Detection of the anti-Hu antibody in the serum of patients with small cell lung cancer - A quantitative Western blot analysis

To identify cancer/testis (CT) antigens and immunogenic proteins, immunoscreening of testicular and small-cell lung cancer cell line NCI-H889 cDNA libraries was performed using serum obtained from a small-cell lung cancer (SCLC) patient. We obtained 113 positive cDNA clones comprised of 74 different genes, designated KP-SCLC-1 through KP-SCLC-74. Of these genes, 59 genes were found to be related to cancers by EMBASE analysis. Three of these antigens, including KP-SCLC-29 (NOL4), KP-SCLC-59 (CCDC83), and KP-SCLC-69 (KIF20B), were CT antigens. RT-PCR and western blot analysis showed that NOL4 was frequently present in small-cell lung cancer cell lines (8/9, 8/9). In addition, NOL4 mRNA was weakly, or at a low frequency, or not detected in various cancer cell lines. Our results reveal that NOL4 was expressed at protein levels in small-cell lung cancer tissues (10/10) but not detected in lung adenocarcinoma and squamous cell carcinoma by immunohistochemical analysis. Serological response to NOL4 was also evaluated by western blot assay using NOL4 recombinant protein. A humoral response against NOL4 proteins was detected in 75% (33/44) of small-cell lung cancer patients and in 65% (13/20) of healthy donors by a serological western blot assay. These data suggest that NOL4 is a specific target that may be useful for diagnosis and immunotherapy in SCLC.

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Exosome-Mediated miR-7-5p Delivery Enhances the Anticancer Effect of Everolimus via Blocking MNK/eIF4E Axis in Non-Small Cell Lung Cancer

10.21203/rs.3.rs-880087/v1 ◽

2021 ◽

Author(s):

Sile Liu ◽

Weiyuan Wang ◽

Yue Ning ◽

Hongmei Zheng ◽

Yuting Zhan ◽

...

Keyword(s):

Lung Cancer ◽

Western Blot ◽

Small Cell Lung Cancer ◽

Cell Lung Cancer ◽

Therapeutic Efficacy ◽

Mtor Inhibitors ◽

Small Cell ◽

Nsclc Cell ◽

Luciferase Reporter ◽

Small Cell Lung

Abstract Background Everolimus is a kind of mTOR inhibitors. Activated mitogen-activated protein kinase interacting kinases/eukaryotic translation initiation factor 4E (MNK/eIF4E) axis plays a crucial role in resistance to Everolimus in non-small cell lung cancer (NSCLC). Typically, eIF4E phosphorylation increased by mTOR inhibitors was mainly mediated by MNKs. But the mechanisms are poorly understood. Recently, extensive reprogramming of miRNA profiles has also been found after long-term mTOR inhibitor exposing. Our previous studies have confirmed that tumor suppressor miR-7-5p was decreased in A549 cells after treatment with Everolimus. Exactly, MNK1 is the target of miR-7-5p. Here, we investigated the biological functions and potential molecular mechanisms of miR-7-5p in the NSCLC undergoing treatment with Everolimus. Methods miR-7-5p level and expression of main markers of MNK/eIF4E axis were evaluated by qRT-PCR, western blot, in situ hybridization, and immunohistochemistry on NSCLC cell lines and human NSCLC samples. Proliferation, migration and invasion of NSCLC cells in culture were explored by Colony formation, CCK-8, Wound healing and Transwell assays. NSCLC cell tumorigenicity was assessed by xenotransplants in nude mice. Targeted binding of miR-7-5p to MNK1 was confirmed by the Dual-luciferase reporter assay. And the isolation and identification of exosomes were investigated by Invitrogen™ Total Exosome RNA Isolation Kit, western blot, transmission electron microscopy and Zetasizer Nano ZS90 instrument. Results Everolimus targeted mTORC1 inducing NSCLC cells to secrete miR-7-5p-loaded exosomes in Rab27A and Rab27B dependent manners. Loss of intracellular miR-7-5p induced phosphorylation of MNK/eIF4E axis, but supplement of extra exosomal miR-7-5p could reverse it. Of note, both lower expression of miR-7-5p and elevated MNK1 protein were associated with poor prognosis of NSCLC. Both endogenous miR-7-5p and exo-miR-7-5p enhanced the therapeutic efficacy of Everolimus through inhibiting the proliferation, migration, and metastasis of NSCLC in vitro and vivo. Combination of miR-7-5p with Everolimus induced apoptosis to exhibit a synergistic anticancer therapeutic efficacy via dual abrogation of MNK/eIF4E and mTOR in NSCLC. Conclusion Everolimus decreases the intracellular miR-7-5p levels through release of miR-7-5p loaded exosomes from NSCLC cells in Rab27A and Rab27B dependent manners. Either endogenous miR-7-5p or exo-miR-7-5p combined with Everolimus can enhance the anticancer efficacy via targeting MNK/eIF4E axis and mTOR. Therefore, exosome-mediated miR-7-5p delivery combined with Everolimus may be considered as a promising novel combined therapeutic strategy for NSCLC.

