Non-small Cell Lung Cancer Lung Tissues Research Articles

CircCAMSAP1 promotes non-small cell lung cancer proliferation and inhibits cell apoptosis by sponging miR-1182 and regulating BIRC5

ABSTRACT Recently, various studies have suggested that circular RNAs (circRNAs) are ubiquitous in various malignant events, including non-small cell lung cancer (NSCLC) and are closely related to cell proliferation and apoptosis. Unfortunately, the molecular functions involved in this action still have little overlap. Therefore, this study aimed to identify a novel circCAMSAP1 role in NSCLC. Overexpression of circCAMSAP1 has been demonstrated in NSCLC lung tissues and cell lines. Sequencing and RNase R experiments were planned to determine whether circCAMSAP1 is looped and exists in NSCLC. We also found that downregulated circCAMSAP1 repressed cell proliferation and increased apoptosis of NSCLC cells in vitro and suppressed xenograft tumor growth in vivo. Furthermore, a luciferase assay revealed that circCAMSAP1 could regulate baculoviral inhibitor of apoptosis protein (IAP) repeat containing 5 (BIRC5, also known as survivin) expression by directly binding to miR-1182. However, BIRC5 without 3ʹ untranslated regions (3ʹUTR) could reverse the influence of downregulated circCAMSAP1 on proliferation and apoptosis in NSCLC. Together, our findings reveal a novel mechanism by which the circCAMSAP1/miR-1182/BIRC5 axis promotes NSCLC progression.

Downregulation of LINC00665 confers decreased cell proliferation and invasion via the miR-138-5p/E2F3 signaling pathway in NSCLC

Non-small cell lung cancer (NSCLC) is a type of malignant tumor which threatens human health and life. Recently, some researches on long non-coding RNAs (lncRNAs) in NSCLC has elucidated critical regulatory roles in cell proliferation, migration, and invasion, the relative clinical significance and mechanisms of action are still unclear. This study focuses on the important role of a novel lncRNA LINC00665 in the development of NSCLC. Long intergenic non-protein coding RNA 665 gene (LINC00665) was found through microarray analysis and was measured by real-time quantitative PCR (RT-qPCR). The interactions between LINC00665 and miR-138-5p as well as the interactions between miR-138-5p and E2F3 (E2F transcription factor 3) were explored by bioinformatics analysis and dual-luciferase assays. CCK-8, transwell and mouse xenograft assays were performed to investigate the effects of LINC00665 and miR-138-5p on NSCLC proliferation and invasion. As a result, LINC00665 expression was upregulated in NSCLC lung tissues and cells. Downregulated LINC00665 could arrest A549 and H1299 cell proliferation and invasion in vitro, and this finding was recapitulated in vivo. LINC00665 directly regulated the expression of miR-138-5p. Additionally, E2F3 was one of the targets of miR-138-5p; E2F3 without 3′UTR could reverse the inhibitory effects of downregulated LINC00665 on proliferation and invasion in A549 and H1299 cells. In conclusion, dysregulation of LINC00665 plays a vital role in NSCLC progression, indicating that its downregulation may confer decreased cell proliferation and invasion via the miR-138-5p/E2F3 signaling pathway.

Abstract 955: Musashi-2 regulates EGFR expression in NSCLC, cell proliferation and survival, response to EGFR inhibitors in EGFR-mutant NSCLC

Abstract Musashi-2 (MSI2) is an RNA-binding protein that regulates mRNA translation. We recently established that MSI2 is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression and drives NSCLC metastasis, in part based on activity supporting a TGF-beta/SMAD3/claudin signaling cascade. Here, reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+;P53R172HΔG/+ murine NSCLC cell lines identified a significant upregulation of HER3 (ERBB3) upon MSI2 depletion. Negative MSI2-dependent regulation of ERBB3 protein was confirmed in multiple murine and human NSCLC models, based on analysis of MSI2 depletion or overexpression. Further, MSI2 positively regulated expression of the epidermal growth factor receptor (EGFR) protein in the same models. Comparing EGFR and KRAS driven models, we found that MSI2 depletion significantly impairs cell proliferation and clonogenicity only in EGFR-mutant NSCLC cell lines. Using RNA immunoprecipitation analysis coupled with qPCR, we show that MSI2 directly binds to EGFR and, to a lesser extent, to HER3 mRNA. MSI2 mRNA binding was correlating with the presence of predicted MSI2 binding sites in corresponding mRNAs. Taken together, this data suggest direct regulation of EGFR protein expression by MSI2 at post transcriptional level. NSCLC lung tissue microarray immunohistochemistry analysis revealed that MSI2 total positivity by H-score, 2+/3+ and 3+ positivity correlated with EGFR 3+ staining. Finally, EGFR inhibitors erlotinib and afatinib synergized with MSI2 depletion in EGFR mutant models, suggesting that therapeutic targeting of MSI2 could be of clinical value, especially in EGFR-mutant lung cancer. In vivo experiments of erlotinib treatments of wild type or MSI2 depleted tumors are ongoing and these results will be presented. Citation Format: Alexander Kudinov, Petr Makhov, Bulat Faezov, Igor Bychkov, Alexander Deneka, Emmanuelle Nicolas, Kathy Q Q. Caid, Rohan Brebione, Eleanor Avrilf, Anna S. Nikonova, Ilya G. Serebriiskii, Hossein Borghaei, Erica A. Golemis, Yanis Boumber. Musashi-2 regulates EGFR expression in NSCLC, cell proliferation and survival, response to EGFR inhibitors in EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 955.

