NSCLC Progression Research Articles

Silencing of CD276 suppresses lung cancer progression by regulating integrin signaling.

BackgroundNon-small cell lung cancer (NSCLC) is one of the cancers with the highest morbidity and mortality among the world. Studies have shown that the invasion and metastasis of tumor are biological characteristics of lung cancer, and also the main cause of treatment failure and patient death. In-depth study of lung cancer invasion related genes will help to explore the etiology of lung cancer, molecular typing and individualized treatment of lung cancer. Studies have shown that CD276 molecules are closely related to the prognosis of tumors, but the exact mechanism remains to be unclear.MethodsWe used the UALCAN and KM-plotter databases to investigate the expression of CD276 in human NSCLC and adjacent normal tissues, and its correlation with clinicopathology. In addition, we analyzed the function of CD276 in NSCLC cell by suppressing the expression of CD276 in A549 and H460 cells.ResultsIn this study, we found that CD276 expression was significantly up-regulated in NSCLC tissues, and its expression was positively correlated with tumor stage in NSCLC. Silencing in CD276 inhibited cell invasion and migration by reducing integrin-associated protein expression.ConclusionsOur results indicate functional role of CD276 in the progression of NSCLC.

LncRNA KCNQ1OT1 promotes cell proliferation, migration and invasion via regulating miR-129-5p/JAG1 axis in non-small cell lung cancer

BackgroundNon-small cell lung cancer (NSCLC) is the most deadly cancer worldwide. LncRNA KCNQ1OT1 has been reported to be involved in the progression of various tumors, including NSCLC. However, the precise mechanism of KCNQ1OT1 in NSCLC requires further investigation.MethodsThe expression levels of KCNQ1OT1, miR-129-5p and JAG1 were detected by qRT-PCR or western blot. Kaplan–Meier survival analysis was used to assess the correlation between KCNQ1OT1 expression and the overall survival of NSCLC patients. CCK-8 assay was used to measure cell viability. Cell migration and invasion were detected by transwell assay. The targets of KCNQ1OT1 and miR-129-5p were predicted by bioinformatics, which was confirmed by dual-luciferase reporter assay or pull-down assay.ResultsKCNQ1OT1 expression was significantly enhanced, while miR-129-5p expression was dramatically reduced in NSCLC tissues and cells. Higher KCNQ1OT1 shortened overall survival and was positively associated with tumor stage and lymph node metastasis. KCNQ1OT1 knockdown inhibited proliferation, migration and invasion of NSCLC cells. Inhibition of miR-129-5p attenuated the inhibition of NSCLC cell viability, migration and invasion induced by KCNQ1OT1 knockdown. In addition, JAG1 was confirmed as a target of miR-129-5p. Knockdown of JAG1 reversed the effects of miR-129-5p knockdown on NSCLC progression. KCNQ1OT1 regulated JAG1 expression by sponging miR-129-5p in NSCLC cells.ConclusionKCNQ1OT1 induced proliferation, migration and invasion of NSCLC cells by sponging miR-129-5p and regulating JAG1 expression, indicating that KCNQ1OT1 was a therapeutic target for NSCLC.

LncRNA NEAT1 regulated cell proliferation, invasion, migration and apoptosis by targeting has-miR-376b-3p/SULF1 axis in non-small cell lung cancer.

Recently, lncRNA has been determined to play an important role in cancer formation and development. However, the regulatory mechanism of lncRNA in NSCLC has not been fully explored. The expression of NEAT1, miR-376b-3p, and SULF1 was detected in each group via quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The proteins expression of SULF1, p-MAPK, MAPK, p-Akt, Akt, and GAPDH were measured via Western blot. MTT assay was applied to detect cell proliferation in each group. Transwell assay was used to assess cell invasion and migration of each group. Cell apoptosis was assessed with flow cytometry. The relationship among NEAT1, miR-376b-3p, and SULF1 was determined using Luciferase reporter assay. In this study, the expression of NEAT1 and SULF1 was upregulated in NSCLC tissues and cells. Of note, the knockdown of NEAT1 and SULF1 could inhibit cell proliferation, migration, and invasion and promote cell apoptosis in NSCLC. Moreover, NEAT1 regulated SULF1 expression via binding to miR-376b-3p in NSCLC cells. Otherwise, the effects of NEAT1 on cell growth and apoptosis were reversed by improving the SULF1 expression in NSCLC cells. Meanwhile, si-NEAT1 transfection inhibited MAPK and Akt signaling pathway by modulating SULF1 in NSCLC cells. In this study, we found that lncRNA NEAT1 regulated cell proliferation, invasion, migration, and apoptosis by targeting has-miR-376b-3p/SULF1 axis in NSCLC. Moreover, the regulatory network of NEAT1 participated in the phosphorylation levels of MAPK and Akt to affect cell progression of NSCLC, providing a new regulatory pathway in the pathogenesis of lung cancer.

