EGFR Signaling Research Articles

Topical Ozone Accelerates Diabetic Wound Healing by Promoting Re-Epithelialization through the Activation of IGF1R–EGFR Signaling

Ozonated oil increases the healing of chronic diabetic wounds, but the underlying mechanisms remain unclear. We investigated the effect of topical ozonated oil on wound healing in mice with diabetes with diet-induced obesity and further elucidated the role of EGFR and IGF1R signaling in diabetic wound healing. We found that topical ozonated oil accelerated wound healing; increased phosphorylation of IGF1R, EGFR, and VEGFR; and improved vascularization at the wound leading edge in mice with diabetes with diet-induced obesity. Exposure of normal epidermal keratinocytes to ozonated medium (20 μM for 2 hours daily) increased cell proliferation and migration distance by increasing phosphorylation of IGF1R and EGFR and downstream phosphoinositide 3-kinase, protein kinase B, and extracellular signal-regulated kinase. These findings shed light on the mechanism for topical ozone action in chronic wounds and support its potential therapeutic application.

LaSOM 335, active against bladder cancer cells, interferes with Let-60 (hRas) and reduces CD73 expression/activity.

Bladder cancer is the fourth most common malignancy in men. It can present along the entire continuum of severity, from mild to well-differentiated disease to extremely malignant tumors with low survival rates. Human RAS genes are the most frequently mutated oncogenes in human cancers, and the critical role of aberrant Ras protein function in carcinogenesis is well established. Therefore, considerable efforts have been devoted to the development of anti-Ras inhibitors for cancer treatment. This study presents the biphenyl dihydropyrimidinone LaSOM 335 with high activity against T24 bladder cancer cells (IC50 = 10.73 ± 0.53 μM) and selectivity of cytotoxicity for this cancer cell line compared to two non-cancer cell lines investigated. Furthermore, we also show that this compound reduced vulvar development in the mutant let-60 gene of Caenorhabditis elegans. Let-60 is a homolog of the mammalian Ras gene. In addition, we observed that LaSOM 335 inhibits the enzymatic activity of CD73 and decreases CD73 expression. Possibly, this expression decrease is due to downstream EGFR signaling via the Ras-Raf-ERK pathway, that directly regulates CD73 expression via ERK1/2. Evidence suggests that non-immunomodulating functions of CD73 play an equally important role for cancer cell survival, progression, and migration. Regarding we also notice that LaSOM 335 was safe in the in vivo model of C. elegans. The set of these findings makes this biphenyl dihydropyrimidinone a promising candidate for further investigations in the bladder cancer field.

Abstract B076: IGFBP3 promotes resistance to olaparib via modulating EGFR signaling in advanced prostate cancer

Abstract Background: Castration-resistant prostate cancer (CRPC) is an incurable disease and a leading cause of cancer death in men worldwide. Olaparib (Lynparza) was among the first PARP inhibitors (PARPi) approved for the treatment of CRPC tumors harboring DNA repair defects. However, clinical resistance to PARPi’s has been documented. The mechanisms underlying resistance to PARPi’s remain elusive. To study acquired resistance, we developed olaparib-resistant LN-OlapR and 2B-OlapR cell lines generated through chronic olaparib treatment of the olaparib-sensitive cell lines LNCaP and C4-2B, respectively. RNA-seq revealed IGFBP3 is overexpressed in both OlapR cell lines. IGFBP3 overexpression is correlated with poor clinical outcome and is thought to participate in DNA repair pathways. IGFBP3 plays a key role in nonhomologous end joining (NHEJ) repair through a ternary complex with EGFR and DNA-PKcs. The IGFBP3/EGFR signaling axis is thought to modulate NHEJ repair and could have implications for PARPi sensitivity. We hypothesize that increased IGFBP3 expression promotes PARPi resistance by enhancing DNA repair capacity. Methods: RNA-sequencing and gene set enrichment analysis were used to determine the expression profile changes in resistant cells compared to parental cells. Real time PCR and western blots confirmed the expression of DNA damage repair genes such as γH2AX, EGFR, and DNA-PKcs. ELISA was used to determine IGFBP3 secretion. RNAi was used to inhibit IGFBP3 and EGFR expression. Gefitinib was used to inhibit EGFR activity. Cell viability assays were used to assess cell growth. Results: Transcriptomic profiling revealed that IGFBP3 is highly expressed in resistant models. We verified increased levels of IGFBP3 RNA and protein in both OlapR models. We found that RNAi inhibition of IGFBP3 increases γH2AX and cleaved-PARP protein levels in the resistant models, which suggests accumulation of DNA double strand breaks (DSBs) leading to genomic instability and cell death. We discovered increased phosphorylation of EGFR and DNA-PKcs in the resistant cells. Furthermore, silencing/inhibiting IGFBP3 and EGFR reduces OlapR cell viability and resensitizes resistant cells to treatment. Conclusions: Our findings demonstrated that inhibiting IGFBP3 and EGFR aids in PARPi sensitivity in the resistant setting. Future work will utilize OlapR models to study how the IGFBP3/EGFR/DNA-PKcs protein complex promotes the development of resistance. Understanding the role of IGFBP3 in PARPi resistance will enhance our ability to re-sensitize resistant CRPC to PARPi therapeutics. Citation Format: Amy R. Leslie, Allen C. Gao, Alan P. Lombard, Shu Ning. IGFBP3 promotes resistance to olaparib via modulating EGFR signaling in advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B076.

