EGF Receptor Research Articles

18F-Fluorodeoxyglucose PET/CT for Early Prediction of Outcomes in Patients with Advanced Lung Adenocarcinomas and EGFR Mutations Treated with First-Line EGFR-TKIs.

Simple SummaryEpithelial growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are the first-line therapy for patients with advanced-stage lung adenocarcinoma with EGFR mutations. However, 17–31% of these patients do not respond to therapy, making early evaluation of treatment response crucial. This prospective study investigates the value of 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) for timely prediction of response and survival of these patients. We evaluated 30 patients with stage IIIB/IV lung adenocarcinomas and EGFR mutations, receiving first-line EGFR-TKI therapy. 18F-FDG PET/CT was performed before and two weeks after initiation of treatment. Positron Emission Tomography Response Criteria in Solid Tumors served as an independent predictor of non-progressive disease; baseline and change of metabolic tumor volume represented independent predictors of progression-free survival and overall survival, respectively. Therefore, 18F-FDG PET/CT is an early predictor of outcomes and individual prognosis of patients with stage IIIB/IV lung adenocarcinomas and EGFR mutations receiving first-line EGFR-TKI therapy.This study aims to investigate the role of 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) in early prediction of response and survival following epithelial growth factor receptor (EGFR)–tyrosine kinase inhibitor (TKI) therapy in patients with advanced lung adenocarcinomas and EGFR mutations. Thirty patients with stage IIIB/IV lung adenocarcinomas and EGFR mutations receiving first-line EGFR-TKIs were prospectively evaluated between November 2012 and May 2015. EGFR mutations were quantified by delta cycle threshold (dCt). 18F-FDG PET/CT was performed before and 2 weeks after treatment initiation. PET response was assessed based on PET Response Criteria in Solid Tumors (PERCIST). Baseline and percentage changes in the summed standardized uptake value, metabolic tumor volume (bsumMTV and ΔsumMTV, respectively), and total lesion glycolysis of ≤5 target lesions/patient were calculated. The association between parameters (clinical and PET) and non-progression disease after 3 months of treatment in CT based on the Response Evaluation Criteria in Solid Tumors Version 1.1 (nPD3mo), progression-free survival (PFS), and overall survival (OS) were tested. The median follow-up time was 19.6 months. The median PFS and OS were 12.0 and 25.3 months, respectively. The PERCIST criteria was an independent predictor of nPD3mo (p = 0.009), dCt (p = 0.014) and bsumMTV (p = 0.014) were independent predictors of PFS, and dCt (p = 0.014) and ΔsumMTV (p = 0.005) were independent predictors of OS. 18F-FDG PET/CT achieved early prediction of outcomes in patients with advanced lung adenocarcinomas and EGFR mutations receiving EGFR-TKIs.

Dynamic EGFR interactomes reveal differential association of signaling modules with wildtype and Exon19-del EGFR in NSCLC cell lines

Protein-protein interaction networks (PPIs) govern the majority of biological processes, but how oncogenic mutations impact these interactions and their functions at a network scale is poorly understood. Mutations of epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) is a pre-requisition for EGFR tyrosine kinase inhibitor (TKI) treatment. Identification of interaction partners that bind to mutated EGFR can help understand the mechanism of action and pathways that mediate drug resistance. In this study, we characterized the dynamic interaction network of a pair of EGFR wildtype and mutant NSCLC cell lines. We performed immunoprecipitation of endogenous EGFR at various time points following EGF treatment and analyzed the associated proteins by quantitative mass spectrometry. Our results showed that the core signaling modules and key downstream pathways are maintained in the mutant cell line, but receptor internalization and intracellular trafficking in the mutant is delayed. Furthermore, we identified mutant EGFR-associated proteins that could affect EGFR functions in lung adenocarcinoma. SignificanceWe analyzed the dynamic EGFR interaction network in NSCLC cell lines expressing wild-type and mutant EGFR. By comparing the similarities and differences in the EGFR proteome, we gained a better understanding of EGFR signal transduction network, and identified new factors for further functional characterizations and clinical significance assessment.

Platelet-Released Growth Factors Influence Wound Healing-Associated Genes in Human Keratinocytes and Ex Vivo Skin Explants.

