug enhanced MDR xenograft sensitivity to chemotherapies in vivo. Molecularly, targeting CGA and GATA2 with miR-107 inhibited EGFR downstream signaling, as evidenced by the reduced phosphorylation of ERK and AKT. These results suggest that miR-107 may contribute to the development of a promising therapeutic approach for the treatment of MDR in GC., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

ADAM17 is upregulated in many cancers and in turn activates signaling pathways, including EGFR/ErbB, as well as those underlying resistance to targeted anti-EGFR therapies. Due to its central role in oncogenic pathways and drug resistance mechanisms, specific and efficacious monoclonal antibodies against ADAM17 could be useful for a broad patient population with solid tumors. Hence, we describe here an inhibitory anti-ADAM17 monoclonal antibody, named D8P1C1, that preferentially recognizes ADAM17 on cancer cells. D8P1C1 inhibits the catalytic activity of ADAM17 in a fluorescence-based peptide cleavage assay, as well as the proliferation of a range of cancer cell lines, including breast, ovarian, glioma, colon and the lung adenocarcinoma. In mouse models of triple-negative breast cancer and ovarian cancer, treatment with the mAb results in 78% and 45% tumor growth inhibition, respectively. Negative staining electron microscopy analysis of the ADAM17 ectodomain in complex with D8P1C1 reveals that the mAb binds the ADAM17 protease domain, consistent with its ability to inhibit the ADAM17 catalytic activity. Collectively, our results demonstrate the therapeutic potential of the D8P1C1 mAb to treat solid tumors.

Background: Bladder cancer (BLCA) is one of the most common malignancies worldwide. One of the main reasons for the unsatisfactory management of BLCA is the complex molecular biological mechanism. Annexin A1 (ANXA1), a Ca2+-regulated phospholipid-binding protein, has been demonstrated to be implicated in the progression and prognosis of many cancers. However, the expression pattern, biological function and mechanism of ANXA1 in BLCA remain unclear. Methods: The clinical relevance of ANXA1 in BLCA was investigated by bioinformatics analysis based on TCGA and GEO datasets. Immunohistochemical (IHC) analysis was performed to detect the expression of ANXA1 in BLCA tissues, and the relationships between ANXA1 and clinical parameters were analyzed. In vitro and in vivo experiments were conducted to study the biological functions of ANXA1 in BLCA. Finally, the potential mechanism of ANXA1 in BLCA was explored by bioinformatics analysis and verified by in vitro and in vivo experiments. Results: Bioinformatics and IHC analyses indicated that a high expression level of ANXA1 was strongly associated with the progression and poor prognosis of patients with BLCA. Functional studies demonstrated that ANXA1 silencing inhibited the proliferation, migration, invasion and epithelial–mesenchymal transition (EMT) of BLCA cells in vitro, and suppressed the growth of xenografted bladder tumors in vivo. Mechanistically, loss of ANXA1 decreased the expression and phosphorylation level of EGFR and the activation of downstream signaling pathways. In addition, knockdown of ANXA1 accelerated ubiquitination and degradation of P-EGFR to downregulate the activation of EGFR signaling. Conclusions: These findings indicate that ANXA1 is a reliable clinical predictor for the prognosis of BLCA and promotes proliferation and migration by activating EGFR signaling in BLCA. Therefore, ANXA1 may be a promising biomarker for the prognosis of patients with BLCA, thus shedding light on precise and personalized therapy for BLCA in the future. [ABSTRACT FROM AUTHOR]