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Identification and Validation of Afatinib Potential Drug Resistance Gene BIRC5 in Non-Small Cell Lung Cancer

Frontiers in Oncology ◽

10.3389/fonc.2021.763035 ◽

2021 ◽

Vol 11 ◽

Author(s):

Xiaoxi Zhu ◽

Renyu Zhou ◽

Yuanzhi Lu ◽

Ying Zhang ◽

Qiang Chen ◽

...

Keyword(s):

Lung Cancer ◽

Cell Cycle ◽

Western Blot ◽

Small Cell Lung Cancer ◽

Cell Lung Cancer ◽

Apoptotic Cell ◽

Small Cell ◽

Small Cell Lung ◽

Repeat Protein ◽

Cell Death Pathway

IntroductionResistance to second-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), afatinib, is the most significant challenge in the clinical management of non-small cell lung cancer (NSCLC), and the underlying mechanisms remain unclear.MethodsGenomic signatures that may confer afatinib resistance in NSCLC were identified via data mining of public databases and integrative bioinformatic analyses. Furthermore, acquired afatinib-resistant lung adenocarcinoma cell lines (HCC827 AR) were established by long-term exposure under afatinib in vitro for stepwise escalation. The expression of baculovirus IAP repeat protein 5 (BIRC5) was detected by western blot, and cellular viability of HCC827 AR was determined by CCK8.ResultsThrough integrative bioinformatic analyses of public datasets, overexpression of baculovirus IAP repeat protein 5 (BIRC5) was identified in both afatinib-resistant NSCLC cells and tissues, and BIRC5 overexpression was positively correlated with lymph node metastasis as well as pathological stage in NSCLC. Furthermore, NSCLC patients with BIRC5 overexpression showed poor survival outcomes. Immune infiltration analysis suggested that BIRC5 expression was significantly inversely correlated with tumor-infiltrating cell numbers and immune biomarker expression in NSCLC. The functions of genes co-expressed with BIRC5 were mainly enriched in cell cycle mitotic phase transition, double-strand break repair, and negative regulation of the cell cycle process signaling pathway. In addition, overexpression of BIRC5 protein was detected in afatinib-resistant cells by western blot, while BIRC5-expressing cells treated with BIRC5 inhibitor, YM155, were sensitive to afatinib.ConclusionsIn this study, we showed that overexpression of BIRC5 resulted in resistance to afatinib in NSCLC and BIRC5-specific inhibitors may overcome the resistant phenotype, indicating that dysregulation of the apoptotic cell death pathway may be the key mechanism underlying TKI resistance in the development of NSCLC.

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miR-202 Enhances the Anti-Tumor Effect of Cisplatin on Non-Small Cell Lung Cancer by Targeting the Ras/MAPK Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000495835 ◽

2018 ◽

Vol 51 (5) ◽

pp. 2160-2171 ◽

Cited By ~ 13

Author(s):

Wei Sun ◽

Wei Ping ◽

Yitao Tian ◽

Wenbin Zou ◽

Jiawei Liu ◽

...

Keyword(s):