Platelet-released growth factors (PRGFs) or other thrombocyte concentrate products, e.g., Platelet-Rich Fibrin (PRF), have become efficient tools of regenerative medicine in many medical disciplines. In the context of wound healing, it has been demonstrated that treatment of chronic or complicated wounds with PRGF or PRF improves wound healing in the majority of treated patients. Nevertheless, the underlying cellular and molecular mechanism are still poorly understood. Therefore, we aimed to analyze if PRGF-treatment of human keratinocytes caused the induction of genes encoding paracrine factors associated with successful wound healing. The investigated genes were Semaphorin 7A (SEMA7A), Angiopoietin-like 4 (ANGPLT4), Fibroblast Growth Factor-2 (FGF-2), Interleukin-32 (IL-32), the CC-chemokine-ligand 20 (CCL20), the matrix-metalloproteinase-2 (MMP-2), the chemokine C-X-C motif chemokine ligand 10 (CXCL10) and the subunit B of the Platelet-Derived Growth Factor (PDGFB). We observed a significant gene induction of SEMA7A, ANGPLT4, FGF-2, IL-32, MMP-2 and PDGFB in human keratinocytes after PRGF treatment. The CCL20- and CXCL10 gene expressions were significantly inhibited by PRGF therapy. Signal transduction analyses revealed that the PRGF-mediated gene induction of SEMA7A, ANGPLT4, IL-32 and MMP-2 in human keratinocytes was transduced via the IL-6 receptor pathway. In contrast, EGF receptor signaling was not involved in the PRGF-mediated gene expression of analyzed genes in human keratinocytes. Additionally, treatment of ex vivo skin explants with PRGF confirmed a significant gene induction of SEMA7A, ANGPLT4, MMP-2 and PDGFB. Taken together, these results describe a new mechanism that could be responsible for the beneficial wound healing properties of PRGF or related thrombocytes concentrate products such as PRF.

Pregnancy-associated breast cancer: the influence of gestational age.

Whether pregnancy-associated breast cancer (PABC) arise before or during pregnancy and whether this histopathology is affected by gestational age are currently unclear. The present study assesses the influence of gestational age and lactation on the histopathologic profile of PABC. We identified 744 patients with PABC (defined as breast cancer during pregnancy or 6 months following delivery). Histopathologic features were compared between pregnant and postpartum patients. We found that age at diagnosis was 34.2 years, and a majority of cancers were diagnosed during pregnancy (71.3%). Within pregnant patients, tumors were significantly more often estrogen receptor (ER)-negative in second and third trimesters (57.4%), as compared to first trimesters (41.9%) (P = 0.036). Similarly, a progesterone receptor (PR)-negative status was reported significantly less often within first trimesters (38.0%) compared to second and third trimesters (57.1%) (P = 0.032). For human EGF receptor 2 status, no significant differences were observed between gestational trimesters or lactating vs non-lactating patients. In postpartum patients, grade III tumors were found in over 80%, with high percentages of ER-negative tumors reaching 63% in those lactating vs 49% in non-lactating patients. This study demonstrates the varying histopathologic profile of PABC by gestational age and lactation status. Second- and third-trimester cancers display most typically the common ER/PR-negative phenotype, which is commonly reported in the literature. The increased ER-negative status and percentage grade III tumors in lactating vs non-lactating patients also suggest the presence of additional factors further diversifying histology. This indicates the need for clear definitions of PABC and the role of potential subgroups, which may provide a stepping stone for further in-depth research into PABC-carcinogenesis.

In vitro anticancer effects of alpelisib against PIK3CA‑mutated canine hemangiosarcoma cell lines.

Hemangiosarcoma (HSA) is a malignant neoplasm that occurs in humans and canines with a poor prognosis owing to metastatic spread, despite effective treatment. The frequency of spontaneous HSA development is higher in canines than in humans. Therefore, canine HSA is a useful model of intractable human disease, which requires early detection and an effective therapeutic strategy. A high frequency of the p110α phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations is detected in a comprehensive genome-wide analysis of canine cases of HSA. The present cloned the full-length cDNA of canine PIK3CA and identified a mutation in codon 1047 from canine cases of HSA and cell lines that were established from these. The enforced expression of the 1047th histidine residue (H1047)R or L mutants of canine PIK3CA in HeLa cells enhanced epidermal growth factor receptor (EGFR) signaling via Akt phosphorylation. PIK3CA mutant canine HSA cell lines exhibited the hyperphosphorylation of Akt upon EGF stimulation as well. Alpelisib, a molecular targeted drug against PIK3CA activating mutations, exerted a significant antitumor effect in canine PIK3CA-mutated HSA cell lines. By contrast, it had no significant effect on canine mammary gland tumor cell lines harboring PIK3CA mutations. On the whole, the findings of the present study suggest that alpelisib may be highly effective against PIK3CA mutations that occur frequently in canine HSA.