Summary: The highly lethal brain cancer glioblastoma (GBM) poses a daunting challenge because the blood-brain barrier renders potentially druggable amplified or mutated oncoproteins relatively inaccessible. Here, we identify sphingomyelin phosphodiesterase 1 (SMPD1), an enzyme that regulates the conversion of sphingomyelin to ceramide, as an actionable drug target in GBM. We show that the highly brain-penetrant antidepressant fluoxetine potently inhibits SMPD1 activity, killing GBMs, through inhibition of epidermal growth factor receptor (EGFR) signaling and via activation of lysosomal stress. Combining fluoxetine with temozolomide, a standard of care for GBM, causes massive increases in GBM cell death and complete tumor regression in mice. Incorporation of real-world evidence from electronic medical records from insurance databases reveals significantly increased survival in GBM patients treated with fluoxetine, which was not seen in patients treated with other selective serotonin reuptake inhibitor (SSRI) antidepressants. These results nominate the repurposing of fluoxetine as a potentially safe and promising therapy for patients with GBM and suggest prospective randomized clinical trials.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that couples the binding of extracellular ligands, such as EGF and transforming growth factor-α (TGF-α), to the initiation of intracellular signaling pathways. EGFR binds to EGF and TGF-α with similar affinity, but generates different signals from these ligands. To address the mechanistic basis of this phenomenon, we have carried out cryo-EM analyses of human EGFR bound to EGF and TGF-α. We show that the extracellular module adopts an ensemble of dimeric conformations when bound to either EGF or TGF-α. The two extreme states of this ensemble represent distinct ligand-bound quaternary structures in which the membrane-proximal tips of the extracellular module are either juxtaposed or separated. EGF and TGF-α differ in their ability to maintain the conformation with the membrane-proximal tips of the extracellular module separated, and this conformation is stabilized preferentially by an oncogenic EGFR mutation. Close proximity of the transmembrane helices at the junction with the extracellular module has been associated previously with increased EGFR activity. Our results show how EGFR can couple the binding of different ligands to differential modulation of this proximity, thereby suggesting a molecular mechanism for the generation of ligand-sensitive differential outputs in this receptor family.

Background: Epidermal growth factor receptor (EGFR) and its signaling pathways play a vital role in pathogenesis of lung cancer. By disturbing EGFR signaling, mutations of EGFR may lead to progression of cancer or the emergence of resistance to EGFR-targeted drugs. Results: We investigated the correlation between EGFR mutations and EGFR-receptor tyrosine kinase (RTK) crosstalk in the signaling network, in order to uncover the drug resistance mechanism induced by EGFR mutations. For several EGFR wild type (WT) or mutated proteins, we measured the EGFR-RTK interactions using several computational methods based on molecular dynamics (MD) simulations, including geometrical characterization of the interfaces and conventional estimation of free energy of binding. Geometrical properties, namely the matching rate of atomic solid angles in the interfaces and center-of-mass distances between interacting atoms, were extracted relying on Alpha Shape modeling. For a couple of RTK partners (c-Met, ErbB2 and IGF-1R), results have shown a looser EGFR-RTK crosstalk for the drug-sensitive EGFR mutant while a tighter crosstalk for the drug-resistant mutant. It guarantees the genotype-determined EGFR-RTK crosstalk, and further proposes a potential drug resistance mechanism by amplified EGFR-RTK crosstalk induced by EGFR mutations. Conclusions: This study will lead to a deeper understanding of EGFR mutation-induced drug resistance mechanisms and promote the design of innovative drugs. [ABSTRACT FROM AUTHOR]

Overexpressed EGFR and mutant K-Ras play vital roles in therapeutic resistance in colorectal cancer patients. To search for an effective therapeutic protocol is an urgent task. A secondary metabolite in the sponge Hippospongia sp., Heteronemin, has been shown to induce anti-proliferation in several types of cancers. A thyroxine-deaminated analogue, tetrac, binds to integrin αvβ3 to induce anti-proliferation in different cancers. Heteronemin- and in combination with tetrac-induced antiproliferative effects were evaluated. Tetrac enhanced heteronemin-induced anti-proliferation in HT-29 cells (KRAS WT CRC) and HCT-116 cells (KRAS MT CRC). Heteronemin and tetrac arrested cell cycle in different phases. Combined treatment increased the cell accumulation in sub-G1 and S phases. The combined treatment also induced the inactivation of EGFR signaling and downregulated the phosphorylated ERK1/2 protein in both cell lines. Heteronemin and the combination showed the downregulation of the phosphorylated and total PI3K protein in HT-29 cells (KRAS WT CRC). Results by NanoString technology and RT-qPCR revealed that heteronemin and combined treatment suppressed the expression of EGFR and downstream genes in HCT-116 cells (KRAS MT CRC). Heteronemin or combined treatment downregulated genes associated with cancer progression and decreased cell motility. Heteronemin or the combined treatment suppressed PD-L1 expression in both cancer cell lines. However, only tetrac and the combined treatment inhibited PD-L1 protein accumulation in HT-29 cells (KRAS WT CRC) and HCT-116 cells (KRAS MT CRC), respectively. In summary, heteronemin induced anti-proliferation in colorectal cancer cells by blocking the EGFR-dependent signal transduction pathway. The combined treatment further enhanced the anti-proliferative effect via PD-L1 suppression. It can be an alternative strategy to suppress mutant KRAS resistance for anti-EGFR therapy.