Silencing Sirtuin 6 induces cell cycle arrest and apoptosis in non-small cell lung cancer cell lines

Sirtuins (SIRT1–7), are NAD-dependent deacetylases and ADP-ribosyl transferases, plays a major part in carcinogenesis. The previous report suggests that in cancer, sirtuins gained tremendous interest and critical regulators of the unusual processes. In carcinogenesis, sirtuins possess either tumor suppressor or promoter. However, in lung cancer condition the studies of sirtuins are less studied. Hence, this designed study investigates the impact of multifaceted sirtuins in NSCLC cells. We evaluated the mRNA and protein expressions of sirtuins by RTPCR and western blot. We found SIRT6 significantly overexpressed in NCI-H520, A549, and NCI-H460 compared with the normal BEAS-2B cell line. Silencing of SIRT6 by siRNA in NSCLC cells caused activation of p53/p21 mediated inhibition of cell proliferation leading to arrest in cell cycle and apoptosis induction. Our results implied that SIRT6 is a tumor promoter in NSCLC development, progression, and regulation. The silencing of SIRT6 to be a novel therapy for lung cancer.

The lncRNA LINC01194/miR-486-5p Axis Facilitates Malignancy in Non-Small Cell Lung Cancer via Regulating CDK4.

BackgroundThis experimental design was based on lncRNA LINC01194 to explore the pathogenesis of NSCLC.MethodsRT-qPCR was used to detect the expression of lncRNA LINC01194 and miR-486-5p in NSCLC tissues and cell lines. CCK-8, colony formation, and transwell assays were used to examine the effects of lncRNA LINC01194 and miR-486-5p on NSCLC cell proliferation and migration invasiveness. For target gene prediction and screening, luciferase reporter assays were used to verify downstream target genes for lncRNA LINC01194 and miR-486-5p. The protein expression of CDK4 was detected using Western blotting. The tumor changes in mice were detected by in vivo experiments in nude mice.ResultsLncRNA LINC01194 was highly expressed in NSCLC tissues and NSCLC lines (A549, H1299, H460 cells, H1975), and lncRNA LINC01194 significantly promoted cell proliferation and migration of NSCLC cells. MiR-486-5p was identified as a potential target for LINC01194, and miR-486-5p was expressed at a low level in NSCLC tissues and NSCLC lines (A549, H1299, H460 cells, H1975). CDK4 was identified as a potential target for miR-486-5p. LncRNA LINC01194 was able to inhibit miR-486-5p expression and upregulate the expression level of CDK4. Finally, the results of in vivo animal models confirmed that lncRNA LINC01194 promoted NSCLC progression by modulating the miR-486-5p/CDK4 axis.ConclusionLncRNA LINC01194 promoted the progression of NSCLC by modulating the miR-486-5p/CDK4 axis.

Circ_0072083 interference enhances growth-inhibiting effects of cisplatin in non-small-cell lung cancer cells via miR-545-3p/CBLL1 axis

BackgroundNon-small-cell lung cancer (NSCLC) is one of the common cancers in the world. Circular RNA 0072083 (circ_0072083, circZFR) has been reported to be associated with the progression of NSCLC. In this study, we intended to explore the role and the potential mechanism of circ_0072083 in NSCLC.MethodsQuantitative real time polymerase chain reaction (qRT-PCR) was performed to detect the expression of circ_0072083, its matching linear RNA (zinc finger RNA binding protein (ZFR)) and microRNA-545-3p (miR-545-3p) in NSCLC cells. The ability of colony formation in NSCLC cells was detected by colony formation assay. The apoptosis and cell cycle were measured by flow cytometry. The metastasis was determined by transwell migration and invasion assays. The protein expression of E-cadherin, N-cadherin, Vimentin and Cbl proto-oncogene like 1 (CBLL1) was examined by western blot assay. The interaction between miR-545-3p and circ_0072083 or CBLL1 was predicted by starBase or Targetscan software. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were applied to validate these interactions. Nude mice bearing tumors were used to confirm the role of circ_0072083 and cisplatin (DDP) in vivo.ResultsThe level of circ_0072083 was higher in NSCLC tissues and cells relative to that in adjacent non-tumor tissues and normal lung cells. The transfection of si-circ_0072083 inhibited colony formation, cell cycle and metastasis while promoted the apoptosis of NSCLC cells stimulated by DDP. MiR-545-3p was a direct functional target of circ_0072083 in NSCLC cells. CBLL1 could bind to miR-545-3p in NSCLC cells. Circ_0072083 promoted the progression of NSCLC induced by DDP through sponging miR-545-3p and enhancing the enrichment of CBLL1 in vivo and in vitro.ConclusionCirc_0072083 depletion contributed to DDP-triggered inhibition of NSCLC tumor through miR-545-3p/CBLL1 axis.