Development and molecular characterization of doxorubicin-resistant canine mammary gland tumour cells

ABSTRACT Canine mammary gland tumour (CMT) commonly affects the female dog. The objective of this study was to develop a doxorubicin-resistant CMT cell line and determine its in vitro and in vivo characteristics, including mRNA and microRNA (miRNA) expression profiles. Doxorubicin-resistant CMT-Star cells were developed from CMT-Stylo cells. The cells were characterized, including tumorigenicity in NOD/SCID mouse models. MiRNA and mRNA expression of the two cell lines were profiled and clustered. ATP binding cassette subfamily B member 1 (ABCB1) and subfamily G member 2 (ABCG2) expressions were significantly increased in the CMT-Star cell line. CMT-Star cells also had altered expression of 785 genes and 14 miRNAs. Downregulating plasminogen (PLG) and plasminogen activator urokinase (PLAU) while upregulating transforming growth factor beta receptor 3 (TGFBR3), epidermal growth factor receptor 1 (EGFR1) and ABCB1 rendered CMT-Star cells less proliferative, less invasive and more resistant to chemotherapeutic drugs. The upregulated miRNAs in CMT-Star cells include miRNA-191, -29a, -107, -99b, -874, -93 and -210, while the downregulated miRNAs include miRNAs-106a, -92a, -92b, -155 and -15b. TGFβR, EGF receptor 1 and Wnt signalling are enriched in doxorubicin-resistant CMT-Star cells and could be potential therapeutic targets in dogs with doxorubicin-resistant CMT.

Abstract P4-01-12: Short-term trastuzumab treatment increases oncogenic fitness in HER2 overexpressing breast cancer models

Abstract Overexpression of Human EGF Receptor 2 (HER2) is found in up to 20% of primary invasive breast cancers and is a marker of aggressive metastatic disease and poor prognosis. HER2 is a member of the transmembrane tyrosine kinase receptors family which includes EGFR (HER1), HER3 and HER4. Phosphorylation of tyrosine residues in the cytoplasmic domain upon homo- or heterodimerization results in stimulation of cellular proliferation, migration, angiogenesis, and inhibition of apoptosis. HER2 is deemed a preferred heterodimer partner and presents an excellent target for personalized medicine. Anti-HER2 monoclonal antibody trastuzumab (TZM) has been used in the clinic over the last decades and it is considered as one of the most successful targeted anti-cancer therapies. However, a large fraction of eligible patients displays either primary or acquired resistance to TZM treatment. In this study, we report that even relatively short 24h TZM exposure (TZM-priming) of AU565 HER2-overexpressing breast cancer cells results in rapid and profound alterations in HER receptors’ expression level and signaling, as well as upregulation of markers associated with drug resistance. We provide several lines of evidence to support this claim: Firstly, Western blot analysis of both monolayer AU565 cells incubated in the presence or absence of 20 μg/mL TZM (TZM priming) or human IgG (control), as well as of liquid overlay AU565 spheroids generated from TZM-primed or control cells, consistently shows significant increase of HER3 and pHER2 levels in the TZM-primed samples. Secondly, immunofluorescent analysis strongly indicates that TZM priming substantially upregulates HER3 and pHER2 levels both in 2D and 3D (spheroids) models. Importantly, this process is accompanied by rearrangement of endocytic markers Rab4, CD63, Sorl1 as well as Extra domain B fibronectin, found at the cells’ surface together with HER2 and HER3 upon TZM priming. A similar trend was also found in HER2-overexpressing ovarian cancer cells SKOV-3 subjected to short-term TZM treatment. Furthermore, spheroids made with TZM-primed AU565 cells display significantly faster cell proliferation compared to IgG-primed or untreated control counterparts, as measured via optical coherent tomography imaging and Imaris 3D rendering software. Finally, tumor xenografts implanted in athymic nude mice using TZM-primed AU565 cells display continuous growth in contrast to those generated from untreated cells. These results suggest that short-term TZM treatment may inadvertently increase oncogenic fitness via adaptation to TZM-HER2 cellular binding and subsequent disruption of dimerization and signaling. These results are consistent with reports indicating that regional HER2 expression heterogeneity appears to increase with TZM treatment. Moreover, these observations are especially concerning because radiolabeled TZM probes have been employed in PET imaging in HER2+ cancer patients. In summary, our results suggest that short-term TZM treatment may result in priming and selection for more aggressive cancer phenotype. Citation Format: Alena Rudkouskaya, Cassandra L Roberge, Lauren Elder, Kailie Matteson, David T Corr, Margarida Barroso. Short-term trastuzumab treatment increases oncogenic fitness in HER2 overexpressing breast cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-12.