Nanoparticle albumin-bound (nab) paclitaxel in combination with nivolumab as treatment of recurrent or metastatic head and neck squamous-cell carcinoma (RM-HNSCC) that progressed on a PD-1 inhibitor: A single-arm, phase 2 trial.

6023 Background: Drugs bound to albumin selectively target cancer cells with upregulated macropinocytosis, an endocytic nutrient-scavenging process driven by constitutive hyper-activation of the EGFR, RAS, PIK3CA and Syndecan1 signaling pathways that frequently occur in HNSCC. nab-paclitaxel is an active drug in HNSCC and has immunologic effects that can prime an immune response to PD-1 inhibitors, potentially reversing resistance to these agents. These effects include decreased TREGs, increased pro-inflammatory macrophages and differentiation of MDSC to dendritic cells, increased HLA class I expression and antigen presentation, and recruitment of CD8+ CTLs into tumor. Methods: In a single-arm phase 2 trial, patients with RM-HNSCC that progressed on a PD-1 inhibitor received 28-day cycles of nab-paclitaxel (125 mg/m2 IV days 1, 8, and 15) and nivolumab (480 mg IV day 1). Treatment continued until discontinuation criteria were met. The primary endpoint was objective response, assessed with RECIST1.1 by an independent radiologist. The primary hypothesis was that this regimen would result in a higher objective response rate (ORR) than historically reported with standard chemotherapy or cetuximab in similar patients. A Simon optimal two-stage design tested the null hypothesis (H0: ORR≤30%) versus the alternative hypothesis (H1: ORR=50%) at the type I error rate of 5% and power 0.80. In the first stage, 15 patients were to be accrued. If ≥6 responses occurred, 31 additional patients were to be accrued. The null hypothesis will be rejected if ≥19 responses are observed in these 46 patients. We report the results of the primary endpoint for the first stage of the trial. Results: 15 evaluable patients enrolled into the first stage of the trial. Key tumor characteristics and prior treatment history included HPV status (positive: 8; negative: 7), PD-L1 status (CPS 1-19: 7; ≥20: 8), # of lines of prior systemic treatment for RM disease (1-2: 12; ≥3: 3), interval (months) from prior PD-1 inhibitor (<6: 11; ≥6: 4), duration (months) on prior PD-1 inhibitor (median: 6, range: 1-16), and best response to prior PD-1 inhibitor (PR: 4; SD: 6; PD: 5). Tumor response to nab-paclitaxel and nivolumab occurred in 7 of 15 patients (ORR 47%). The best tumor response was PR (7), SD (5) and PD (3). Conclusions: Among patients with RM-HNSCC that progressed on a PD-1 inhibitor, the ORR with nab-paclitaxel and nivolumab was 47% during the first stage of the phase 2 trial. Enrollment to the second stage is ongoing. Clinical trial information: NCT04831320 .

FZKA reverses gefitinib resistance by regulating EZH2/Snail/EGFR signaling pathway in lung adenocarcinoma

Ethnopharmacological relevanceFuzheng Kang-Ai (FZKA) decoction is mainly composed of 12 components with different types of herbs. In the last decade, FZKA has been used as an adjuvant treatment for lung cancer in clinical practice. Our previous studies have confirmed that FZKA shows a strong anti-cancer activity, significantly increases the clinical efficacy of gefitinib and reverses gefitinib resistance in non-small cell lung cancer (NSCLC). However, the molecular mechanism still needs to be further elucidated. Aim of the studyThe aim of this study was to investigate the role and mechanism by which FZKA inhibited the cell growth, proliferation and invasion of lung adenocarcinoma(LUAD) and reversed the acquired resistance of gefitinib for the therapy in LUAD. Materials and methodsCell viability assay and EDU assay were used for detecting of cell viability and cell proliferation. Transwell assay was performed to measure cell invasion. Western Blot and qRT-PCR were used for protein and gene expression test. The gene promoter activity was determined by dul-luciferase reporter assay. The in situ expression of protein was measured by cell immunofluorescence. Stabilized cell lines were established for stable overexpression of EZH2. Transient transfection assay was used for gene silence and overexpression. Xenograft tumors and bioluminescent imaging were used for in vivo experiments. ResultsFZKA significantly inhibited the cell viability, proliferation and cell invasion of LUAD, the combination of FZKA and gefitinib had a great synergy on the above processes. Moreover, FZKA significantly decreased EZH2 mRNA and protein expression, FZKA reversed the resistance of gefitinib by down-regulation of EZH2 protein. ERK1/2 kinase mediated the down-regulation of EZH2 reduced by FZKA. In addition, FZKA decreased the expression of Snail and EGFR by decreasing EZH2. Overexpression of Snail and EGFR significantly reversed the effect of FZKA-inhibited cell invasion and cell proliferation. More important, the combination of FZKA and gefitinib enhanced the inhibitory effect on EZH2, Snail and EGFR proteins. Furthermore, the growth inhibition and reversal of gefitinib resistance induced by FZKA were further validated in vivo. Finally, the expression and clinical correlation of EZH2,EGFR and Snail in cancer patients were further validated using bioinformatics analysis. ConclusionsFZKA significantly suppressed tumor progression and reversed gefitinib resistance by regulating the p-ERK1/2-EZH2-Snail/EGFR signaling pathway in LUAD.