Lung Cancer ◽

Drug Resistance ◽

Western Blot ◽

Small Cell Lung Cancer ◽

Cell Lung Cancer ◽

Mapk Pathway ◽

Small Cell ◽

Luciferase Reporter ◽

Small Cell Lung ◽

Kras Gene

Background/Aims: KRas is usually mutated in non-small cell lung cancer (NSCLC). The mutated KRas gene is a negative prognostic indicator that promotes tumor proliferation, metastasis, and drug resistance in NSCLC, and thus has become a target for cancer therapy. This study is focused on the effects of the microRNA (miR)-202/KRas axis in regulating chemosensitivity in NSCLC. Methods: Quantitative reverse transcriptase real-time PCR analysis was performed to examine the expression of miR-202. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were performed to evaluate the sensitivity of cisplatin against NSCLC cells. The miR-202/KRas axis was confirmed by western blot and luciferase reporter assays. Cell apoptosis was measured by flow cytometry. KRas expression, MEK1/2 and ERK1/2 phosphorylation, and activation of caspase-9 and caspase-3 were detected by western blot. Results: A significant decrease in miR-202 expression was observed in NSCLC cells both in vivo and in vitro. In addition, miR-202 expression was associated with drug resistance. Recovery of miR-202 expression levels was found to increase the sensitivity of both NCI-H441 and A549 NSCLC cells to cisplatin treatment. Mechanically, as the Ras/mitogen-activated protein kinase (MAPK) pathway was aberrantly activated in NCI-H441 and A549 NSCLC cells, the overexpression of miR-202 was found to inhibit the Ras/MAPK pathway by targeting the KRas gene. As a result, increased miR-202 expression expanded apoptosis signaling induced by cisplatin in NSCLC cells. Conclusion: The miR-202/KRas axis controlled the chemosensitivity of NSCLC by mediating the Ras/MAPK pathway. Thus, the combination of platinum-based drugs with miR-202 may represent a novel strategy to enhance the anti-tumor effect against NSCLC.

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MicroRNA-103a Curtails the Stemness of Non-Small Cell Lung Cancer Cells by Binding OTUB1 via the Hippo Signaling Pathway

Technology in Cancer Research & Treatment ◽

10.1177/1533033820971643 ◽

2020 ◽

Vol 19 ◽

pp. 153303382097164

Author(s):

Zhenzhen Hu ◽

Dan Xiao ◽

Tingting Qiu ◽

Jun Li ◽

Zhentian Liu

Keyword(s):

Lung Cancer ◽

Western Blot ◽

Small Cell Lung Cancer ◽

Cell Lung Cancer ◽

Quantitative Polymerase Chain Reaction ◽

Small Cell ◽

Luciferase Reporter ◽

Hippo Signaling ◽

Small Cell Lung ◽

Luciferase Reporter Gene

Objective: Although microRNA-103a (miR-103a) dysfunction has been implicated in various cancers, its relevance to non-small cell lung cancer (NSCLC) has not been clarified. This study was conducted to examine the molecular mechanism underlying the regulatory role of miR-103a in NSCLC. Methods: Kaplan–Meier analysis was carried out to assess the relationship between overall survival of NSCLC patients and miR-103a expression. Reverse-transcription quantitative polymerase chain reaction and western blot analyses were applied to evaluate the expression of relevant genes in tissues and cells. Sphere formation, MTS, flow cytometry, and Transwell assays were performed to characterize stemness. Dual luciferase reporter gene assays were used to clarify the binding relationship between miR-103a and ovarian tumor domain-containing ubiquitin aldehyde binding protein 1 (OTUB1). Finally, western blot analysis was used to assess the involvement of the Hippo pathway in NSCLC. Results: In NSCLC tissues and cells, miR-103a was expressed at low levels, whereas OTUB1 was expressed at high levels. Higher miR-103 expression levels were associated with a better prognosis for patients with NSCLC. When miR-103a was overexpressed, cell viability and stemness decreased, whereas apoptosis and cell cycle arrest were facilitated. The expression of phosphorylated YAP also decreased significantly. Opposite trends were observed after miR-103a silencing. OTUB1 expression and YAP phosphorylation decreased in the presence of miR-103a, and OTUB1 overexpression blocked the inhibitory effects of miR-103a on NSCLC cells. Conclusion: The miR-103a/OTUB1/Hippo axis may play a role in modulating the malignant behavior and stemness of cancer stem cells and thus could be a potential therapeutic target for the management of NSCLC.

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The MicroRNA-130a-5p/RUNX2/STK32A Network Modulates Tumor Invasive and Metastatic Potential in Non-Small Cell Lung Cancer

10.21203/rs.3.rs-22562/v1 ◽

2020 ◽

Author(s):

Fang Ma ◽

Yangchun Xie ◽

Yiyu Lei ◽

Zengshuyu Kuang ◽

Xianling Liu

Keyword(s):