CHAF1B promotes proliferation and reduces apoptosis in 95‑D lung cancer cells and predicts a poor prognosis in non‑small cell lung cancer.

Chromatin assembly factor 1 subunit B (CHAF1B) participates in DNA synthesis and repair. High CHAF1B expression has been associated with a poor prognosis in several types of cancers. However, no study has evaluated the clinical significance and biological function of CHAF1B in non‑small cell lung cancer (NSCLC). In the present study, we aimed to investigate CHAF1B expression and its role in NSCLC. In the present study, it was revealed that CHAF1B was highly expressed in NSCLC lung tissues and 95‑D cells. Kaplan‑Meier survival analysis indicated that high CHAF1B expression in tumour tissue was associated with poor clinical outcomes in NSCLC patients. Multivariate Cox analyses revealed that lymph node metastasis, tumour‑node‑metastasis (TNM) stage and CHAF1B expression were independent prognostic factors in NSCLC patients. Moreover, CHAF1B knockdown in 95‑D cells markedly inhibited tumour proliferation, reduced colony formation, induced cell cycle arrest and promoted apoptosis. In vivo studies demonstrated that CHAF1B knockdown inhibited the growth of transplanted tumours. Furthermore, our results revealed that the mechanism by which CHAF1B induced apoptosis was mediated by the activation of the p53‑dependent apoptotic signalling pathway (BAK/Bcl‑2/caspase‑3) in 95‑D cells. These data indicated that CHAF1B plays an important role in tumourigenesis and may be a therapeutic molecular target to counter NSCLC progression.

Abstract 4452: Musashi-2 regulates EGFR/HER3 expression in NSCLC, cell proliferation and response to EGFR inhibitors in EGFR-mutant NSCLC

Abstract Musashi-2 (MSI2) is an RNA-binding protein that regulates mRNA translation. We recently established that MSI2 is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression and drives NSCLC metastasis, in part based on activity supporting a TGF-beta/SMAD3/claudin signaling cascade. Here, reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+;P53R172HΔG/+ murine NSCLC cell lines identified a significant ~2.7-fold upregulation of HER3 (ERBB3) upon MSI2 depletion. Negative MSI2-dependent regulation of ERBB3 protein was confirmed in multiple NSCLC models, based on analysis of MSI2 depletion or overexpression. Further, MSI2 positively regulated expression of the epidermal growth factor receptor (EGFR) protein in the same models. Comparing EGFR and KRAS driven models, we found that MSI2 depletion significantly impairs cell proliferation only in EGFR-mutant NSCLC cell lines. Using RNA immunoprecipitation analysis coupled with qPCR, we show that MSI2 directly binds to EGFR and, to a lesser extent, to HER3 mRNA. MSI2 mRNA binding was approximately correlating with the presence of predicted MSI2 binding sites in corresponding mRNAs. NSCLC lung tissue microarray analysis revealed that MSI2 total positivity by H-score, 2+/3+ and 3+ positivity correlated with EGFR 3+ staining. Finally, EGFR inhibitors erlotinib and afatinib synergized with MSI2 depletion in EGFR mutant models, suggesting that therapeutic targeting of MSI2 could be of clinical value, especially in EGFR-mutant lung cancer. Citation Format: Peter Makhov, Alexander Kudinov, Alexander Deneka, Brian L. Egleston, Emmanuelle Nicolas, Kathy Q. Cai, Rohan Brebion, Eleanor Avril, Mark Hitrik, Anna S. Nikonova, Ilya G. Serebriiskii, Vladimir Khazak, Hossein Borghaei, Erica A. Golemis, Yanis Boumber. Musashi-2 regulates EGFR/HER3 expression in NSCLC, cell proliferation and response to EGFR inhibitors in EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4452.