Synovial fluid from end-stage osteoarthritis induces proliferation and fibrosis of articular chondrocytes via MAPK and RhoGTPase signaling

Alterations in the composition of synovial fluid have been associated with adverse effects on cartilage integrity and function. Here, we examined the phenotypic and proliferative behavior of human articular chondrocytes when cultured in vitro for 13 days with synovial fluid derived from end-stage osteoarthritis patients. Chondrocyte proliferation and phenotypical changes induced by osteoarthritic synovial fluid were analyzed using DNA staining, RT-qPCR, immunostainings, and immunoblotting. The molecular mechanisms by which osteoarthritic synovial fluid induced fibrosis and proliferation were studied using a phospho-protein antibody array and luciferase-based transcription factor activity assays. Specific pathway inhibitors were used to probe the involvement of pathways in fibrosis and proliferation. Prolonged stimulation with osteoarthritic synovial fluid sustained chondrocyte proliferation and induced profound phenotypic changes, favoring a fibrotic over a chondrogenic or hypertrophic phenotype. A clear loss of chondrogenic markers at both the transcriptional and protein level was observed, while expression of several fibrosis-associated markers were upregulated over time. Phospho-kinase analysis revealed activation of MAPK and RhoGTPase signaling pathways by osteoarthritic synovial fluid, which was confirmed by elevated transcriptional activity of Elk-1 and SRF. Inhibitor studies revealed that ERK played a central role in the loss of chondrocyte phenotype, while EGFR and downstream mediators p38, JNK and Rac/Cdc42 were essential for fibrosis-associated collagen expression. Finally, we identified EGF signaling as a key activator of chondrocyte proliferation. Osteoarthritic synovial fluid promoted chondrocyte fibrosis and proliferation through EGF receptor activation and downstream MAPK and RhoGTPase signaling.

Proanthocyanidins Promote Endothelial Cell Viability and Angiogenesis.

Botanic drugs are reportedly effective in treating ischemic conditions by improving vascular circulation. However, it has been very rare for biomaterial researchers to look into the possibility of using such products in the context of tissue regeneration. This work studied 4 botanic drugs to explore their effects on vascular endothelial cell growth. Human umbilical endothelial cells were cultured in the presence of different doses of astragalus powder extract, astragalus injection, puerarin injection, and proanthocyanidin (PAC). Among the 4 drugs, PAC showed a potent effect on cell viability and stimulated cell growth in a dose-dependent manner. In particular, the PAC under test was able to maintain a high level of cell viability/proliferation comparable with the cells supplemented with the endothelial cell growth medium, at both low and normal serum conditions. Blocking either endothelial cell growth factor receptors or epithelial cell growth factor receptors was ineffective in reducing the stimulatory effect. The PAC released from polyvinyl alcohol cryogels stimulated HUVECs proliferation. The chick embryo chorioallantoic membrane model was further used to test the angiogenicity of PAC, showing that this botanic drug was potent in stimulating vasculature development. This work therefore demonstrates for the first time that PAC is capable of upregulating endothelial cell activity and growth in vitro in the absence of growth factors and that PAC can be loaded and released from drug carriers and can stimulate angiogenesis. These findings suggest the application of PAC in angiogenesis and tissue regeneration.

Inhibition of DCLK1 sensitizes resistant lung adenocarcinomas to EGFR-TKI through suppression of Wnt/β-Catenin activity and cancer stemness

Lung adenocarcinoma is the most common form of lung cancer, accounting for 60% of non-small cell lung cancer (NSCLC) cases in Asian patients. Importantly, nearly half of these patients have epithelial growth factor receptor (EGFR) mutations. Though EGFR-tyrosine kinase inhibitors (EGFR-TKIs) are recommended as the first-line therapy for NSCLC patients, the development of resistance reduces their efficiency and limits their application. As the complicated and heterogeneous mechanism of acquired resistance among individuals, the efficiency of anti-angiogenesis therapy, immune checkpoint inhibitors, or chemo-radiotherapies is rather less promising. In this research, we investigated the role of the tumor stem cell marker DCLK1 in EGFR-TKI resistance of lung adenocarcinoma. We discovered that DCLK1 was critical in maintaining the stemness of tumor cells through the Wnt/β-Catenin pathway, which was conducive to the development of EGFR-TKI resistance. Inhibiting DCLK1 activity restored the sensitivity of TKI-resistant tumor cells and organoids. Moreover, our study showed that DCLK1 inhibitor had a synergistic effect in controlling tumor growth when combined with EGFR-TKIs. Overall, our study provides new insights into EGFR-TKI resistant lung adenocarcinoma through inhibition of DCLK1 expression.