Loss-of-function mutations in the human oligophrenin-1 (OPHN1) gene cause intellectual disability, a prevailing neurodevelopmental condition. However, the role OPHN1 plays during neuronal development is not well understood. We investigated the role of the Drosophila OPHN1 ortholog Graf in the development of the mushroom body (MB), a key brain structure for learning and memory in insects. We show that loss of Graf causes abnormal crossing of the MB β lobe over the brain midline during metamorphosis. This defect in Graf mutants is rescued by MB-specific expression of Graf and OPHN1. Furthermore, MB α/β neuron-specific RNA interference experiments and mosaic analyses indicate that Graf acts via a cell-autonomous mechanism. Consistent with the negative regulation of epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) signaling by Graf, activation of this pathway is required for the β-lobe midline-crossing phenotype of Graf mutants. Finally, Graf mutants have impaired olfactory long-term memory. Our findings reveal a role for Graf in MB axon development and suggest potential neurodevelopmental functions of human OPHN1. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) have been suggested to play important roles in some biological processes. However, the detailed mechanisms are not fully understood. We previously identified an antisense lncRNA, Bmdsx-AS1, that is involved in pre-mRNA splicing of the sex-determining gene Bmdsx in the silkworm. In this study, we analyzed the changes in the male external genitalia of transgenic overexpressed Bmdsx-AS1 silkworm lines and analyzed downstream and upstream responses. We found that Bmdsx-AS1 transgenic silkworms, compared with wild type, showed more claspers in the male external genitalia. Quantitative real-time PCR (qPCR) results indicated that overexpression of Bmdsx-AS1 decreased the expression of genes in the EGFR signaling pathway. Knockdown of Bmdsx-AS1 increased the activity of the EGFR pathway. Through promoter prediction, promoter truncation and electrophoretic mobility shift assay (EMSA) analyses, we found that the protein encoded by the Hox gene BmAbd-B specifically binds to the promoter of Bmdsx-AS1. Moreover, overexpression of BmAbd-B in the silkworm BmE cell line indicated that BmAbd-B negatively regulates the mRNA expression of Bmdsx-AS1. Our study provides insights into the regulatory mechanism of the lncRNA in the silkworm.

The Janus-kinase/signal transducer and activator of transcription (JAK/ STAT) pathway regulates the anterior posterior axis of the Drosophila follicle cells. In the anterior, it activates the bone morphogenetic protein (BMP) signaling pathway through expression of the BMP ligand decapentaplegic (dpp). In the posterior, JAK/STAT works with the epidermal growth factor receptor (EGFR) pathway to express the T-box transcription factor midline (mid). Although MID is necessary for establishing the posterior fate of the egg chamber,we showthat it is not sufficient to determine a posterior fate. The ETS-transcription factor pointed (pnt) is expressed in an overlapping domain to mid in the follicle cells. This study shows that pnt is upstream of mid and that it is sufficient to induce a posterior fate in the anterior end, which is characterized by the induction of mid, the prevention of the stretched cells formation and the abrogation of border cell migration. We demonstrate that the anterior BMP signaling is abolished by PNT through dpp repression. However, ectopic DPP cannot rescue the anterior fate formation, suggesting additional targets of PNT participate in the posterior fate determination. [ABSTRACT FROM AUTHOR]

To bridge the gap between qualitative and quantitative analyses of the epidermal growth factor receptor (EGFR) in tissues, we generated an sfGFP-tagged EGF receptor (EGFR-sfGFP) in Drosophila. The homozygous fly appears similar to wild type with EGFR expression and activation patterns that are consistent with previous reports in the ovary, early embryo, and imaginal discs. Using ELISA, we quantified an average of 1100, 6200 and 2500 receptors per follicle cell (FC) at stages 8/9, 10 and ≥11 of oogenesis, respectively. Interestingly, the spatial localization of the EGFR to the apical side of the FCs at early stages depended on the TGFα-like ligand Gurken. At later stages, EGFR localized to basolateral positions of the FCs. Finally, we followed the endosomal localization of EGFR in the FCs. The EGFR colocalized with the late endosome, but no significant colocalization of the receptor was found with the early endosome. The EGFR-sfGFP fly is an exciting new resource for studying cellular localization and regulation of EGFR in tissues. [ABSTRACT FROM AUTHOR]