PP4C facilitates lung cancer proliferation and inhibits apoptosis via activating MAPK/ERK pathway

PurposeProtein phosphatase 4 catalytic subunit (PP4C) has been shown to play crucial regulatory roles in biological process and is frequently upregulated in cancer such as breast and colorectal carcinoma. However, the function and potential molecular mechanism of PP4C in lung cancer remains unclear. MethodsBioinformatic analysis was used to detect the expression level and prognosis of patients. Western blot, quantitative real-time PCR (qRT-PCR), CCK8, 5-Ethynyl-2′-deoxyuridine (Edu) proliferation assay and flow cytometric were used to explore the function in lung cancer cells. ResultsIn this study, we found that PP4C was upregulated in lung cancer tissues as compared with that in normal lung tissues. Furthermore, patients with high expression level of PP4C were correlated with a poor prognosis in lung cancer patients. In vitro, CCK8, Edu proliferation assays and flow cytometry analysis showed that PP4C could promote lung cancer cell growth and inhibit apoptosis. Mechanistic investigations revealed that PP4C may interact with PP4R1 and promote ERK activation. Additionally, PP4C depletion resulted in lower tumor growth in vivo. ConclusionsTaken together, these data showed the oncogenic of PP4C in NSCLC tumorigenesis and provide a new insight of PP4C in the progression of NSCLC.

Down-regulation of MBNL1-AS1 contributes to tumorigenesis of NSCLC via sponging miR-135a-5p

Lung cancer remains a big threat to human health. Growing evidence has reported the crucial regulatory effect of lncRNAs on NSCLC progression. Nevertheless, the detailed function of lncRNA MBNL1-AS1 involved in NSCLC development is poorly known. In our research, we confirmed that MBNL1-AS1 was significantly reduced in NSCLC patient tissues and NSCLC cells. Meanwhile, we reported that overexpression of MBNL1-AS1 obviously repressed A549 and H1975 cell proliferation, blocked cell cycle and inhibited the migration and invasion. Moreover, A549 and H1975 cell apoptosis was increased by the overexpression of MBNL1-AS1. Then, we predicted that miR-135a-5p was a potential target of MBNL1-AS1 and its level was correlated with MBNL1-AS1 in NSCLC negatively. Our previous study indicated miR-135a-5p could induce lung cancer progression through regulating LOXL4. Here, we found that MBNL1-AS1 was able to regulate miR-135a-5p expression negatively. The direct binding association between MBNL1-AS1 and miR-135a-5p was proved using dual-luciferase reporter assay and RIP experiment. Subcutaneous xenotransplanted tumor model was set up and it was confirmed increased MBNL1-AS1 remarkably restrained tumorigenic ability of NSCLC through sponging miR-135a-5p in vivo. To sum up, our data revealed the significance of the MBNL1-AS1 and miR-135a-5p in NSCLC. In conclusion, MBNL1-AS1 could be a new therapeutic target to treat NSCLC.

PREDICTIVE VALUE OF THE DYNAMIC DETERMINATION OF CIRCULATING TUMOR DNA ON PFS IN PATIENTS WITH EGFR MUTATED NSCLC, WITH OSIMERTINIB THERAPY

Aim: study of the predictive value of determining ctDNA during treatment with osimertinib in patients with NSCLC with EGFR mutation. Methods: The study included patients with metastatic EG-FR-associated NSCLC, in whom, with progression against the background of 1st - 2nd generation TKIs, the T790M mutation was detected. Patients received osimertinib therapy 80 mg/ day, daily, until progression. Before treatment, and then every 2 months, whole blood was taken to conduct a qualitative assessment of ctDNA in dynamics by the RT-PCR method. Results: From 2016 to 2019 in St. Petersburg Clinical Scientific and Practical Center of Specialized Types of Medical Care (Oncology), 22 patients were identified T790M associated progression of EGFR NSCLC. 81.9% (18/22) are women, 18.1% (4/22) are men. The average age is 61.2 years (50-75). 1/22 had smoking experience for more than 30 years. The molecular genetic profile in 16 is represented by ex19del, 5 L858R, 1 -a combination of rare mutations G719S+S768I. The effect of therapy was evaluated in 20/22 patients. PR and SD were registered in 9/20 (45%) and 10/20 (50%) patients, respectively. Median PFS - 16.7 months (cI 95%, 11,4-22,0). In 12/22 patients was observed the disappearance of ctDNA T790M after 2 months of osimertinib therapy. PFS is 18,9 months (95% CI, 14,8-19,7), in patients with no mutation detected in the second month of treatment compared with the group of patients in which the ctDNA was determined (PFS 8.0 months) (CI 95%, 4,2-11,8) (p=0.015). Correlation analysis did not reveal any clinical factors associated with the disappearance of ctDNA. Conclusions: The disappearance of ctDNA in plasma after 2 months of treatment with osimertinib is associated with an increase in PFS and can be considered as a predictive marker in patients with metastatic NSCLC EGFR T790M.