Anti-colorectal cancer mechanism of Astragali Radix-Curcumae Rhizoma-Paridis Rhizoma based on network pharmacology and experimental verification

The present study explored the underlying mechanism of Astragali Radix-Curcumae Rhizoma-Paridis Rhizoma(AR-CR-PR) in the treatment of colorectal cancer(CRC) by network pharmacology and molecular docking and animal tests and verified the core targets based on the orthotopic transplantation model in nude mice. The active components of AR-CR-PR were retrieved from databases such as TCMSP. The targets of drugs and the disease were obtained from PubChem, SwissTargetPrediction, TTD, and DrugBank, and the intersection targets were imported into STRING for the analysis of the protein-protein interaction(PPI). Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) analyses were performed through DAVID. AutoDock Vina was used to perform molecular docking and binding ability prediction between the active components and the core targets. The effects of AR-CR-PR on tumor growth, metastasis, and phosphorylation of core target proteins in tumor tissues based on the orthotopic transplantation model in nude mice. As revealed by network pharmacology, AR-CR-PR contained nine core components, such as quercetin, curcumin, and β-ecdysone, and the key targets included protein kinase B(AKT1), mitogen-activated protein kinase 3(MAPK3), MAPK1, and epithelial growth factor receptor(EGFR), which was indicated that the anti-CRC effect of AR-CR-PR was presumedly achieved by regulating tumor cell proliferation, apoptosis, migration, and angiogenesis through PI3 K-AKT, MAPK and other signaling pathways. The results of molecular docking showed that the nine core components had strong binding abilities to AKT1 and MAPK3. The results in vivo showed that AR-CR-PR could reduce the volume of the orthotopic tumor, inhibit liver metastasis, and decrease the phosphorylation of AKT1 and MAPK3 in the CRC model. The mechanism of AR-CR-PR in the intervention of CRC may be related to the activation of PI3 K-AKT and MAPK signaling pathway. This study provides a scientific basis for the clinical application of AR-CR-PR in the treatment of CRC and ideas for modern research on AR-CR-PR.

Safety and efficacy of nivolumab plus recombinant human endostatin in previously treated advanced non-small-cell lung cancer.

BackgroundEvidence of the efficacy of immune checkpoint inhibitors (ICIs) plus antiangiogenic drugs in previously treated patients with advanced non-small-cell lung cancer (NSCLC) is still insufficient, so we investigated the safety and efficacy of nivolumab plus recombinant human (rh)-endostatin in such patients.MethodsPatients without epithelial growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) targetable mutations in advanced NSCLC who did not respond to previous treatment were enrolled. Eligible patients received nivolumab (3 mg/kg, i.v. drip, day 1) every 2 weeks and rh-endostatin (210 mg, continuous i.v. infusion for 168 h) every 4 weeks until disease progression or discontinuation. The primary endpoint was the objective response rate (ORR). The secondary endpoints included disease control rate (DCR), duration of response (DOR), clinical benefit response rate (CBR), progression-free survival (PFS), overall survival (OS) and safety.ResultsA total of 34 patients received a median of 4 cycles of therapy. In all, 14 patients achieved confirmed partial response (PR) with an ORR of 41.2% [14/34; 95% confidence interval (CI): 23.7–58.6%], DCR of 64.7% (22/34; 95% CI: 47.8–81.6%), CBR of 44.1% (95% CI: 26.5–61.7%), and a DOR of 6.9 (95% CI: 4.4–9.4) months. Median follow-up was 12.2 (range, 2.3–18.1) months. Median PFS (mPFS) was 6.8 (95% CI: 1.1–12.1) months, median OS (mOS) was 17.1 (95% CI: 6.6–27.6) months, and 12-month survival rate of 64.4% (95% CI: 46.2–82.6%). In all, 18 (18/34, 52.9%) patients experienced at least one treatment-related adverse event (TRAE), and Grade 3 TRAEs occurred in 4 (4/34, 11.8%) of them.ConclusionsThis study is first to assess nivolumab plus rh-endostatin in previously treated patients with advanced NSCLC. In view of its favorable efficacy and safety profile, this combination represents a promising treatment regimen in this patient population.

Cell HE staining smears and paired cell paraffin sections in detection of epithelial growth factor receptor gene of pleural fluid specimens.