Oxidative stress influences stem cell behavior by promoting the differentiation, proliferation, or apoptosis of stem cells. Thus, characterizing the effects of reactive oxygen species (ROS) on stem cell behavior provides insights into the significance of redox homeostasis in stem cell-associated diseases and efficient stem cell expansion for cellular therapies. We utilized the Drosophila testis as an in vivo model to examine the effects of ROS on germline stem cell (GSC) maintenance. High levels of ROS induced by alteration in Keap1/Nrf2 activity decreased GSC number by promoting precocious GSC differentiation. Notably, high ROS enhanced the transcription of the EGFR ligand spitz and the expression of phospho-Erk1/2, suggesting that high ROS-mediated GSC differentiation is through EGFR signaling. By contrast, testes with low ROS caused by Keap1 inhibition or antioxidant treatment showed an overgrowth of GSC-like cells. These findings suggest that redox homeostasis regulated by Keap1/Nrf2 signaling plays important roles in GSC maintenance.

Glucose metabolism is an important metabolic pathway in the auditory system. Chronic alcohol exposure can cause metabolic dysfunction in auditory cells during hearing loss. While alcohol exposure has been linked to hearing loss, the mechanism by which impaired glycolysis promotes cytotoxicity and cell death in auditory cells remains unclear. Here, we show that the inhibition of epidermal growth factor receptor (EGFR)-induced glycolysis is a critical mechanism for alcohol exposure-induced apoptosis in HEI-OC1 cells. The cytotoxicity via apoptosis was significantly increased by alcohol exposure in HEI-OC1 cells. The glycolytic activity and the levels of hexokinase 1 (HK1) were significantly suppressed by alcohol exposure in HEI-OC1 cells. Mechanistic studies showed that the levels of EGFR and AKT phosphorylation were reduced by alcohol exposure in HEI-OC1 cells. Notably, HK1 expression and glycolytic activity was suppressed by EGFR inhibition in HEI-OC1 cells. These results suggest that impaired glycolysis promotes alcohol exposure-induced apoptosis in HEI-OC1 cells via the inhibition of EGFR signaling. [ABSTRACT FROM AUTHOR]

Ganglioside GT1b is well-known for its role in cytokine production and in activating epidermal growth factor receptor (EGFR)-mediated signaling pathways in cancer cells. However, there are no reports that clearly elucidate the role of GT1b in EGFR-mediated signaling pathways in porcine oocytes during the process of in vitro maturation (IVM). In this study, we investigated the role of GT1b in EGFR-mediated activation of the ERK1/2 pathway in porcine cumulus-oocyte complexes (COCs) at 44 h of IVM. Our data show that expression of the ST3GAL2 protein significantly increased in porcine COCs at 44 h irrespective of treatment with EGF. Meiotic maturation and mRNA levels of factors (HAS2, TNFAIP6, and PTX3) related to cumulus cell expansion significantly increased in COCs treated with 2 μM GT1b during IVM in the absence of EGF. They also increased in COCs treated with EGF/GT1b as compared to that in the other groups. Interestingly, protein levels of EGFR, phospho-EGFR, ERK1/2, and phospho-ERK1/2 dramatically increased in COCs treated with EGF/GT1b. Moreover, the rate of fertilization and the developmental competence of blastocyst were significantly higher in EGF/GT1b-treated COCs. Taken together, these results suggest that exogenous GT1b improves meiotic maturation and cumulus cell expansion in porcine COCs via activation of EGFR-mediated ERK1/2 signaling. [ABSTRACT FROM AUTHOR]