ZNF300 Interacting with E2F3 Promoted the Malignant Progression of NSCLC by Regulating Proliferation, Differentiation, and Cell Cycle

Background: Chemo-resistance induced by cisplatin (DDP) has become the major impediment to lung cancer chemotherapy. The study aimed to unearth an alternative route for reversing chemo-resistance and suppressing the malignant progression of tumors. Methods: Gene expression profile was integrated with DNA methylation profile of A549/DDP cells of human adenocarcinoma. Immunohistochemistry were used to analyze the association of gene with characteristics of NSCLC patients. Cell viability, apoptosis, cell cycle, migration, engulfment, and senescence were assessed between the different groups of cells. Genes related with differentiation and cell cycle, screened by microarrays, were examined by immunoblotting. Xenograft model was utilized to validate the results in vitro. Findings: ZNF300 was association with poor overall survival of NSCLC patients. Multidrug-resistant cells exhibited a more malignant behavior related to chemo-sensitive cells. Most of genes related with differentiation, cyclin-dependent kinase inhibitor, and MAPK/ERK pathways were downregulated, while most of genes related with cell cycle and cancer stem cell were upregulated in multidrug-resistant cells compared to chemo-sensitive cells. ICA improved the antitumor effect of cisplatin on multidrug-resistant cells. Interpretation: ZNF300 promoted the malignant behavior of multidrug-resistant cells through inhibiting differentiation via supressing MAPK/ERK pathways and enhancing cancer stem cell-like properties via upregulating Nanog and OCT4. Additionally, CDK1 activated by ZNF300 via inhibiting WEE1 and MYT1 and modulating AURKA/BORA/PLK1/CDC25 cascade promoted cells to enter cell cycle and escape the apoptosis caused by chemotherapeutic drug. ICA improved the antitumor effect of cisplatin on multidrug resistant cells by inducing differentiation. Funding Statement: This work was supported by the National Natural Scientific Fund of China (№:81372499), the Cultivating Project for Young Scholar at Hubei University of Medicine ( № :2018QDJZR01 and 2018QDJZR11), and the Open Project of Hubei Key Laboratory of Wudang Local Chinese Medicine Research (Hubei University of Medicine) (№:WDCM2018002). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The informed consents were obtained from all the patients undertaking surgery and participating in this study. All animal experiments were authorized by the Experimental Animal Ethics Committee of Xinqiao Hospital of the Army Medical University [SYXK(YU)2012-0011] and Hubei University of Medicine [SYXK(E)2016-0031].

LncRNA SNHG16 promotes non‐small cell lung cancer development through regulating EphA2 expression by sponging miR‐520a‐3p

BackgroundRecent evidence has found that lncRNA small nucleolar RNA host gene 16 (SNHG16) was associated with cell carcinogenesis in NSCLC. Here, we further investigated the precise functions and mechanisms of SNHG16 in NSCLC progression.MethodsThe expression of SNHG16, microRNA (miR)‐520a‐3p and EPH Receptor A2 (EphA2) was measured using quantitative real‐time polymerase chain reaction and western blot, respectively. Cell proliferation was determined using 3‐(4, 5)‐dimethylthiahiazo (−z‐y1)‐3, 5‐di‐phenytetrazoliumromide (MTT) assay. The migrated and invaded cells were measured by Transwell assay. Flow cytometry was used to detect apoptotic cells. The interaction between miR‐520a‐3p and SNHG16 or EphA2 was confirmed using a dual‐luciferase reporter assay.ResultsWe found that SNHG16 was upregulated in NSCLC tissues and cell lines, knockdown of SNHG16 inhibited cell proliferation, migration, invasion and induced apoptosis in vitro as well as suppressed tumor growth in vivo. MiR‐520a‐3p directly bound to SNHG16 and miR‐520a‐3p, and SNHG16 acted as a ceRNA in regulating EphA2 through competitively binding to miR‐520a‐3p. Additionally, rescue assay exhibited the anticancer activity mediated by SNHG16 knockdown on NSCLC could be reversed by miR‐520a‐3p inhibition or EphA2 overexpression.ConclusionSNHG16 promoted NSCLC development by regulating the miR‐520a‐3p/EphA2 axis, suggesting novel insights for the pathogenesis of NSCLC and new potential therapeutic targets for the treatment of NSCLC.Key points Knockdown of SNHG16 inhibited NSCLC cell proliferation, migration, invasion and induced apoptosis in vitro as well as suppressed tumor growth in vivo. SNHG16 directly interacted with miR‐520a‐3p.EphA2 was a target of miR‐520a‐3p.SNHG16 could regulate the expression of EphA2 by binding to miR‐520a‐3p.SNHG16 promoted NSCLC development by regulating the miR‐520a‐3p/EphA2 axis.