The advanced non-small cell lung cancer (NSCLC) patients with pleural effusion have no opportunity for surgery treatment. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the first-line drugs for these patients with EGFR-sensitive mutation. However, the disease progression and drug update during or after treatment of EGFR-TKIs bring more challenges and puzzles to clinical diagnosis and treatment, which inevitably requires archived pleural cell samples for EGFR re-examination or comparative study. Understanding the DNA quality of archived pleural fluid samples and effectively using archival data of pleural fluid cells are of great significance for tracing the origin of cases and basic medical research. This study aims to evaluate the consistency of EGFR mutant gene expression between the 2 methods, and to explore a reliable way for preserving cytological data and making full use of cytological archival data via cell HE staining smear and cell paraffin section. A total of 57 pleural fluid cytology cases in the Department of Pathology of China Aerospace Center Hospital from October 2014 to April 2021 were selected. Tumor cells were detected by cell HE staining smears and immunohistochemical staining for TTF-1 and Napsin A in the paired cell paraffin sections. There were more than 200 tumor cells in cell HE staining smear and the proportion of tumor cells were ≥70% in matched cell paraffin sections. Patients with 2 cell smears (one for cell data retention and the other for DNA extraction) were selected as the research subjects, and 57 pleural fluid samples were enrolled. EGFR gene mutation was detected by amplification refractory mutation system-polymerase chain reaction in 57 paired cell HE staining smears and cell paraffin sections. DNA concentration was 2 ng/μL. Cell HE smear was amplified side-by-side with DNA samples from paired cell paraffin sections. Result determination was according to the requirements of the reagent instructions. The external control cycle threshold (Ct) value of the No. 8 well of the samples to be tested was between 13 and 21, which was considered as successful and reliable samples. When the Ct value of EGFR gene mutation was <26, it was considered as positive; when the Ct value was between 26 and 29, it was critical positive; when the Ct value was equal or more than 29, it was negative. ΔCt value was the difference between mutant Ct value and externally controlled Ct value. The smaller the ΔCt value was, the better the quality of DNA of the detected sample was. Among the 57 pleural effusion samples, 42 patients were hospitalized with pleural effusion as the first symptom, accounting for 73.7% (42/57). EGFR mutation was detected in 37 samples [64.9% (37/57)]. The mutation rate for 19del was 37.8% (14/37) while for L858R was 48.6% (18/37). Females were 56.7% (21/37) of mutation cases. The mutation consistency rate of cell HE staining smear and matched cell paraffin sections was 100%. The ΔCt values of cell HE staining smears were less than those of matched cell paraffin sections. The mutation Ct values of 37 cytological samples were statistically analyzed according to the preservation periods of the years of 2014-2015, 2016-2017, 2018-2019, and 2020-2021. There were significant differences in cell paraffin section in the years of 2014-2015 and 2016-2017 compared with the years of 2018-2019 and 2020-2021, while no significant differences were found in cell HE staining smear. Statistical analysis of externally controlled Ct values of 57 cytological samples showed that there were significant differences between cell HE staining smears and cell paraffin section in the years of 2014-2015 and 2016-2017, compared with the years of 2018-2019 and 2020-2021. The mutational Ct values of 37 paired cell blocks and smears were all <26, and the externally controlled Ct values of 57 paired cell paraffin sections and HE staining smears were all between 13 and 21. The DNA quality of cell HE smears and matched cell paraffin section met the qualified requirements. Two methods possess show an excellent consistency in detecting EGFR mutation in NSCLC pleural fluid samples. The DNA quality of cell HE staining smear is better than that of cell paraffin sections, so cell HE staining smear can be used as important supplement of the gene test source. It should be noted that the limitation of cell HE staining smears is non-reproducibility, so multiple smears of pleural fluid are recommended to be prepared for multiple tests.

Lactobacillus rhamnosus GG colonization in early life regulates gut-brain axis and relieves anxiety-like behavior in adulthood

Evidence reveals that gut dysbiosis is involved in bidirectional interactions in gut-brain axis and participates in the progress of multiple disorders like anxiety. Gut microbes in early life are crucial for establishment of host health. We aimed to investigate whether early life probiotics Lactobacillus rhamnosus GG (LGG) colonization could relieve anxiety in adulthood through regulation of gut-brain axis. Live or fixed LGG was gavaged to C57BL/6 female mice from day 18 of pregnancy until natural birth, and newborn mice from day 1 to day 5 respectively. In this study, we found that live LGG could be effectively colonized in the intestine of offspring. LGG colonization increased intestinal villus length and colonic crypt depth, accompanied with barrier function protection before weaning. Microbiota composition by 16S rRNA sequencing showed that some beneficial bacteria, such as Akkermansia and Bifidobacteria, were abundant in LGG colonization group. The protective effect of LGG on gut microbiota persisted from weaning to adulthood. Intriguingly, behavioral results assessed by elevated plus mazed test and open field test demonstrated relief of anxiety-like behavior in adult LGG-colonized offspring. Mechanically, LGG colonization activated epithelial growth factor receptor (EGFR) and enhanced serotonin transporter (SERT) expression and modulated serotonergic system in the intestine, and increased brain-derived neurotrophic factor and γ-aminobutyric acid receptor levels in the hippocampus and amygdala. Blocking EGFR blunted LGG-induced the increased SERT and zonula occludens-1 expression. Collectively, early life LGG colonization could protect intestinal barrier of offspring and modulate gut-brain axis in association with relief of anxiety-like behavior in adulthood.

Evaluation of fluorine-18-fluorodeoxyglucose PET/computed tomography and human epithelial growth factor receptor 2 expression in treatment-naive patients with lung adenocarcinoma.