Analysis of primary and acquired resistance to cetuximab from multiomic data in the New EPOC trial.

3577 Background: The use of cetuximab (cetux) with chemotherapy (chemo) in patients with operable liver metastases from colorectal cancer (CRC) conferred a survival disadvantage in New EPOC. Whilst genetic alterations such as emergent sub-clonal mutations in RAS/ BRAF, ERBB2 and MET are recognized as mechanisms of resistance to EGFR inhibition (EGFRi), divergent mechanisms such as transcriptomic changes may also be responsible. Of the CRC intrinsic subtypes (CRIS), CRIS-C has elevated EGFR signalling and is thought to have greater sensitivity to EGFRi. Methods: Resected primary tumors (n = 205; pre neoadjuvant treatment) and/or liver metastases (n = 144; post neoadjuvant treatment) from 223 patients, wild type for KRAS (codons 12/13/61) at trial entry, randomized to chemo +/- cetux underwent targeted NGS and/or transcriptome profiling. Primary clinical endpoints were progression free (PFS) and overall survival (OS). Results: Repeat NGS identified additional RAS/BRAF mutations in primary tumors from 49 patients (25 KRAS, 15 NRAS, 9 BRAF V6000E). Of the 148 patients with confirmed RAS/BRAF wild type primary tumors , 46% were classified as CRIS-C with the remainder being CRIS-A (9.5%), CRIS-B (11%), CRIS-D (8.8%) or CRIS-E (8.1%). CRIS-C patients had a similar PFS with ChemoCetux vs Chemo (HR 0.84 95% CI 0.47-1.5 p = 0.55) whereas non-CRIS-C had a shorter PFS (HR 2.12 95%CI 1.24-3.63 p = 0.006; p interaction 0.021). OS results were similar (p interaction 0.067). In liver metastases, genetic changes were comparable (Chemo vs ChemoCetux): KRAS (4.1% v 2.1%), NRAS (4.1% v 0%), BRAF V600E (0% v 0%), ERBB2 gain/amplification (20% v 21%) and MET gain/amplification (25% v 18%). However, gain/amplification of MET resulted in shorter OS with ChemoCetux vs Chemo (HR 5.13 95%CI 1.38-19.15 p = 0.015) whereas those without MET gain/amplification had a similar OS (HR 1.32 95%CI 0.76-2.28 p = 0.32; p interaction 0.029). The same association was found after excluding liver metastases with RAS/BRAF mutations (n = 101, p interaction 0.030). Differential expression/mutation analyses did not identify other stratifiers after false discovery correction. Conclusions: The shorter survival with ChemoCetux was restricted to patients with a non CRIS-C subtype before treatment supporting the use of this transcriptomic classifier to stratify for EGFRi. After neoadjuvant treatment, gain/amplification of MET was associated with a shorter survival with ChemoCetux. By contrast MAPK pathway mutations were largely unchanged supporting earlier findings that alternative resistance mechanisms predominate when EGFRi is combined with chemotherapy. In summary these results highlight the heterogeneity and differential responses to EGFRi that exist within RAS/RAF wild type CRC and provide insights into the substantial survival detriment observed in the New EPOC trial. Clinical trial information: ISRCTN22944367 .

Effect of the Knockout of FGL1 by CRISPR on the GefitinibSensitivity of PC9/GR Cells

This paper will investigate the effect of the Knockout of FGL1 by CRISPR on the EGFR signaling analyzed phosphorERK western blot and how it may control the gefitinib sensitivity of PC9/GR cells measured by the MTT Assay. Thisexperiment will knock out FGL1 using CRISPR. The Knockout will then be verified through Phospho-ERK westernblot. Moreover, the sensitivity of the gefitinib will be measured by the MTT Assay. The possible results generated fromthe reports’ experiments may provide insight into future clinical trials of FGL1 Knockout consequences on gefitinibsensitivity. Gefitinib is a potential drug for lung cancer. Therefore, investigating this drug may help scientists betterunderstand how the FGL1 may increase the sensitivity of Gefitinib cells and potentially discover a breakthrough to curecancer.

