Suppressed Non-small Cell Lung Cancer Cell Research Articles

Knockdown of circPRKCA Restrained Cell Growth, Migration, and Invasion of NSCLC Cells Both in vitro and in vivo via Regulating miR-330-5p/PDK1/AKT Pathway.

BackgroundProtein kinase Cα (PRKCA) is an oncogene in multiple cancers including non-small-cell lung cancer (NSCLC) and can be transcribed into a number of circular PRKCAs (circPRKCAs). Here, we aimed to elaborate the role and mechanism of circPRKCA_024 (circPRKCA) in malignant progression of NSCLC.MethodsExpression of circPRKCA, miRNA (miR)-330-5p and 3-phosphoinositide-dependent protein kinase-1 (PDK1) was measured by real-time quantitative PCR and Western blotting, and their relationship was testified by dual-luciferase reporter assay, RNA immunoprecipitation, and RNA pull-down assay. Cell behaviors were evaluated by cell counting kit (CCK)-8, flow cytometry, and transwell assays. AKT activity was confirmed by Western blotting. Xenograft experiment assessed tumor growth.ResultsExpression of circPRKCA and PDK1 was upregulated, and miR-330-5p was downregulated in NSCLC tissues and cell lines. High circPRKCA was correlated with TNM stage and lymph node metastasis of NSCLC patients. Silencing circPRKCA could suppress cell viability, migration, and invasion in A549 and H1299 cells, accompanied with apoptosis rate promotion. Moreover, circPRKCA knockdown retarded tumor growth of A549 cells in vivo. Molecularly, miR-330-5p was sponged by circPRKCA, and PDK1 was a target of miR-330-5p. Inhibiting miR-330-5p could attenuate the suppression of circPRKCA knockdown on cell growth, migration, and invasion; contrarily, promoting miR-330-5p caused inhibition on those cell behaviors by downregulating PDK1. Analogously, AKT activity was suppressed by circPRKCA downregulation and miR-330-5p upregulation in NSCLC cells both in vitro and in vivo.ConclusionDepleting circPRKCA inhibited PDK1 to suppress NSCLC cell malignant behaviors through miR-330-5p/PDK1/AKT pathway.

Oxygen-Enhanced Optoacoustic Tomography Reveals the Effectiveness of Targeting Heme and Oxidative Phosphorylation at Normalizing Tumor Vascular Oxygenation.

Multispectral optoacoustic tomography (MSOT) is an emerging noninvasive imaging modality that can detect real-time dynamic information about the tumor microenvironment in humans and animals. Oxygen enhanced (OE)-MSOT can monitor tumor vasculature and oxygenation during disease development or therapy. Here, we used MSOT and OE-MSOT to examine in mice the response of human non-small cell lung cancer (NSCLC) xenografts to a new class of antitumor drugs, heme-targeting agents heme-sequestering peptide 2 (HSP2) and cyclopamine tartrate (CycT). HSP2 inhibits heme uptake, while CycT inhibits heme synthesis in NSCLC cells, where heme is essential for ATP generation via oxidative phosphorylation. HSP2 and CycT can inhibit ATP generation and thereby suppress NSCLC cell tumorigenic functions. MSOT showed that treatment of NSCLC tumors with HSP2 or CycT reduced total hemoglobin, increased oxygen saturation, and enhanced the amplitude of response to oxygen gas breathing challenge. HSP2 and CycT normalized tumor vasculature and improved tumor oxygenation, where levels of several hypoxia markers in NSCLC tumors were reduced by treatment with HSP2 or CycT. Furthermore, treatment with HSP2 or CycT reduced levels of angiogenic factor VEGFA, its receptor VEGFR1, and vascular marker CD34. Together, our data show that heme-targeting drugs HSP2 and CycT elicit multiple tumor-suppressing functions, such as inhibiting angiogenic function, normalizing tumor vasculature, alleviating tumor hypoxia, and inhibiting oxygen consumption and ATP generation. SIGNIFICANCE: Heme-targeting agents HSP2 and CycT effectively normalize tumor vasculature and alleviate tumor hypoxia, raising the possibility of their combination with chemo-, radio-, and immunotherapies to improve antitumor efficacy.See related commentary by Tomaszewski, p. 3461.

Long noncoding RNA Linc00210 promotes non-small cell lung cancer progression via sponging miR-16-5p/PTK2 axis.