Non-Small Cell Lung Cancer Downregulates miR-495 and miR-5688 Under Hypoxia to Maintain Interleukin-11 Expression

Background: Hypoxia is one of the hallmarks of cancer and is associated with poor prognosis. However, the molecular mechanism by which hypoxia promotes tumor progression remains unclear. MicroRNAs participate in almost all biological processes through posttranscriptional regulation of the expression of multiple genes. In thecurrent study, we found that two miRNAs, miR-495 and miR-5688, participate in NSCLC progression under hypoxia. Methods: The expression of miR-495 and miR-5688 in human tissue specimens was measured by quantitative RT-PCR (qRT-PCR). Colony formation, MTS, wound healing, and transwell assays and a xenograft model were used to evaluate the role of miR-495 and miR-5688 in tumor progression. Two algorithms, PicTar and TargetScan, were used to predict miRNA targets, and a dual-luciferase reporter assay was used to confirm the target. Findings: MiR-495 and miR-5688 were downregulated in NSCLC tissues compared with normal tissues. We determinedthat hypoxia led to the downregulation of miR-495 and miR-5688 in NSCLC cells. In addition, miR-495 and miR-5688 suppressed cell proliferation, migration and invasion in vitro. The NSCLC xenograft model showed that miR-495 and miR-5688 inhibited tumor formation in vivo. Interestingly, we found that miR-495 and miR-5688 had the same target, IL-11. Furthermore, a series of rescue experiments demonstrated that miR-495 and miR-5688 executed their tumor suppressive role by repressing IL-11 expression. Interpretation: We identified two new players, miR-495 and miR-5688, that participate in NSCLC progression under hypoxia. Mechanistically, hypoxia downregulates miR-495 and miR-5688 in NSCLC to enhance IL-11 expression and tumor progression. Our study indicates that the miR-495/miR-5688/IL-11 axis may serve as a therapeutic target and potential biomarker for NSCLC. Funding Statement: This work was supported by the National Natural Science Foundation of China (Grant No. 81602026) and the Natural Science Foundation of Tianjin (Grant No. 18JCQNJC81600 and 18JCZDJC32600). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: All samples were obtained with informed consent, and the study was approved by the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital. All mouse studies were approved by the Animal Ethics Committee of Tianjin Medical University.

Hsa_circ_0002483 inhibited the progression and enhanced the Taxol sensitivity of non-small cell lung cancer by targeting miR-182-5p

In this study, we identified a novel circRNA, circ_0002483, and further investigated its functions in the progression and Taxol resistance of NSCLC. We found that circ_0002483 was expressed at low levels in NSCLC tissues and cell lines. Functional assays indicated that circ_0002483 overexpression significantly inhibited NSCLC cell proliferation and invasion in vitro and in vivo and enhanced the sensitivity of NSCLC cells to Taxol. Mechanistically, circ_0002483 was identified to sponge multiple miRNAs including miR-182-5p (also named miR-182), miR-520q-3p, miR-582-3p, miR-587, and miR-655. In addition, circ_0002483 was also demonstrated to regulate the expression of GRB2, FOXO1, and FOXO3, three target genes of miR-182-5p, by sponging miR-182-5p. Circ_0002483 was demonstrated to inhibit NSCLC progression in vitro and in vivo and enhanced the sensitivity of NSCLC cells to Taxol by sponging miR-182-5p to release the inhibition on GRB2, FOXO1, and FOXO3 mRNAs.

LncRNA ZEB2-AS1 Aggravates Progression of Non-Small Cell Lung Carcinoma via Suppressing PTEN Level.