Human epithelial growth factor receptor 2 (HER2) is overexpressed in several types of cancers. The correlation between tumor glucose activity and HER2 expression can vary. This study is a retrospective investigation of fluorine-18-fluorodeoxyglucose PET/computed tomography (18F-FDG PET/CT) and HER2 expression status in patients with lung adenocarcinoma. The maximum standard uptake value (SUVmax) of 18F-FDG PET/CT was compared with the HER2 expression status in pretreated patients with lung adenocarcinoma. Moreover, clinicopathological characteristics, including age, gender, smoking, serum tumor markers, tumor location, size, stage and genetic mutation, were also evaluated in groups with different HER2 expressions. Patients' progression-free survival (PFS) and overall survival (OS) were also analyzed. Ninety-six patients with HER2 expression, including 54 patients with HER2 overexpression and 30 patients without HER2 expression were enrolled in this study. The primary pulmonary lesion was single in all patients, and all lesions were FDG-avid on PET/CT. SUVmax had no significant association with HER2 expression or overexpression in lung adenocarcinoma. Moreover, elevated serum CYFRA211 levels were obviously associated with HER2 expression but not associated with HER2 overexpression. There were no significant differences in other clinicopathological characteristics in groups with different HER2 expressions. Furthermore, multivariate Cox regression analysis revealed that SUVmax, HER2 expression and tumor node metastasis stage were independent predictors of PFS, and SUVmax, CYFRA211 and epidermal growth factor receptor mutation were independent predictors of OS. SUVmax had no significant association with the HER2 expression status in lung adenocarcinoma. 18F-FDG PET/CT and HER2 expression could provide valuable prognostic information for treatment-naive patients with lung adenocarcinoma.

FOXO3/TGF-β signal-dependent ciliogenesis and cell functions during differentiation of temperature-sensitive mouse cochlear precursor hair cells.

The transcription factor FOXO3 is necessary to preserve cochlear hair cells. Growth factors, including TGF-β, closely contribute to cochlear hair cell regeneration. In the present study, to investigate the roles of FOXO3 in the ciliogenesis and cell functions of cochlear hair cells, UB/OC-2 temperature-sensitive mouse cochlear precursor hair cells were treated with TGF-β receptor type 1 inhibitor EW-7197 or EGF receptor inhibitor AG-1478 after transfection with or without siRNA-FOXO3a. GeneChip analysis revealed that treatment with EW-7197 increased Foxo3 genes and decreased genes of Smads. During cell differentiation, treatment with EW-7197 or AG-1478 induced an increase in length of cilia-like structures that were positive for acetylated tubulin and inhibited cell migration. Treatment with EW-7197 also increased cell metabolism measured as mitochondrial basal respiration (oxygen consumption rate). The effects of EW-7197 were stronger than those of AG-1478. Knockdown of FOXO3 prevented the growth of cilia-like structures induced by EW-7197 or AG-1478 and induced cell migration under treatment with EW-7197. No change of the epithelial cell polarity molecule PAR3 was observed with any treatment. Treatment with the antimicrobial agent amikacin prevented the growth of cilia-like structures induced by EW-7197 and induced apoptosis. Pretreatment with the glucocorticoid dexamethasone inhibited the apoptosis induced by amikacin. This in vitro model of mouse cochlear hair cells suggests that FOXO3/TGF-β signaling plays a crucial role in ciliogenesis and cell functions during differentiation of cochlear hair cells. This model is useful for analysis of the mechanisms of hearing loss and to find therapeutic agents to prevent it.

Sapienic Acid Metabolism Influences Membrane Plasticity and Protein Signaling in Breast Cancer Cell Lines.

The importance of sapienic acid (6c-16:1), a monounsaturated fatty acid of the n-10 family formed from palmitic acid by delta-6 desaturase, and of its metabolism to 8c-18:1 and sebaleic acid (5c,8c-18:2) has been recently assessed in cancer. Data are lacking on the association between signaling cascades and exposure to sapienic acid comparing cell lines of the same cancer type. We used 50 μM sapienic acid supplementation, a non-toxic concentration, to cultivate MCF-7 and 2 triple-negative breast cancer cells (TNBC), MDA-MB-231 and BT-20. We followed up for three hours regarding membrane fatty acid remodeling by fatty acid-based membrane lipidome analysis and expression/phosphorylation of EGFR (epithelial growth factor receptor), mTOR (mammalian target of rapamycin) and AKT (protein kinase B) by Western blotting as an oncogenic signaling cascade. Results evidenced consistent differences among the three cell lines in the metabolism of n-10 fatty acids and signaling. Here, a new scenario is proposed for the role of sapienic acid: one based on changes in membrane composition and properties, and the other based on changes in expression/activation of growth factors and signaling cascades. This knowledge can indicate additional players and synergies in breast cancer cell metabolism, inspiring translational applications of tailored membrane lipid strategies to assist pharmacological interventions.