Phase 2 trial of epidermal growth factor (EGF) vaccine CIMAvax in combination with pembrolizumab in first line and maintenance setting in advanced non–small cell lung cancer patients.

TPS2677 Background: CIMAvax-EGF is a novel growth-factor depleting immunotherapy consisting of human recombinant EGF conjugated to recombinant P64k derived from Neisseria meningitidis, that elicits an anti-EGF antibody response, resulting in reduction of circulating EGF levels. A randomized phase 3 study of CIMAvax-EGF administered as switch maintenance therapy after first-line platinum-based chemotherapy in patients with advanced NSCLC demonstrated overall survival (OS) advantage in comparison to best supportive care alone, particularly in patients with high baseline serum EGF levels. A phase 1 trial combining CIMAvax-EGF with nivolumab, an anti-PD1 immune checkpoint inhibitor, showed that the combination is safe and appears to induce higher rate of good anti-EGF antibody response within the first 8 weeks of therapy compared to historical comparison group receiving CIMAvax-EGF alone. CIMAvax-EGF is currently being investigated in combination with pembrolizumab in a multi-arm phase II trial as 1L therapy in combination with pembrolizumab for patients with EGFR/ALK wildtype NSCLC and PD-L1 > 50% (Cohort C) and as maintenance therapy after completing 1L chemoimmunotherapy for patients with PD-L1 < 50% (Cohorts D and E). Accrual to this trial is ongoing (NCT02955290). Methods: This is an open-label, non-randomized, multi-site, phase 2 basket trial. CIMAvax-EGF (2.4 mg IM every 2 weeks x 4 doses, then every 4 weeks thereafter) is being tested as first-line therapy in combination with pembrolizumab (200 mg IV every 4 weeks) in patients with advanced NSCLC with PD-L1 ≥ 50% (Cohort C), using a Simon two stage design (n up to 41). A safety lead-in consisting of the first 6 NSCLC patients was completed. There were no G3 or higher adverse events attributed to the combination within the first 4 weeks of protocol treatment. Patients with advanced squamous NSCLC (Cohort D, n=50) and non-squamous NSCLC (without EGFR/ALK/ROS-1/KRAS mutations; Cohort E, n=45) with PD-L1 expression <50% and no evidence of progression of disease by RECIST 1.1 criteria after at least 4 cycles of induction platinum-based doublet with pembrolizumab, prior to initiation of standard of care maintenance therapy will be enrolled to receive protocol treatment with pembrolizumab in combination with CIMAvax-EGF as maintenance therapy. The primary endpoint is to evaluate objective response rate (Cohort C), and 12-month PFS in Cohorts D and E. Secondary endpoints include 12-month overall survival in all cohorts. Exploratory objectives include characterizing tissue-based immune and EGFR signaling profile as well as serum EGF levels, anti-EGF antibody levels and other blood-based biomarkers in relation to clinical outcomes. Clinical trial information: NCT02955290 .

A first-in-human, open-label, dose escalation and expansion study of orally administered NX-019 in patients with advanced EGFR mutant cancer.

TPS9159 Background: CNS involvement, brain metastases and/or leptomeningeal disease, is common in patients with EGFR mutant NSCLC and occurs in 25-50% of patient’s journeys. Despite the activity and increased CNS penetration of later generation EGFR inhibitors, management of CNS disease remains a clinical challenge. NX-019 is a broad-spectrum EGFR inhibitor targeting both common and rare mutations including EGFR ex20ins, G719S, and L861Q. NX-019 exhibits selective activity towards mutant vs. wt. EGFR in preclinical studies. Substantial CNS exposure has been demonstrated in animal models, as well as potent anti-tumor activity toward intracranial xenografts with prolonged inhibition of EGFR signaling. Similar drug levels compared to plasma were measured in the brains of rats and mice. NX-019 was readily detectable in the CSF of cynomolgus monkeys, dogs and rats at concentrations similar to the estimated plasma free fraction. Methods: NT019-101 is a first-in-human, international, open-label study designed to evaluate single-agent NX-019 in patients with EGFR-mutant, locally advanced or metastatic NSCLC that has progressed following prior treatment. Patients with ECOG performance status 0–2 and EGFR mutant cancers including stable, asymptomatic brain metastases are eligible for Dose Escalation. Patients with known EGFR C797S mutations and secondary drivers, e.g., MET amplification and RET fusions, are excluded. Primary endpoints are to determine recommended phase 2 dose (RP2D), maximum tolerated dose (MTD), safety and tolerability (part 1), and to evaluate antitumor activity at the RP2D by RECIST v1.1 and RANO-BM (part 2). Secondary endpoints are to evaluate pharmacokinetics (PK) and antitumor activity by RECIST v1.1 (part 1) and PK, safety, tolerability, and CNS antitumor activity (part 2). Dose escalation will utilize a 3+3 design with up to 6 patients per cohort in part 1. Part 2 will enroll patients in 5 cohorts (n = 10 or 29 each), including patients with NSCLC who have (1) new or progressing CNS metastases including leptomeningeal disease after treatment with osimertinib, (2) active CNS disease and ex20ins mutations, (3) ex20ins mutations without CNS disease and (4) other rare mutations, based on a Simon 2-stage design. First patient was treated in October 2022 and enrollment is ongoing. The study is planned for approximately 35 centers in North America and Asia-Pacific region. Clinical trial information: NCT05514496 .