BackgroundThe aim of this study was to assess the involvement of lncRNA ZEB2-AS1 in the development of NSCLC and to explore the potential mechanism involved.Material/MethodsZEB2-AS1 expressions in 48 paired NSCLC tissues and paracancerous tissues were examined by qRT-PCR. ZEB2-AS1 level in NSCLC patients affected by tumor staging and lymphatic metastasis was examined as well. Regulatory effects of ZEB2-AS1 on proliferative, migratory, and invasive properties of NCI-H1650 and HCC827 cells were evaluated. The interaction between ZEB2-AS1 and EZH2 was identified through RIP assay. Subsequently, the binding of EZH2 on PTEN promoter region was tested by ChIP. Finally, rescue experiments were conducted to assess the involvement of PTEN in the development of NSCLC.ResultsZEB2-AS1 was upregulated in NSCLC tissues and cell lines. Its level was higher in NSCLC patients with T3–T4 or accompanied with lymphatic metastasis relative to those with T1–T2 or without metastatic loci. Knockdown of ZEB2-AS1 suppressed proliferative, migratory, and invasive properties of NCI-H1650 and HCC827 cells. PTEN level was elevated after knockdown of ZEB2-AS1 or EZH2 in HCC827 cells. Subsequently, RIP assay proved the interaction between ZEB2-AS1 and EZH2. Knockdown of ZEB2-AS1 markedly reduced the binding of EZH2 on the PTEN promoter region. Notably, knockdown of PTEN reversed the effects of EZB2-AS1 on regulating proliferative, migratory, and invasive properties of NSCLC cells.ConclusionslncRNA ZEB2-AS1 is upregulated in NSCLC, which elevates the viability and malignant degree of NSCLC cells by downregulating PTEN, thus aggravating the progression of NSCLC.

Targeting ZEB2 By microRNA-129 In Non-Small Cell Lung Cancer Suppresses Cell Proliferation, Invasion And Migration Via Regulating Wnt/β-Catenin Signaling Pathway And Epithelial-Mesenchymal Transition.

IntroductionNon-small cell lung cancer (NSCLC) is a common cause of deaths all over the world. Emerging evidence has indicated that microRNA (miR) play key roles in NSCLC progression. We aimed to determine the functions of miR-129 in NSCLC. miR-129 was dramatically downregulated in NSCLC tissue samples and cells. The decreased miR-129 was found to be associated with poorer prognosis and malefic phenotype of NSCLC patients. We demonstrated that miR-129 upregulation could inhibit NSCLC cell growth. Furthermore, we also sought the molecular mechanism by which miR-129 repressed NSCLC development.MethodsQRT-PCR was applied to detect the expressions of miR-129 in 51 pairs of NSCLC tissue samples. We further performed the Kaplan–Meier analysis to determine the association between miR-129 expressions and the survival rate of NSCLC patients. We then measured the expression levels of miR-129 in NSCLC cell lines. After that, MTT assays were performed to determine the influence of miR-129 on A549 cell proliferation. Transwell assay was then conducted to explore the biological functions of miR-129 in invasion and migration of NSCLC cells.ResultsResults showed that ZEB2 was directly targeted by miR-129 in NSCLC cell lines. Moreover, miR-129 restoration could inhibit EMT and Wnt/β-catenin in NSCLC cell lines.ConclusionIn short, all these results indicated that miR-129/ZEB2 axis maybe a useful diagnostic and prognostic biomarker for NSCLC treatment.

Inhibiting Pathways Predicted From a Steroid Hormone Gene Signature Yields Synergistic Antitumor Effects in NSCLC.

Mounting evidence supports a role for estrogen signaling in NSCLC progression. We previously reported a seven-gene signature that predicts prognosis in estrogen receptor β positive (ERβ+) NSCLC. The signature defines a network comprised of ER and human EGFR-2/3 (HER2/HER3) signaling. We tested the efficacy of combining the pan-HER inhibitor, dacomitinib, with the estrogen antagonist, fulvestrant, in ERβ+ NSCLC models with differing genotypes. We assessed the potency of this combination on xenograft growth and survival of host mice, and the ability to reverse the gene signature associated with poor outcome. Synergy was observed between dacomitinib and fulvestrant in three human ERβ+ NSCLC models: 201T (wild-type EGFR), A549 (KRAS mutant), and HCC827 (EGFR 19 deletion) with combination indices of 0.1-0.6. The combination, but not single agents, completely reversed the gene signature associated with poor prognosis in a mechanism that is largely mediated by activator protein 1 downregulation. Invivo, the combination also induced tumor regression and reversed the gene signature. In HCC827 xenografts treated with the combination, survival of mice was prolonged after therapy discontinuation, tumors that recurred were less aggressive, and two mechanisms of HER inhibitor resistance involving c-Met activation and PTEN loss were blocked. The combination of an ER blocker and a pan-HER inhibitor provides synergistic efficacy in different models of ERβ+ NSCLC. Our data support the use of this combination clinically, considering its ability to induce potent antitumor effects and produce a gene signature that predicts better clinical outcomes.