Brain tumors are a heterogeneous group of neoplasms ranging from almost benign to highly aggressive phenotypes. The malignancy of these tumors mostly relies on gene expression reprogramming, which is frequently accompanied by the aberrant regulation of RNA processing mechanisms. In brain tumors, defects in alternative splicing result either from the dysregulation of expression and activity of splicing factors, or from mutations in the genes encoding splicing machinery components. Aberrant splicing regulation can generate dysfunctional proteins that lead to modification of fundamental physiological cellular processes, thus contributing to the development or progression of brain tumors. Herein, we summarize the current knowledge on splicing abnormalities in brain tumors and how these alterations contribute to the disease by sustaining proliferative signaling, escaping growth suppressors, or establishing a tumor microenvironment that fosters angiogenesis and intercellular communications. Lastly, we review recent efforts aimed at developing novel splicing-targeted cancer therapies, which employ oligonucleotide-based approaches or chemical modulators of alternative splicing that elicit an impact on brain tumor biology. [ABSTRACT FROM AUTHOR]

Breast cancers that express epidermal growth factor (EGF) receptors (EGFRs) are associated with poor prognosis. Our group recently showed in breast cancer patients that EGFR expression is strongly correlated with high tumor uptake of the glucose analogue, 18F-fluorodeoxyglucose (FDG). Here, we explored the cellular mechanism and signaling pathways that can explain the relation between EGFR and breast cancer cell glucose metabolism. FDG uptake, lactate production and hexokinase (HK) activity were measured, and proliferation assays and western blots were performed. EGF stimulated an increase of FDG uptake in EGFR-positive T47D and MDA-MB-468 cells, but not in MCF-7 cells. In T47D cells, the effect was dose-dependent and was accompanied by increased lactate production, indicating a shift toward glycolytic flux. This metabolic response occurred through enhanced HK activity and upregulated glucose transporter 1 (GLUT1) expression. EGFR stimulation also increased T47D cell proliferation. Blocking EGFR activation with BIBX1382 or gefitinib completely abolished both FDG uptake and proliferation effects. EGFR stimulation induced MAP kinase (MAPK) and PI3 kinase (PI3K) activation. Increased cell proliferation by EGFR stimulation was completely abolished by MAPK inhibition with PD98059 or by PI3K inhibition with LY294002. Increased FDG uptake was also completely abrogated by PI3K inhibition but was uninfluenced by MAPK inhibition. These findings suggest that the association between breast tumor EGFR expression and high FDG uptake might be contributed by stimulation of the PI3K pathway downstream of EGFR activation. This was in contrast to EGFR-mediated cell proliferation that required MAPK as well as PI3K signaling. [ABSTRACT FROM AUTHOR]

The host limits adenovirus infections by mobilizing immune systems directed against infected cells that also represent major barriers to clinical use of adenoviral vectors. Adenovirus early transcription units encode a number of products capable of thwarting antiviral immune responses by co-opting host cell pathways. Although the EGF receptor (EGFR) was a known target for the early region 3 (E3) RIDα protein encoded by nonpathogenic group C adenoviruses, the functional role of this host-pathogen interaction was unknown. Here we report that incoming viral particles triggered a robust, stress-induced pathway of EGFR trafficking and signaling prior to viral gene expression in epithelial target cells. EGFRs activated by stress of adenoviral infection regulated signaling by the NFκB family of transcription factors, which is known to have a critical role in the host innate immune response to infectious adenoviruses and adenovirus vectors. We found that the NFκB p65 subunit was phosphorylated at Thr254, shown previously by other investigators to be associated with enhanced nuclear stability and gene transcription, by a mechanism that was attributable to ligand-independent EGFR tyrosine kinase activity. Our results indicated that the adenoviral RIDα protein terminated this pathway by co-opting the host adaptor protein Alix required for sorting stress-exposed EGFRs in multivesicular endosomes, and promoting endosome-lysosome fusion independent of the small GTPase Rab7, in infected cells. Furthermore RIDα expression was sufficient to down-regulate the same EGFR/NFκB signaling axis in a previously characterized stress-activated EGFR trafficking pathway induced by treatment with the pro-inflammatory cytokine TNF-α. We also found that cell stress activated additional EGFR signaling cascades through the Gab1 adaptor protein that may have unappreciated roles in the adenoviral life cycle. Similar to other E3 proteins, RIDα is not conserved in adenovirus serotypes associated with potentially severe disease, suggesting stress-activated EGFR signaling may contribute to adenovirus virulence. [ABSTRACT FROM AUTHOR]