Downregulation of long non-coding RNA LET predicts poor prognosis and increases Notch signaling in non-small cell lung cancer

Long non-coding RNAs (lncRNAs) have been found to be dysregulated in a variety of tumors. The lncRNA-Low Expression in Tumor (LET) is a recently identified lncRNA, but its expression pattern and biological significance in human non-small cell lung cancer (NSCLC) are still largely unknown. In this study, we found that lncRNA-LET was significantly downregulated in human NSCLC lung tissues and cell lines. Decreased lncRNA-LET expression was strongly associated with advanced tumor stages and poorer overall survival of NSCLC patients. Functionally, overexpression of lncRNA-LET in NSCLC H292 cells significantly suppressed cell proliferation, migration and invasion, and promoted cell cycle arrest and apoptosis, while knockdown of lncRNA-LET in NSCLC H1975 cells showed an opposite effect, pointing to a tumor-suppressive role for lncRNA-LET in NSCLC. Mechanistically, we demonstrated that lncRNA-LET overexpression significantly reduced the expression of Notch1 intracellular Domain (NICD1) in H292 cells while knockdown of lncRNA-LET increased NICD1 expression in H1975 cells. Similarly, NSCLC lung tissues with high levels of lncRNA-LET had lower NICD1 expression. Thus, our results provide a strong rationale for lncRNA-LET to be used as a prognostic indicator and a potent therapeutic target for NSCLC patients, and highlight a novel lncRNA-LET/Notch axis in regulating NSCLC cell fate and tumor progression.

Correlation analysis between expression of NF-κB and quartz's deposit in non-small cell lung cancer in Xuanwei, Yunnan province

Objective To evaluate the relationship between quartz's deposit and expression of NF-κB in non-small cell lung cancer (NSCLC) lung tissues in Xuanwei, Yunnan Province, and to clarify the role of quartz in Xuanwei NSCLC's carcinogenic mechanism. Methods As research objects, the lung tissues of NSCLC and lung benign lesions after surgical resection were collected from July 2009 to September 2015 at the Third Affiliated Hospital of Kunming Medical University. Firstly, the transmission electron microscopic (TEM) with energy dispersive X-ray analyzer (EDS) was used for observation of crystalline deposit and local pathological changes. Secondly, expression level of NF-κB had been analysed and a correlation analysis with particle size of SiO2 crystal in the same lung sample was made. Results The occurrence rates of quartz in Xuanwei NSCLC lung tissues were above non-Xuanwei NSCLC and benign lung tissues (P 0.05). The expression level of NF-κB was generally increasing when quartz's size became smaller. Conclusion Quartz's deposit may play a certain role in carcinogenic mechanism of lung cancer in Xuanwei, the smaller the particle size, the greater the cytotoxicity. Key words: Carcinoma, non-small-cell lung; Quartz; NF-kappa B

Differentially expressed and activated proteins associated with non small cell lung cancer tissues.

BackgroundLung cancer is a leading cause of mortality. The most common cancer subtype, non small cell lung cancer (NSCLC), accounts for 85-90 % all cases and is mainly caused by environmental and genetic factors. Mechanisms involved in lung carcinogenesis include deregulation of several kinases and molecular pathways affecting cell proliferation, apoptosis and differentiation. Despite advances in lung cancer detection, diagnosis and staging, survival rate still remains poor and novel biomarkers for both diagnosis and therapy need to be identified. In the present study, we have explored the potential of novel specific biomarkers in the diagnosis of NSCLC, and the over-expression/activation of several kinases involved in disease development and progression.MethodLung tumor tissue specimens and adjacent cancer-free tissues from 8 NSCLC patients undergoing surgery were collected. The differential activation status of ERK1/2, AKT and IKBα/NF-κβ was analyzed. Subsequently, protein expression profile of NSCLC vs normal surrounding tissue was compared by a proteomic approach using LC-MS MS. Subsequently, MS/MS outputs were analyzed by the Protein Discoverer platform for label-free quantitation analysis. Finally, results were confirmed by western blotting analysis.ResultsThis study confirms the involvement of ERK1/2, AKT, IKBα and NF-κβ proteins in NSCLC demonstrating a significant over-activation of all tested proteins. Furthermore, we found significant differential expression of 20 proteins (Rsc ≥ 1.50 or ≤ −1.50) of which 7 are under-expressed and 13 over-expressed in NSCLC lung tissues. Finally, we validated, by western blotting, the two most under-expressed NSCLC tissue proteins, carbonic anhydrase I and II isoforms.ConclusionOur data further support the possibility of developing both diagnostic tests and innovative targeted therapy in NSCLC. In addition to selective inhibitors of ERK1/2, AKT, IKBα and NF-κβ, as therapeutic options, our data, for the first time, indicates carbonic anhydrase I and II as attractive targets for development of diagnostic tools enabling selection of patients for a more specific therapy in NSCLC.