Role of EGF Receptor Regulatory Networks in the Host Response to Viral Infections.

In this review article, we will first provide a brief overview of EGF receptor (EGFR) structure and function, and its importance as a therapeutic target in epithelial carcinomas. We will then compare what is currently known about canonical EGFR trafficking pathways that are triggered by ligand binding, versus ligand-independent pathways activated by a variety of intrinsic and environmentally induced cellular stresses. Next, we will review the literature regarding the role of EGFR as a host factor with critical roles facilitating viral cell entry and replication. Here we will focus on pathogens exploiting virus-encoded and endogenous EGFR ligands, as well as EGFR-mediated trafficking and signaling pathways that have been co-opted by wild-type viruses and recombinant gene therapy vectors. We will also provide an overview of a recently discovered pathway regulating non-canonical EGFR trafficking and signaling that may be a common feature of viruses like human adenoviruses which signal through p38-mitogen activated protein kinase. We will conclude by discussing the emerging role of EGFR signaling in innate immunity to viral infections, and how viral evasion mechanisms are contributing to our understanding of fundamental EGFR biology.

The cell cycle‐related genes RHAMM, AURKA, TPX2, PLK1, and PLK4 are associated with the poor prognosis of breast cancer patients

Breast cancer is the third most common type of cancer diagnosed. Cell cycle is a complex but highly organized and controlled process, in which normal cells sense mitogenic growth signals that instruct them to enter and progress through their cell cycle. This process culminates in cell division generating two daughter cells with identical amounts of genetic material. Uncontrolled proliferation is one of the hallmarks of cancer. In this study, we analyzed the expression of the cell cycle-related genes receptor for hyaluronan (HA)-mediated motility (RHAMM), AURKA, TPX2, PLK1, and PLK4 and correlated them with the prognosis in a collective of 3952 breast cancer patients. A high messenger RNA expression of all studied genes correlated with a poor prognosis. Stratifying the patients according to the expression of hormonal receptors, we found that in patients with estrogen and progesterone receptor-positive and human epithelial growth factor receptor 2-negative tumors, and Luminal A and Luminal B tumors, the expression of the five analyzed genes correlates with worse survival. qPCR analysis of a panel of breast cancer cell lines representative of major molecular subtypes indicated a predominant expression in the luminal subtype. In vitro experiments showed that radiation influences the expression of the five analyzed genes both in luminal and triple-negative model cell lines. Functional analysis of MDA-MB-231 cells showed that small interfering RNA knockdown of PLK4 and TPX2 and pharmacological inhibition of PLK1 had an impact on the cell cycle and colony formation. Looking for a potential upstream regulation by microRNAs, we observed a differential expression of RHAMM, AURKA, TPX2, PLK1, and PLK4 after transfecting the MDA-MB-231 cells with three different microRNAs. Survival analysis of miR-34c-5p, miR-375, and miR-142-3p showed a different impact on the prognosis of breast cancer patients. Our study suggests that RHAMM, AURKA, TPX2, PLK1, and PLK4 can be used as potential targets for treatment or as a prognostic value in breast cancer patients.

Potential applications of clickable probes in EGFR activity visualization and prediction of EGFR-TKI therapy response for NSCLC patients

The epithelial growth factor receptor (EGFR) is abnormally overexpressed on the cell surface of cancer cells and is strongly associated with cancer cell proliferation, migration, differentiation, apoptosis, and angiogenesis. Tools enabling the visualization of EGFR in a structure-function approach are highly desirable to predict EGFR mutations and guide EGFR tyrosine kinase inhibitor (TKI) treatment making. Here, we describe the design, synthesis, and application of new, potent and selective clickable probes 13 (HX03), 20 (HX04) and 24 (HX05) by introducing an alkyne ligation handle to visualize EGFR activity in living cancer cells and tissue slices. These clickable probes are versatile chemical tools based on the key pharmacophore (4-anilinoquinazoline) of EGFR-TKIs (e.g., canertinib, dacomitinib and afatinib) and are able to irreversibly target the kinase domain of EGFR. Among them, 13 exhibits the highest reactivity towards EGFR kinase, particularly to EGFR kinase with primary mutations. Using activity-based protein profiling strategy, 13 showed high sensitivity and selectivity in labeling of endogenous EGFR in a native cellular context. Moreover, 13 was applied to visualize EGFR mutant activity in tumour tissues from non-small-cell lung cancer (NSCLC) xenograft mouse models, and patients with NSCLC for the prediction of EGFR-TKI sensitivity. These results demonstrate that strategically designed EGFR-TKI-based probes allow discriminating EGFR mutations in human tissues and hold promise as useful diagnostic tools in predicting EGFR-TKI therapy response.