Lung Cancer ◽

Transcription Factor ◽

Western Blot ◽

Small Cell Lung Cancer ◽

Cell Lung Cancer ◽

Small Cell ◽

Cell Counting ◽

Luciferase Reporter ◽

Small Cell Lung

Abstract Background Non-small cell lung cancer (NSCLC) remains a huge health burden for human health and life worldwide. Our study here was to illuminate the relevance of microRNA-130a-5p (miR-130a-5p) on growth and epithelial mesenchymal transition (EMT) in NSCLC cells as along with metastasis in vivo, and to explore the underlying mechanism.Methods RT-qPCR was carried out for miR-130a-5p expression determination in NSCLC cells and tissue samples. Dual luciferase reporter gene assay, RT-qPCR and western blot were carried out to study the potential targets of miR-130a-3p. Effects of miR-130a-5p, runt-related transcription factor 2 (RUNX2) and encoding serine/threonine kinase 32A (STK32A) on NSCLC proliferation, migration, invasion as well as EMT processes were assessed by cell counting kits-8, colony formation, Transwell and western blot assays.Results miR-130a-5p was diminished in NSCLC tissues and cells versus their counterparts. miR-130a-5p exerted its repressive role in NSCLC by curtailing cell viability, migration, invasion as well as EMT, while facilitating apoptosis. miR-130a-5p directly targeted RUNX2, a transcription factor, and conversely regulated its expression. RUNX2 was found to interacted with STK32A to promote its expression. Following the validation of the tumor-supporting role of STK32A in NSCLC cells, RUNX2 overexpression was monitored to reverse miR-130a-5p-inhibited NSCLC tumor volume and weight through enhancing STK32A expression in vivo.Conclusions miR-130a-5p diminished the growth and EMT of NSCLC cells by regulating the RUNX2/STK32A axis, offering possible targets for the treatment for NSCLC.

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Everolimus-Induced Loss of Exosomal miR-7-5p Activates MNK/eIF4E Axis in Non-Small Cell Lung Cancer

10.21203/rs.3.rs-740536/v1 ◽

2021 ◽

Author(s):

Sile Liu ◽

Weiyuan Wang ◽

Yue Ning ◽

Hongmei Zheng ◽

Yuting Zhan ◽

...

Keyword(s):

Lung Cancer ◽

Tumor Suppressor ◽

Western Blot ◽

Small Cell Lung Cancer ◽

Cell Lung Cancer ◽

Mtor Inhibitors ◽

Small Cell ◽

Translation Initiation Factor ◽

Luciferase Reporter ◽

Small Cell Lung

Abstract Background: Everolimus is a kind of mTOR inhibitors. Activated mitogen-activated protein kinase interacting kinases/eukaryotic translation initiation factor 4E (MNK/eIF4E) axis plays a crucial role in resistance to Everolimus in non-small cell lung cancer cells (NSCLC). Typically, eIF4E phosphorylation increased by mTOR inhibitors was mainly mediated by MNKs. But the mechanisms are poorly understood. Recently, extensive reprogramming of miRNA profiles has also been found after long-term mTOR inhibitor exposing. Our previous studies have found that tumor suppressor miR-7-5p was loss in A549 cells after treatment of Everolimus. Exactly, MNK1 is the target of miR-7-5p. Here, we analyzed levels and functions of miR-7-5p in the usage of Everolimus in NSCLC.Methods: miR-7-5p level and expression of main markers of MNK/eIF4E axis was evaluated by qRT-PCR, in situ hybridization, and immunohistochemistry on human NPC samples, and by RT-PCR, western blot on NPC cell lines. Proliferation, migration and invasion of NPC cells in culture were assessed by Colony formation, CCK-8, Wound healing and Transwell assays. NPC cell tumorigenicity was assessed by xenotransplants in nude mice. Targeted binding of miR-7-5p to MNK1 was confirmed by the Dual-luciferase reporter assay. And the isolation and identification of exosomes were invested by Invitrogen™ Total Exosome RNA Isolation Kit, western blot, transmission electron microscopy and Zetasizer Nano ZS90 instrument.Results: Everolimus stimulatedtherelease of miR-7-5p loaded exosomes from NSCLC cellsinRab27A and Rab27B dependent manners, thereby reducing the intracellular tumor suppressor miR-7-5p to attenuate the inhibition of MNK1 and promote MNK-dependent eIF4E phosphorylation. Of note, both lower miR-7-5p and positive MNK1 could act as independent poor prognostic biomarkers for NSCLC. And delivery of miR-7-5p would inhibit the poor prognosis of it. Bothendogenous miR-7-5p-5p or exo-miR-7-5p collaborated with Everolimus, not only inhibited the proliferation, migration, and metastasisof NSCLC in vitro and in vivo, but also promoted the apoptosis of NSCLC viatargeting MNK/eIF4E axis.Conclusion: Everolimus-induced loss of exosomal miR-7-5p activates MNK/eIF4E axis to blunt effectiveness of itself in NSCLC. Therefore, delivery of miR-7-5p could act as a combined therapeutic strategy for mTOR-targeted cancer therapy and prognosis.

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