Statistical Analysis of Long-Chain Non-Coding RNA SNHG1 Influencing the Proliferation of Non-Small Cell Lung Cancer A549 Cells by Competitive Binding to microRNA-101

Objective: To explore the effect of LncRNA SNHG1 on the proliferation of non-small cell lung cancer A549 cells by competitive binding to miR-101 using bioinformatics analysis. Methods: We used quantitative real-time PCR analyzed the differential expression of SNHG1 in non-small cell lung to analyze cancer tissues, adjacent nontumorous tissues, non-small cell lung cancer cell lines (A549, SPC-A1) and normal human lung epithelial cells (BEAS-2B). Transient transfected A549 cells with siRNA SNHG1 plasmid and detected the proliferation and apoptosis of cells in vitro or in vivo by MTT assay, apoptosis test and model tumor of athymic mouse. SNHG1 plasmid vector containing miR-101 predicted binding site to investigate whether SNHG1 was a functional target of miR-101 by luciferase reporter assay. The interaction between SNHG1 and miR-101 on the proliferation of A549 cells was investigated by transfection of miR-101-ih and then monitored the expression of SNHG1. Results: We found that expression of SNHG1 was up-regulated in NSCLC tissues and A549 cell lines. Furthermore, function assays showed that SNHG1 inhibition suppressed NSCLC cell proliferation both in vitro and outside vivo, and accelerated the apoptosis in vitro. We also found that the luciferase activity was significantly decreased by the co-transfection of miR-101-mim and SNHG1-Wt. In addition, we found that inhibition of SNHG1 led to significantly increased expression of miR-101. The expression of SNHG1 and the proliferation of A549 cells in cells that transfected with miR-101-ih was significantly higher than control group. Conclusions: LncRNA SNHG1 had a direct interaction with miR-101, and miR-101 was an inhibitory target for SNHG1 in NSCLC progression. The inhibition of miR-101 could enhanced the expression of SNHG1 and promoted the proliferation of A549 cells. This can be verified using bioinformatics analysis.

An assessment of the relationship between the expression of CCR7/CCL19 axis and selected regulatory miRNAs in non-small cell lung cancer

CC chemokine receptor type 7 (CCR7) and its ligands has been implicated in the occurrence and progression of NSCLC. Previous studies have revealed that the diagnostic value of CCR7/CCL19 axis in lung tumorigenesis remains controversial. The present study evaluates the relationship between the mRNA expression of CCR7/CCL19 axis and selected regulatory miRNAs in NSCLC patients. It analyzes the expression level of CCR7 mRNA and its ligand in tumor tissue in relation to expression level of two miRNAs: miR let-7a and miR-335, as transcriptional regulators of study genes. Twenty-seven patients (n = 27) were enrolled. The expression of the studied genes and miRNAs was evaluated by qPCR. Tumour tissue fragments, adjacent macroscopically-unchanged lung tissue (control) and patient serum were used as biological material for study. Elevated expression of CCR7 and CCL19 mRNA was observed in patients with metastasis to lymph nodes. We noticed upregulated miR-335 expression and downregulated miR let-7a expression in patient serum with regard to AJCC tumor staging. Higher miR-335 expression and lower miR let-7a expression level was observed in patients with metastasis to lymph node. The presence of changes observed in the expression level of miR-335 and miR let-7a in the serum of NSCLC patients in relation to lymph node metastases and tumor stage may serve as a non-invasive molecular biomarker of tumor progression; however, this observation requires further investigation.

Synaptotagmin 7 in twist-related protein 1-mediated epithelial – Mesenchymal transition of non-small cell lung cancer

BackgroundTwist-related protein 1 (TWIST1) plays an essential role in the carcinogenesis and metastasis of NSCLC. Our aims were to identify the molecule at the downstream of TWIST1 and to evaluate its potential as a diagnostic and a prognostic marker in NSCLC. MethodsThe functional genes at the downstream of TWIST1 were obtained via microarray gene expression analyses in the NSCLC cell line. The expression levels of synaptotagmin 7 (SYT7) in a cohort of patients with NSCLC (n = 154) were examined using immunohistochemistry staining and assessed by Kaplan-Meier survival analysis and Cox regression analysis. The effects of SYT7 on the tumorigenesis and metastasis of NSCLC were measured in NSCLC cells. In vivo xenograft lung cancer models were used to study the tumorigenesis role of SYT7. FindingsWe discovered that SYT7 is significantly altered by TWIST1 expression. We further confirmed that SYT7 protein was significantly higher in NSCLC than that in the adjacent normal lung tissue, and higher SYT7 expression was associated with poor survival of NSCLC patients. The protein level of SYT7 was positively correlated with TWIST1 in NSCLC tissue. Functional experiments indicated that SYT7 promoted proliferation, invasion, and metastasis and inhibited cell apoptosis of NSCLC cells in vitro. In vivo experiments showed that shSYT7 inhibited the xenograft tumor growth of NSCLC cells. Knocking down of SYT7 increased the expression of E-cadherin and decreased the level of N-cadherin and Vimentin in cultured cells. InterpretationOur data indicate that SYT7 is an important promoter for EMT and tumor progression in NSCLC. FundThis project was supported by grants from the Major Scientific and Technological Innovation Project of Shandong Province (2018CXGC1212), Science and Technology Foundation of Shandong Province (2014GSF118084, 2016GSF121043), Medical and Health Technology Innovation Plan of Jinan City (201805002) and the National Natural Science Foundation of China (81372333).