Human respiratory syncytial virus (RSV) is the leading viral cause of acute pediatric lower respiratory tract infections worldwide, with no available vaccine or effective antiviral drug. To gain insight into virus-host interactions, we performed a genome-wide siRNA screen. The expression of over 20,000 cellular genes was individually knocked down in human airway epithelial A549 cells, followed by infection with RSV expressing green fluorescent protein (GFP). Knockdown of expression of the cellular ATP1A1 protein, which is the major subunit of the Na+,K+-ATPase of the plasma membrane, had one of the strongest inhibitory effects on GFP expression and viral titer. Inhibition was not observed for vesicular stomatitis virus, indicating that it was RSV-specific rather than a general effect. ATP1A1 formed clusters in the plasma membrane very early following RSV infection, which was independent of replication but dependent on the attachment glycoprotein G. RSV also triggered activation of ATP1A1, resulting in signaling by c-Src-kinase activity that transactivated epidermal growth factor receptor (EGFR) by Tyr845 phosphorylation. ATP1A1 signaling and activation of both c-Src and EGFR were found to be required for efficient RSV uptake. Signaling events downstream of EGFR culminated in the formation of macropinosomes. There was extensive uptake of RSV virions into macropinosomes at the beginning of infection, suggesting that this is a major route of RSV uptake, with fusion presumably occurring in the macropinosomes rather than at the plasma membrane. Important findings were validated in primary human small airway epithelial cells (HSAEC). In A549 cells and HSAEC, RSV uptake could be inhibited by the cardiotonic steroid ouabain and the digitoxigenin derivative PST2238 (rostafuroxin) that bind specifically to the ATP1A1 extracellular domain and block RSV-triggered EGFR Tyr845 phosphorylation. In conclusion, we identified ATP1A1 as a host protein essential for macropinocytic entry of RSV into respiratory epithelial cells, and identified PST2238 as a potential anti-RSV drug. [ABSTRACT FROM AUTHOR]

The resolution of Shigella flexneri infection-associated hyperinflammation is crucial for host survival. Using in vitro and in vivo models of shigellosis, we found that S. flexneri induces the expression of indoleamine 2,3-dioxygenase 1 (IDO1) through the nucleotide oligomerization domain 2 (NOD2) and epidermal growth factor receptor (EGFR) signaling pathway. Congruently, abrogation of NOD2 or EGFR compromises the ability of S. flexneri to induce IDO1 expression. We observed that the loss of IDO1 function in vivo exacerbates shigellosis by skewing the inflammatory cytokine response, disrupting colon epithelial barrier integrity and consequently limiting the host life-span. Interestingly, administration of recombinant EGF rescued mice from IDO1 inhibition-driven aggravated shigellosis by restoring the cytokine balance and subsequently restricting bacterial growth. This is the first study that underscores the direct implication of the NOD2-EGFR axis in IDO1 production and its crucial homeostatic contributions during shigellosis. Together, these findings reveal EGF as a potential therapeutic intervention for infectious diseases. [ABSTRACT FROM AUTHOR]

Lung cancer is among the common and deadly cancers. Although the treatment options for late-stage cancer patients have continued to increase in numbers, the overall survival rates for these patients have not shown significant improvement. This highlights the need for new targets and drugs to more effectively treat lung cancer patients. In this study, we characterize the MCL-1 inhibitor maritoclax alone or in combination with a BCL-2/xL inhibitor in a panel of lung cancer cell lines. BCL-2 family proteins, phosphorylated proteins, and apoptosis were monitored following the treatments. We found that maritoclax was effective at inhibiting growth in these lung cancer cells. We also establish that cell lines with EGFR mutations were most sensitive to the combined inhibition of MCL-1 and BCL-2/xL. In addition, a high level of phosphorylated AKT (S473) was identified as a marker for sensitivity to the combination treatment. This work has defined EGFR mutations and AKT phosphorylation as markers for sensitivity to combined MCL-1 and BCL-2/xL targeted therapy and establishes a rationale to explore multiple BCL-2 family members in patients who are refractory to EGFR inhibitor treatment. Our data support the design of a clinical trial that aims to employ inhibitors of the BCL-2 family of proteins in lung cancer patients. [ABSTRACT FROM AUTHOR]