Relationship between EGFR inhibition in lung adenocarcinoma, cell surface expression of the placental antigen ALPP, and efficacy of ALPP-ADC therapy.

e15018 Background: In normal tissues placental alkaline phosphatases (ALPP) and ALPPL2 are exclusively expressed in the placenta. Their discovery in the cancer surfaceome has opened the possibility of targeting ALPP/ALPPL2. However, their low expression in most cancer types limits their therapeutic potential. In this study, we report a novel mechanism of ALPP upregulation in cancer cells that renders these cells more susceptible to ALPP-targeting therapies, which we evidence in the context of lung adenocarcinoma. Methods: Surface expression of ALPP and ALPPL2 was determined following biotinylation tagging of the cancer surfaceome, isolation of biotinylated proteins using monomeric avidin and mass spectrometry of the biotinylated trypsin digested fraction. ALPP antibody was conjugated with Monomethylauristatin F (ALPP-ADC-MMAF). For orthotopic xenograft models of LUAD were injected. For gefitinib treatment, Mice xenotransplanted with H1650 were treated with gefitinib (50mg/kg) daily for 10 days starting from Day 17 post cancer cell inoculation. For the gefitinib/ALPP-ADC combinational treatment, mice received anti-ALPP-MMAF (5mg/kg) at Day 19 and Day 26. Results: We assessed ALPP and ALPPL2 surfaceome protein levels in 158 cancer cell lines and their mRNA expression levels in 10,967 tumors representing 32 cancer types from The Cancer Genome Atlas (TCGA), which revealed ALPP, and to a lesser extent ALPPL2, to be variably expressed in several cancer types including lung adenocarcinoma (LUAD). We further confirmed surface expression of ALPP by tissue microarray analysis of 204 lung tumors. We report that treatment of LUAD cells with EGFR inhibitors or induction of cancer cell quiescence greatly enhanced ALPP surface expression. Mechanistically, repression of EGFR signaling resulted in activation of the transcription factor FoxO3a, which triggered expression of ALPP. Using xenograft models of LUAD, we further demonstrate that gefitinib treatment significantly upregulated surface expression of ALPP in LUAD tumors but not normal tissue. Combination therapy with gefitinib and an ALPP-ADC-MMAF resulted in superior anti-cancer efficacy compared with gefitinib or ALPP-ADC-MMAF alone. Conclusions: Our study provides mechanistic insights into ALPP upregulation in cancer cells and establishes FoxO3a as a transcriptional regulator of ALPP. Importantly, our findings have yielded a novel ‘two-hit’ combinational therapeutic strategy with enhanced ALPP targeting for greater efficacy.

Integration of Multi-omic Data in a Molecular Tumor Board Reveals EGFR-Associated ALK-Inhibitor Resistance in a Patient With Inflammatory Myofibroblastic Cancer.

Inflammatory myofibroblastic tumors (IMTs) are intermediate-grade mesenchymal neoplasms commonly characterized by chromosomal rearrangements causing constitutive activation of anaplastic lymphoma kinase (ALK) and/or ALK mutations causing reduced sensitivity to ALK tyrosine kinase inhibitors (TKI). We present a patient with an IMT who initially responded to first-line alectinib, but who later suffered disease relapse and presently survives with moderate residual disease after receiving second-line lorlatinib. Biopsy specimens were analyzed using next generation sequencing (DNA-seq and RNA-seq) and reverse phase protein microarray (RPPA) as part of an institutional Molecular Tumor Board (MTB) study. An EML4-ALK rearrangement and EGFR activation (pEGFRY1068) were present in both the primary and recurrent tumors, while a secondary ALK I1171N mutation was exclusive to the latter. EGFR signaling in the background of a secondary ALK mutation is correlated with reduced ALK TKI sensitivity in vitro, implicating an important mechanism of drug resistance development in this patient. The RPPA results also critically demonstrate that ALK signaling (ALKY1604) was not activated in the recurrent tumor, thereby indicating that standard-of-care use of third- or fourth-line ALK TKI would not likely be efficacious or durable. These results underscore the importance of real-time clinical integration of functional protein drug target activation data with NGS in the MTB setting for improving selection of patient-tailored therapy.

Genome-Wide Mapping Implicates 5-Hydroxymethylcytosines in Diabetes Mellitus and Alzheimer's Disease.