Modulation of NF-κB/miR-21/PTEN pathway sensitizes non-small cell lung cancer to cisplatin.

BackgroundPlatinum-based chemotherapy is a standard strategy for non-small cell lung cancer (NSCLC), while chemoresistance remains a major therapeutic challenge in current clinical practice. Our present study was aimed to determine whether inhibition of the NF-κB/miR-21/PTEN pathway could increase the sensitivity of NSCLC to cisplatin.MethodsThe expression of miR-21 in NSCLC tissues was determined using in situ hybridization. Next, the effect of miR-21 on the sensitivity of A549 cells to cisplatin was determined in vitro. Whether miR-21 regulated PTEN expression was assessed by luciferase assay. Furthermore, whether NF-κB targeted its binding elements in the miR-21 gene promoter was determined by luciferase and ChIP assay. Finally, we measured the cell viability and apoptosis under cisplatin treatment when NF-κB was inhibited.ResultsAn elevated level of miR-21 was observed in NSCLC lung tissues and was related to a short survival time. Exogenous miR-21 promoted cell survival when exposed to cisplatin, while miR-21 inhibition could reverse this process. The RNA and protein levels of PTEN were significantly decreased by exogenous miR-21, and the 3′-untranslated region of PTEN was shown to be a target of miR-21. The expression of miR-21 was regulated by NF-κB binding to its element in the promoter, a finding that was verified by luciferase and ChIP assay. Hence, inhibition of NF-κB by RNA silencing protects cells against cisplatin via decreasing miR-21 expression.ConclusionModulation of the NF-κB/miR-21/PTEN pathway in NSCLC showed that inhibition of this pathway may increase cisplatin sensitivity.

Expressions of GRP78 and Bax associate with differentiation, metastasis, and apoptosis in non-small cell lung cancer

The purpose of this study was to detect the expressions of GRP78 and Bax in human non-small cell lung cancer (NSCLC) tissues, to analyze their correlations with carcinogenesis and the development of NSCLC, and to investigate the relationship of GRP78 expression to metastasis and apoptosis in the NSCLC cell line HCC827. The positive expression rates of GRP78 and Bax in NSCLC lung tissues were 59.7% and 34.7% by RT-PCR, respectively. The mRNA and protein expression levels of GRP78 in NSCLC tissues were significantly higher than that in the relatively normal surrounding lung tissues (p < 0.05); the lesser the degree of tumor differentiation was, the higher the mRNA and protein expression levels of GRP78 were (p < 0.05). The mRNA and protein expression levels of GRP78 from patients in advanced pathological stages (III-IV) were significantly higher than the corresponding levels in patients in early pathological stages (I-II) (p < 0.05); the mRNA and protein expression levels of GRP78 in patients with positive lymph node metastasis were significantly higher than those in patients with negative lymph node metastasis (p < 0.05). The mRNA and protein expression levels of Bax in the above cases showed the opposite trend of the mRNA and protein expression levels of GRP78. However, the mRNA and protein expression levels of both GRP78 and Bax were independent of the patient’s sex, the patient’s age, the tumor size and the histological type (adenocarcinoma or squamous cell carcinoma) of NSCLC (p > 0.05). The mRNA expression level of GRP78 and the mRNA expression level of Bax in human NSCLC tissues were negatively correlated (r = -0.353, p = 0.002). After transfection of GRP78 siRNA in HCC827 cells, the GRP78 protein expression level was significantly decreased (p < 0.01), while the Bax protein expression level was significantly increased (p < 0.01); the number of cells that passed through the Transwell chamber was significantly less in the non-transfected control group compared to the transfected control group (p < 0.01). The number of apoptotic cells was significantly greater in the non-transfected control group compared to the transfected control group (p < 0.01). The expression levels of GRP78 and Bax were related to the carcinogenesis, development and metastasis of NSCLC. GRP78 expression with siRNA interference in the human NSCLC cell line HCC827 can reduce metastasis and promote apoptosis in HCC827 cells.