Abstract CT034: Osimertinib plus selumetinib for patients (pts) with EGFR-mutant (EGFRm) NSCLC following disease progression on an EGFR-TKI: Results from the Phase Ib TATTON study

Abstract The EGFR T790M mutation is the most common cause of resistance in pts receiving a first or second-generation EGFR-TKI for EGFRm NSCLC. Other resistance mechanisms include upregulation of the RAS/RAF/MEK/ERK signaling pathway. The Phase Ib TATTON study (NCT02143466) assessed osimertinib in combination with novel therapeutics including the oral, potent and selective MEK1/2 inhibitor, selumetinib (AZD6244, ARRY-142886). Here we present data from the dose-finding (Part A) and dose expansion (Part B) portions of this study. Adults with advanced EGFRm NSCLC and disease progression on prior EGFR-TKI, including third-generation agents, were eligible regardless of T790M or KRAS status. In Part A, pts received osimertinib 80 mg QD plus intermittent or continuous selumetinib. Asian pts received continuous selumetinib 25/50 mg BID, while other pts received continuous selumetinib 50/75 mg BID, or intermittent selumetinib 75 mg BID 4 days on/3 days off (4/3), or on days 1 and 4 of each week of treatment. In Part B, pts received osimertinib plus intermittent selumetinib 75 mg BID 4/3. Primary objectives were safety, tolerability and preliminary efficacy (objective response rate [ORR]). Secondary objectives included duration of response (DoR) and pharmacokinetics (PK). At data cut-off (Feb 2018), 36 and 47 pts received treatment in Parts A and B, respectively. In Part A, most pts were white (18, 50%) or Asian (17, 47%), and 26 (72%) had a baseline exon 19 deletion. In Part B, 44 (94%) pts were Asian, and 30 (64%) had a baseline exon 19 deletion. The most common treatment-related adverse events (TRAEs) in Part A were diarrhea (27, 75%), nausea (14, 39%) and fatigue (12, 33%). Six pts had dose-limiting toxicities (DLTs) with continuous selumetinib (all Grade 3): ALT and AST increase (50 mg); diarrhea, asthenia and dizziness (50 mg); diarrhea (n=2, 75 mg); diarrhea and nausea (75 mg); and pneumonitis (75 mg). No DLTs were reported with intermittent dosing, so the selumetinib 4/3 schedule was selected for Part B. The most common TRAEs in Part B were diarrhea (38, 81%), stomatitis (15, 32%), and paronychia (14, 30%). In Part A, 15 (42%) pts had confirmed partial response (PR); 14 (39%) had stable disease at 6 weeks (SD); 3 (8%) had progressive disease (PD); 2 (6%) died; and 2 (6%) were not evaluable (NE). Median DoR was 16.6 months; 77% remained in response at 12 months. In Part B, 16 (34%) pts had confirmed PR; 16 (34%) had SD; 11 (23%) had PD; 2 (4%) died; and 2 (4%) were NE. Median DoR was 9.1 months; 31% remained in response at 12 months. Osimertinib and selumetinib PK parameters were similar to those previously observed with monotherapy. Osimertinib plus intermittent selumetinib had an acceptable safety profile and demonstrated preliminary anti-tumor activity in pts with disease progression on a prior EGFR-TKI. Responses were durable and this combination warrants further investigation. Citation Format: Suresh S. Ramalingam, Hideo Saka, Myung-Ju Ahn, Helena Yu, Helena Yu, Leora Horn, Toyoaki Hida, Mireille Cantarini, Remy Verheijen, Jonathan Wessen, Geoffrey Oxnard, Yuichiro Ohe. Osimertinib plus selumetinib for patients (pts) with EGFR-mutant (EGFRm) NSCLC following disease progression on an EGFR-TKI: Results from the Phase Ib TATTON study [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 CT034.