Diabetes mellitus (DM) is a recognized risk factor for dementia. Because DM is a potentially modifiable condition, greater understanding of the mechanisms linking DM to the clinical expression of Alzheimer's disease dementia may provide insights into much needed dementia therapeutics. In this feasibility study, we investigated DM as a dementia risk factor by examining genome-wide distributions of the epigenetic DNA modification 5-hydroxymethylcytosine (5hmC). We obtained biologic samples from the Rush Memory and Aging Project and used the highly sensitive 5hmC-Seal technique to perform genome-wide profiling of 5hmC in circulating cell-free DNA (cfDNA) from antemortem serum samples and in genomic DNA from postmortem prefrontal cortex brain tissue from 80 individuals across four groups: Alzheimer's disease neuropathologically defined (AD), DM clinically defined, AD with DM, and individuals with neither disease (controls). Distinct 5hmC signatures and biological pathways were enriched in persons with both AD and DM versus AD alone, DM alone, or controls, including genes inhibited by EGFR signaling in oligodendroglia and those activated by constitutive RHOA. We also demonstrate the potential diagnostic value of 5hmC profiling in circulating cfDNA. Specifically, an 11-gene weighted model distinguished AD from non-AD/non-DM controls (AUC = 91.8%; 95% CI, 82.9-100.0%), while a 4-gene model distinguished DM-associated AD from AD alone (AUC = 87.9%; 95% CI, 77.5-98.3%). We demonstrate in this small sample, the feasibility of detecting and characterizing 5hmC in DM-associated AD and of using 5hmC information contained in circulating cfDNA to detect AD in high-risk individuals, such as those with diabetes.

Eicosapentaenoic acid mitigates ulcerative colitis-induced by acetic acid through modulation of NF-κB and TGF-β/ EGFR signaling pathways

BackgroundUlcerative colitis (UC) is a chronic mucosal inflammation of the large intestine that mostly affects the rectum and colon. The absence of safe and effective therapeutic agents encourages the discovery of novel therapeutic agents to effectively treat UC and its complications. The purpose of this research was to examine the protective impact of Eicosapentaenoic acid (EPA) in rats with UC induced by acetic acid (AA). MethodAA (2 ml, 3 % v/v) was injected intrarectally to cause UC. Before administering AA, EPA (300 and 1000 mg/kg) was given orally for 28 days. ResultsEPA inhibited AA-induced UC by enhancing colonic histopathological changes like inflammation, goblet cell loss, glandular hyperplasia and mucosal ulceration, concomitant with a reduction in colon weight, colon weight/length ratio, C-reactive protein (CRP), and serum lactate dehydrogenase (LDH). EPA also effectively restored the imbalance between oxidants and antioxidants caused by AA. In addition, EPA increased the levels of trefoil factor-3 (TFF-3) and glucagon-like peptide-1 (GLP-1), while significantly reducing the expression of nuclear factor kappa B (NF-κB), interferon-γ (IFN-γ), and interleukin-6 (IL-6), transforming growth factor-1(TGF-β1), and phosphorylated epidermal growth factor receptor (P-EGFR), phosphatidylinositol-3-kinase (PI3K) and protein kinase B (AKT) expression in colonic tissues. ConclusionEPA inhibited AA-induced UC in rats by modulating the TGF-β/P-EGFR and NF-κB inflammatory pathways, regulating the oxidant/antioxidant balance, and enhancing the colon barrier integrity.

Lysyl oxidase-like 3 restrains mitochondrial ferroptosis to promote liver cancer chemoresistance by stabilizing dihydroorotate dehydrogenase

To overcome chemotherapy resistance, novel strategies sensitizing cancer cells to chemotherapy are required. Here, we screen the lysyl-oxidase (LOX) family to clarify its contribution to chemotherapy resistance in liver cancer. LOXL3 depletion significantly sensitizes liver cancer cells to Oxaliplatin by inducing ferroptosis. Chemotherapy-activated EGFR signaling drives LOXL3 to interact with TOM20, causing it to be hijacked into mitochondria, where LOXL3 lysyl-oxidase activity is reinforced by phosphorylation at S704. Metabolic adenylate kinase 2 (AK2) directly phosphorylates LOXL3-S704. Phosphorylated LOXL3-S704 targets dihydroorotate dehydrogenase (DHODH) and stabilizes it by preventing its ubiquitin-mediated proteasomal degradation. K344-deubiquitinated DHODH accumulates in mitochondria, in turn inhibiting chemotherapy-induced mitochondrial ferroptosis. CRISPR-Cas9-mediated site-mutation of mouse LOXL3-S704 to D704 causes a reduction in lipid peroxidation. Using an advanced liver cancer mouse model, we further reveal that low-dose Oxaliplatin in combination with the DHODH-inhibitor Leflunomide effectively inhibit liver cancer progression by inducing ferroptosis, with increased chemotherapy sensitivity and decreased chemotherapy toxicity.

