RNA N6-methyladenosine modulates endothelial atherogenic responses to disturbed flow in mice

Atherosclerosis preferentially occurs in atheroprone vasculature where human umbilical vein endothelial cells (HUVECs) are exposed to disturbed flow. Disturbed flow is associated with vascular inflammation and focal distribution. Recent studies have revealed the involvement of epigenetic regulation in atherosclerosis progression. N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic mRNA, but its function in endothelial atherogenic progression remains unclear. Here, we show that m6A mediates the EGFR signaling pathway during EC activation to regulate the atherosclerotic process. Oscillatory stress (OS) reduced the expression of METTL3, the primary m6A methyltransferase. Through m6A sequencing and functional studies, we determined that m6A mediates the mRNA decay of the vascular pathophysiology gene EGFR which leads to EC dysfunction. m6A modification of the EGFR 3'UTR accelerated its mRNA degradation. Double mutation of the EGFR 3'UTR abolished METTL3-induced luciferase activity. Adenovirus-mediated METTL3 overexpression significantly reduced EGFR activation and endothelial dysfunction in the presence of OS. Furthermore, TSP-1, an EGFR ligand, was specifically expressed in atheroprone regions without being affected by METTL3. Inhibition of the TSP-1/EGFR axis by using shRNA and AG1478 significantly ameliorated atherogenesis. Overall, our study revealed that METTL3 alleviates endothelial atherogenic progression through m6A-dependent stabilization of EGFR mRNA, highlighting the important role of RNA transcriptomics in atherosclerosis regulation.

The efficacy of PI3Kγ and EGFR inhibitors on the suppression of the characteristics of cancer stem cells

Cancer stem cells (CSCs) are capable of continuous proliferation, self-renewal and are proposed to play significant roles in oncogenesis, tumor growth, metastasis and cancer recurrence. We have established a model of CSCs that was originally developed from mouse induced pluripotent stem cells (miPSCs) by proposing miPSCs to the conditioned medium (CM) of cancer derived cells, which is a mimic of carcinoma microenvironment. Further research found that not only PI3K-Akt but also EGFR signaling pathway was activated during converting miPSCs into CSCs. In this study, we tried to observe both of PI3Kγ inhibitor Eganelisib and EGFR inhibitor Gefitinib antitumor effects on the models of CSCs derived from miPSCs (miPS-CSC) in vitro and in vivo. As the results, targeting these two pathways exhibited significant inhibition of cell proliferation, self-renewal, migration and invasion abilities in vitro. Both Eganelisib and Gefitinib showed antitumor effects in vivo while Eganelisib displayed more significant therapeutic efficacy and less side effects than Gefitinib on all miPS-CSC models. Thus, these data suggest that the inhibitiors of PI3K and EGFR, especially PI3Kγ, might be a promising therapeutic strategy against CSCs defeating cancer in the near future.

Annexin A1 promotes the progression of bladder cancer via regulating EGFR signaling pathway

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.

Quercetin Attenuates Podocyte Apoptosis of Diabetic Nephropathy Through Targeting EGFR Signaling

Podocytes injury is one of the leading causes of proteinuria in patients with diabetic nephropathy (DN), and is accompanied by podocytes apoptosis and the reduction of podocyte markers such as synaptopodin and nephrin. Therefore, attenuation of podocyte apoptosis is considered as an effective strategy to prevent the proteinuria in DN. In this study, we evaluated the anti-podocyte-apoptosis effect of quercetin which is a flavonol compound possessing an important role in prevention and treatment of DN and verified the effect by using db/db mice and high glucose (HG)-induced mouse podocytes (MPs). The results show that administration of quercetin attenuated the level of podocyte apoptosis by decreasing the expression of pro-apoptotic protein Bax, cleaved caspase 3 and increasing the expression of anti-apoptotic protein Bcl-2 in the db/db mice and HG-induced MPs. Furthermore, epidermal growth factor receptor (EGFR) was predicted to be the potential physiological target of quercetin by network pharmacology. In vitro and vivo experiments confirmed that quercetin inhibited activation of the EGFR signaling pathway by decreasing phosphorylation of EGFR and ERK1/2. Taken together, this study demonstrates that quercetin attenuated podocyte apoptosis through inhibiting EGFR signaling pathway, which provided a novel approach for further research of the mechanism of quercetin in the treatment of DN.