Long noncoding RNAs (lncRNAs) are important regulators involved in a variety of cancer development. However, the role of Linc00210 in non-small cell lung cancer (NSCLC) remains unknown. This study aims to investigate the clinical value of Linc00210 in NSCLC patients and the biological functions of Linc00210 in NSCLC. Gene expression in NSCLC tissues and cell lines was detected using qRT-PCR or Western blot. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and colony formation assays were conducted to evaluate the effect of Linc00210 on NSCLC cell proliferation. Transwell assay and annexin V-Fluorescein 5-isothiocyanate (FITC)/Propidium Iodide (PI) were done to analyze the effect of Linc00210 on cancer cell invasion and apoptosis, respectively. Luciferase reporter assay and RIP assay were performed to determine the target of Linc00210 and miR-16-5p. Besides, these assays were used to determine reciprocally inhibition of each other-controlled NSCLC cell behaviors. In vivo tumorigenesis experiments were applied to exhibit subcutaneous tumor growth. Linc0021 was highly expressed in NSCLC tissues and cell lines. Knockdown of Linc00210 inhibited cancer cell proliferation and invasion, and increased cell apoptosis, and regulated the expression of Cyclin A1, proliferating cell nuclear antigen (PCNA), E-cadherin, N-cadherin, Bax, and Bcl-2 in NSCLC cells. Further data showed Linc00210 bound to and directly modulated the miR-16-5p levels. Impressively, overexpression of miR-16-5p suppressed NSCLC cell proliferation and invasion, but increased cell apoptosis, and these behaviors could be overturned by overexpression of Linc00210 in vitro and in vivo. Finally, Linc00210 and miR-16-5p cooperatively controlled expression of protein tyrosine kinase 2 (PTK2), a miR-16-5p target. Linc00210/miR-16-5p/PTK2 signaling suggests a promising novel strategy for anti-NSCLC therapy.

LncRNA KTN1-AS1 promotes the progression of non-small cell lung cancer via sponging of miR-130a-5p and activation of PDPK1.

Non-small cell lung cancer (NSCLC) is the major cause of cancer-associated death worldwide, but its underlying mechanisms remain to be fully elucidated. Long noncoding RNAs (lncRNAs) are known to play an important role in the aberrant regulation of gene expression in many cancers, including NSCLC. Here, we investigated the involvement of the lncRNA KTN1-AS1 in NSCLC. We found that KTN1-AS1 expression was upregulated in NSCLC tissue and was positively associated with poor prognosis. KTN1-AS1 knockdown inhibited cell growth and proliferation, increased apoptosis, and modulated the expression of cell cycle- and apoptosis-related proteins (cyclin A1, cyclin-dependent kinase 2, Bcl2, and Bax) in NSCLC cell lines and tumour xenografts in nude mice. KTN1-AS1 bound to and directly regulated the expression of miR-130a-5p. Notably, miR-130a-5p overexpression suppressed NSCLC cell proliferation and increased apoptosis in vitro and in vivo, and this effect was reversed by KTN1-AS1 overexpression. Finally, we showed that KTN1-AS1 modulated the expression of 3-phosphoinositide-dependent protein kinase 1 (PDPK1), a miR-130a-5p target and key regulator of autophagy in NSCLC cells. Taken together, our results suggest that the KTN1-AS1/miR-130a-5p/PDPK1 pathway may be a potential therapeutic target for NSCLC.

Abstract 1443: Inhibiting HMG-CoA reductase suppresses non-small cell lung carcinogenesis

Abstract 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGCR) is a rate-limiting enzyme in the mevalonate pathway and is associated with the development of several tumor types. However, the role of HMGCR in non-small cell lung cancer (NSCLC) is still unknown. In the present study, we found that HMGCR is overexpressed in human lung adenocarcinoma tissues compared with normal tissues. Knockdown of HMGCR in NSCLC cells attenuated growth and induced apoptosis in vitro and in vivo. Furthermore, we found that fluvastatin, an inhibitor of HMGCR, suppressed NSCLC cell growth and induced apoptosis. Intriguingly, fluvastastin functions by inhibiting the HMGCR-driven Braf/MEK/ERK1/2 and Akt signaling pathways. Notably, fluvastatin attenuated tumor growth in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis and in a patient-derived xenograft (PDX) lung tumor model. Overall, our findings suggest that fluvastatin might be promising chemopreventive or potential therapeutic drug against NSCLC tumorigenesis, providing hope for rapid clinical translation. Citation Format: Qiushi Wang, Tianshun Zhang, Ruihua Bai, Keke Wang, Xiang Li, Kangdong Liu, Ting Wang, Xiaoyu Chang, Weiya Ma, Ann Bode, Qingxin Xia, Yongping Song, Zigang Dong. Inhibiting HMG-CoA reductase suppresses non-small cell lung carcinogenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1443.

Abstract 4155: ARID1A loss leads to chemotherapy resistance and is associated with a poor clinic outcome for patients with non-small cell lung cancer