Association of hsa-miR-5571-5p Expression with Clinicopathological Factors besides Identification of Its Hub Target Genes and Key Pathway in Breast Cancer.

miRNAs are small non-coding RNAs; regulate gene expression using RNA degradation or translation repression. Dysregulation of miRNAs is involved in the initiation and progression of many cancers. We aimed to determine the relationship between miR-5571-5p expression and clinical factors and regulatory mechanisms in breast cancer. Histopathologic sections approximately with 25 microns thick from FFPE tissues were achievement of Al-Zahra Hospital (Isfahan, Iran) in 2020-2021 years by Pathologist. miR-5571-5p expression, determined using real-time PCR. For miRNA target genes prediction, integrated miRNA target prediction tools, were used. Gene ontology and KEGG pathway analysis were accomplished to identify the biological function. A PPI network was constructed to display key target genes. For hub genes validation, GEPIA databases were used. miR-5571-5p was upregulated in breast tumor tissues, and its increase was significantly related to a poor prognosis in breast cancer (P<0.0001). At first, 324 target genes were predicted, and then 110 genes with a decrease in expression were selected. GO analysis showed that genes were mainly enriched in the regulation of the ERBB2 and EGFR signaling pathway. KEGG pathway analysis suggested that downregulated genes were enriched in glioma, the ErbB signaling pathway, and breast cancer. Finally, the ten hub genes (EGF, PIK3R1, SOS1, PTEN, SHC1, CBLB, LIFR, LEP, PDE1C, and NT5C2) were detected from the PPI network. miR-5571-5p up-regulation is associated with breast cancer progression and worse survival. The current study identified ten genes associated with breast cancer, which might help to provide candidate targets for the treatment.

Network pharmacology and molecular docking study for biological pathway detection of cytotoxicity of the yellow jasmine flowers

BackgroundThe yellow jasmine flower (Jasminum humile L.) is a fragrant plant belonging to the Oleaceae family with promising phytoconstituents and interesting medicinal uses. The purpose of this study was to characterize the plant metabolome to identify the potential bioactive agents with cytotoxic effects and the underlying mechanism of cytotoxic activity.MethodsFirst, HPLC–PDA-MS/MS was used to identify the potential bioactive compounds in the flowers. Furthermore, we assessed the cytotoxic activity of the flower extract against breast cancer (MCF-7) cell line using MTT assay followed by the cell cycle, DNA-flow cytometry, and Annexin V-FITC analyses alongside the effect on reactive oxygen species (ROS). Finally, Network pharmacology followed by a molecular docking study was performed to predict the pathways involved in anti-breast cancer activity.ResultsHPLC–PDA-MS/MS tentatively identified 33 compounds, mainly secoiridoids. J. humile extract showed a cytotoxic effect on MCF-7 breast cancer cell line with IC50 value of 9.3 ± 1.2 µg/mL. Studying the apoptotic effect of J. humile extract revealed that it disrupts G2/M phase in the cell cycle, increases the percentage of early and late apoptosis in Annexin V-FTIC, and affects the oxidative stress markers (CAT, SOD, and GSH-R). Network analysis revealed that out of 33 compounds, 24 displayed interaction with 52 human target genes. Relationship between compounds, target genes, and pathways revealed that J. humile exerts its effect on breast cancer by altering, Estrogen signaling pathway, HER2, and EGFR overexpression. To further verify the results of network pharmacology, molecular docking was performed with the five key compounds and the topmost target, EGFR. The results of molecular docking were consistent with those of network pharmacology.ConclusionOur findings suggest that J. humile suppresses breast cancer proliferation and induces cell cycle arrest and apoptosis partly by EGFR signaling pathway, highlighting J. humile as a potential therapeutic candidate against breast cancer.

Inositol possesses antifibrotic activity and mitigates pulmonary fibrosis

BackgroundMyo-inositol (or inositol) and its derivatives not only function as important metabolites for multiple cellular processes but also act as co-factors and second messengers in signaling pathways. Although inositol supplementation has been widely studied in various clinical trials, little is known about its effect on idiopathic pulmonary fibrosis (IPF). Recent studies have demonstrated that IPF lung fibroblasts display arginine dependency due to loss of argininosuccinate synthase 1 (ASS1). However, the metabolic mechanisms underlying ASS1 deficiency and its functional consequence in fibrogenic processes are yet to be elucidated.MethodsMetabolites extracted from primary lung fibroblasts with different ASS1 status were subjected to untargeted metabolomics analysis. An association of ASS1 deficiency with inositol and its signaling in lung fibroblasts was assessed using molecular biology assays. The therapeutic potential of inositol supplementation in fibroblast phenotypes and lung fibrosis was evaluated in cell-based studies and a bleomycin animal model, respectively.ResultsOur metabolomics studies showed that ASS1-deficient lung fibroblasts derived from IPF patients had significantly altered inositol phosphate metabolism. We observed that decreased inositol-4-monophosphate abundance and increased inositol abundance were associated with ASS1 expression in fibroblasts. Furthermore, genetic knockdown of ASS1 expression in primary normal lung fibroblasts led to the activation of inositol-mediated signalosomes, including EGFR and PKC signaling. Treatment with inositol significantly downregulated ASS1 deficiency-mediated signaling pathways and reduced cell invasiveness in IPF lung fibroblasts. Notably, inositol supplementation also mitigated bleomycin-induced fibrotic lesions and collagen deposition in mice.ConclusionThese findings taken together demonstrate a novel function of inositol in fibrometabolism and pulmonary fibrosis. Our study provides new evidence for the antifibrotic activity of this metabolite and suggests that inositol supplementation may be a promising therapeutic strategy for IPF.