ZC3H15 promotes glioblastoma progression through regulating EGFR stability

Zinc finger CCCH-type containing 15 (ZC3H15), a highly conserved protein involved in several cellular processes, which was responsible for tumorigenesis and may be a promising marker in myeloid leukemia (AML) and hepatocellular carcinoma (HCC). However, little is known about the biological significance and molecular mechanisms of ZC3H15 in GBM. In this study, we revealed that ZC3H15 was overexpressed in GBM and high ZC3H15 expression was associated with poor survival of patients with GBM. We found that ZC3H15 promoted the proliferation, migration, invasion, and tumorigenesis of GBM cells by activating the EGFR signaling pathway. We also revealed that ZC3H15 reduced EGFR ubiquitination, which was responsible for EGFR protein stabilization. In addition, we demonstrated that ZC3H15 inhibited the transcription of CBL, which was an E3 ubiquitin ligase for EGFR proteasomal degradation. And silencing of CBL could partly abrogate the inhibitory effects on cell proliferation, migration, and invasion of GBM cells induced by ZC3H15 knockdown. Thus, our research revealed the important roles of ZC3H15 in GBM development and provided a brand-new insight for improving the treatment of GBMs.

KIF15 Promotes Progression of Castration Resistant Prostate Cancer by Activating EGFR Signaling Pathway

Castration-resistant prostate cancer (CRPC) continues to be a major clinical problem and its underlying mechanisms are still not fully understood. The epidermal growth factor receptor (EGFR) activation is an important event that regulates mitogenic signaling. EGFR signaling plays an important role in the transition from androgen dependence to castration-resistant state in prostate cancer (PCa). Kinesin family member 15 (KIF15) has been suggested to be overexpressed in multiple malignancies. Here, we demonstrate that KIF15 expression is elevated in CRPC. We show that KIF15 contributes to CRPC progression by enhancing the EGFR signaling pathway, which includes complex network intermediates such as mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. In CRPC tumors, increased expression of KIF15 is positively correlated with EGFR protein level. KIF15 binds to EGFR, and prevents EGFR proteins from degradation in a Cdc42-dependent manner. These findings highlight the key role of KIF15 in the development of CRPC and rationalize KIF15 as a potential therapeutic target.

880 Tetravalent, bispecific innate cell engager AFM24 enhances macrophage mediated tumor cell phagocytosis

BackgroundEnabling innate immunity holds promise to provide a treatment option for patients suffering from various kinds of malignancies. Innate cell engagers (ICE®) derived from the ROCK® (redirected optimized cell killing) platform have demonstrated to induce antibody dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) via bivalent targeting of a unique epitope on CD16A of NK cells and macrophages, respectively. Previously published preclinical and clinical data of ICE® molecules show promising efficacy and safety as monotherapy, as a combination therapy with immuno-oncology drugs such as PD-1/PD-L1, and in combination with adoptive NK cell transfer. AFM24 is a tetravalent bispecific epidermal growth factor (EGFR)- and CD16A-binding ICE® for enhanced targeting and killing of EGFR+ tumor cells currently in clinical development. In contrast to approved EGFR-targeting antibodies, AFM24 does not inhibit the signalling pathway of the EGFR but utilizes this receptor merely as an ”anchor” to direct NK cells and macrophages to attack tumor cells via ADCC and ADCP.MethodsADCP assays were performed with monocyte-differentiated macrophages from healthy donor PBMCs. Target tumor cells were labelled and co-cultures with macrophages, AFM24 and control antibodies. FACS analysis and live-cell imaging (IncuCyte®) were used to measure ADCP events.ResultsWe show that AFM24 enhances macrophage mediated tumor cell phagocytosis i.e., ADCP of tumor cell lines with varying levels of EGFR expression and irrespective of EGFR signaling pathway mutations. The ability of AFM24 to enhance ADCP was further demonstrated in patient-derived xenograft cell lines from various EGFR+ tumor indications. Assays with myeloid-derived suppressor cells, natural killer cells and other immune modulators were designed to address the activity of our ICE® in the context of the suppressive nature of the tumor microenvironment.ConclusionsWe report the ability of our ICE® to enhance ADCP, which might be instrumental to their efficacy, especially in tumors enriched with macrophages. In addition, due to its novel mechanism of action, AFM24 may overcome limitations of existing EGFR-targeting agents, such as dose limiting toxicity, and/or intrinsic or acquired resistance of the tumor. Consequently, AFM24 may be a potential future treatment option for a wide spectrum of patients including those that do not respond or are resistant to current EGFR-directed therapies that inhibit the signaling pathway.