Deciphering Phenotypes from Protein Biomarkers for Translational Research with PIPER.

Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) has widespread clinical use for detection of inborn errors of metabolism, therapeutic drug monitoring, and numerous other applications. This technique detects proteolytic peptides as surrogates for protein biomarker expression, mutation, and post-translational modification in individual clinical assays and in cancer research with highly multiplexed quantitation across biological pathways. LC-MRM for protein biomarkers must be translated from multiplexed research-grade panels to clinical use. LC-MRM panels provide the capability to quantify clinical biomarkers and emerging protein markers to establish the context of tumor phenotypes that provide highly relevant supporting information. An application to visualize and communicate targeted proteomics data will empower translational researchers to move protein biomarker panels from discovery to clinical use. Therefore, we have developed a web-based tool for targeted proteomics that provides pathway-level evaluations of key biological drivers (e.g., EGFR signaling), signature scores (representing phenotypes) (e.g., EMT), and the ability to quantify specific drug targets across a sample cohort. This tool represents a framework for integrating summary information, decision algorithms, and risk scores to support Physician-Interpretable Phenotypic Evaluation in R (PIPER) that can be reused or repurposed by other labs to communicate and interpret their own biomarker panels.

151 TRPA1/V3-Dependent Regulation of Airway Epithelial Cell Damage and Repair Triggered by wood/Biomass Smoke Particulate

Abstract Wood/biomass smoke particulate material (WBSPM) is a pervasive environmental pollutant. Acute high-dose inhalation exposure can cause airway epithelial cell (AEC) damage and respiratory dysfunction. We hypothesized that AEC damage is limited by compensatory signaling to allow epithelial repair. RNA sequencing, proteomics, targeted interventions, and cultured human AECs treated with WBSPM and Transient Receptor Potential Ankyrin-1 (TRPA1) agonists revealed TRPA1-driven endoplasmic reticulum stress (ERS) and oxidative stress (OS) as contributory pathways to cytotoxicity. Alternatively, TRP vanilloid-3 (TRPV3) counteracted these events despite also being activated by WBSPM. Using the TRPA1 agonist AITC (and others) as a surrogate for WBSPM, morphological distortion indicative of AEC damage was observed 2-4h post-treatment; at &amp;gt;6h cells recovered. Strikingly, at 2h, the abundance of only 6 proteins changed including loss of fibronectin and tubulin and increased heme oxygenase 1. At 6h, 36 proteins changed, including ER chaperones and transcriptional/cell cycle regulatory proteins, OS-response, and structural/matrix proteins. Analysis of the phosphoproteome revealed changes in 2672 and 1886 unique phosphosites at 2 and 6h, attributable to PKC, p38 MAPK, EGFR, Akt and other kinase activities based on “phosphosite-GSEA” analysis. A kinase inhibitor screen linked PKC, p38 MAPK, GSK3b and ERK1/2 with cytotoxicity, while EGFR, MEK1/2, JNK1/2/3 and Akt were protective. Considering that TRPV3 expression is regulated by EGFR following WBSPM injury and TRPV3 enhanced adhesion and slowed proliferation via attenuated EGFR signaling, we propose a mechanism whereby the balance of TRPA1, TRPV3, and calcium/growth factor receptor-dependent kinase signaling coordinates WBSPM injury, compensation and repair.

TRPV1 inhibition overcomes cisplatin resistance by blocking autophagy-mediated hyperactivation of EGFR signaling pathway

Cisplatin resistance along with chemotherapy-induced neuropathic pain is an important cause of treatment failure for many cancer types and represents an unmet clinical need. Therefore, future studies should provide evidence regarding the mechanisms of potential targets that can overcome the resistance as well as alleviate pain. Here, we show that the emergence of cisplatin resistance is highly associated with EGFR hyperactivation, and that EGFR hyperactivation is arisen by a transcriptional increase in the pain-generating channel, TRPV1, via NANOG. Furthermore, TRPV1 promotes autophagy-mediated EGF secretion via Ca2+ influx, which activates the EGFR-AKT signaling and, consequentially, the acquisition of cisplatin resistance. Importantly, TRPV1 inhibition renders tumors susceptible to cisplatin. Thus, our findings indicate a link among cisplatin resistance, EGFR hyperactivation, and TRPV1-mediated autophagic secretion, and implicate that TRPV1 could be a crucial drug target that could not only overcome cisplatin resistance but also alleviate pain in NANOG+ cisplatin-resistant cancer.