Abstract Purpose: ARID1A (AT-rich interactive domain 1A) is a member of the switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex, and plays important roles in the regulation of gene transcription and DNA damage responses. ARID1A mutations occur frequently in non-small cell lung cancer (NSCLC), but the functions of ARID1A loss in the initiation, progression and maintenance of NSCLC remains largely unknown. This study aimed to investigate the clinical, pathological, and functional significance of ARID1A in NSCLC. Experimental design: We first analyzed the prognostic value of ARID1A using a gene-expression microarray from the Director's Challenge Lung Study. Kaplan-Meier method and Log-rank tests were used to estimate the correlation between ARID1A expression and overall survival (OS). Univariate and multivariate Cox regression models were used to investigate the relationship between ARID1A and other clinical covariates. We further established stable ARID1A knockdown H1299 and H358 cell lines, and ARID1A exogenously expressing H460 and A549 cell lines. We performed functional assays to study the effects of ARID1A on proliferation, migration, invasion, cell cycle progression, sensitivity to chemotherapy, and in vivo tumor growth. Results: In the Director's Challenge Study, decreased expression of ARID1A was associated with lymph node dissemination, higher T-stage (T3/T4), and worse survival compared with high expression in patients in the whole group (n=440) and subgroup who received surgery alone and no adjuvant therapy (n=328). ARID1A ablation by shRNA suppressed cell proliferation, promoted cell mobility, increased G2/M phase cell cycle distribution, and protected NSCLC cells from chemotherapy; whereas overexpression of ARID1A showed the opposite biological effects. Consistent with in vitro findings, in vivo tumor xenografts demonstrated ARID1A depletion led to reductions in cell growth but resistance of NSCLC to chemotherapy. Conclusions: Our results suggest that loss of ARID1A suppresses NSCLC cell growth perhaps by inducing G2/M arrest, but promotes cell migration/invasion, and protect cells from chemotherapy. Further study of ARID1A is needed to better define its roles in the initiation and progression of NSCLC and response to chemotherapy. Citation Format: Linlin Yang, Changxian Shen, Xiaokui Mo, Terence M. Williams. ARID1A loss leads to chemotherapy resistance and is associated with a poor clinic outcome for patients with non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4155.

Xanthohumol targets the ERK1/2‑Fra1 signaling axis to reduce cyclin D1 expression and inhibit non‑small cell lung cancer.

High expression of cyclin D1 has a crucial role in the maintenance of unlimited cell growth in human cancer cells. The present study indicated that cyclin D1 was overexpressed in human non-small cell lung cancer (NSCLC) tumor tissues and cell lines. Knockout of cyclin D1 suppressed NSCLC cell growth, colony formation and in vivo tumor growth. Of note, the natural product xanthohumol (Xanth) inhibited NSCLC cells via the downregulation of cyclin D1. A further mechanistic study revealed that Xanth suppressed ERK1/2 signaling and reduced the protein levels of FOS-related antigen 1 (Fra1), which eventually inhibited the transcriptional activity of activator protein-1 and decreased the mRNA level of cyclin D1. Furthermore, suppression of ERK1/2 impaired Fra1 phosphorylation and enhanced Xanth-induced Fra1 ubiquitination and degradation. In addition, the S265D mutation compromised Xanth-induced Fra1 degradation. Finally, the in vivo anti-tumor effect of Xanth was validated in a xenograft mouse model. In summary, the present results indicated that targeting ERK1/2-Fra1-cyclin D1 signaling is a promising anti-tumor strategy for NSCLC treatment.

Circ_0007385 served as competing endogenous RNA for miR-519d-3p to suppress malignant behaviors and cisplatin resistance of non-small cell lung cancer cells.

BackgroundCircular RNAs (circRNAs) have been closely implicated in competing endogenous RNA (ceRNA) network among human cancers including non‐small cell lung cancer (NSCLC). However, the role of most circRNAs in NSCLC remains to be determined. Here, we aimed to investigate the role of hsa_circ_0007385 (circ_0007385) in NSCLC cells.MethodsExpression of hsa_circ_0007385 (circ_0007385), miRNA (miR)‐519d‐5p and high‐mobility group box 1 (HMGB1) was measured by real‐time quantitative PCR and western blotting. Functional experiments were evaluated by cell counting kit (CCK)‐8, flow cytometry, fluorescein active caspase‐3 staining kit, transwell assays, western blotting, and xenograft experiment. The relationship among circ_0007385,miR‐519d‐5p and HMGB1 was testified by dual‐luciferase reporter assay. Kaplan‐Meiersurvival curve identified overall survival in NSCLC patients.Resultscirc_0007385 expression was higher in NSCLC tissues and cell lines, and was associated with poor overall survival. Silencing circ_0007385 could suppress cell proliferation, migration and invasion in A549 and H1975 cells, as well as cisplatin (DDP) resistance. Moreover, circ_0007385 silence retarded tumor growth of A549 cells in vivo. Molecularly, there was a direct interaction between miR‐519d‐3p and either circ_0007385 or HMGB1; expression of miR‐519d‐3p was downregulated in NSCLC tumors in a circ_0007385‐correlated manner, and circ_0007385 could indirectly regulate HMGB1 via miR‐519d‐3p. Functionally, both inhibiting miR‐519d‐3p and restoring HMGB1 could overturn the suppressive effect of circ_0007385 knockdown on cell proliferation, migration, invasion, and DDP resistance.ConclusionsCollectively, circ_0007385 deletion could function anti‐tumor role in NSCLC by suppressing malignant behaviors and DDP resistance in vitro and in vivo via circ_0007385/miR‐519d‐3p/HMGB1 axis. These outcomes might enhance our understanding of the molecular mechanisms underlying the malignant progression of NSCLC.Key pointsSignificant findings of the study circ_0007385 was upregulated in NSCLC tissues and cells, and was associated with poor overall survival.Silenced circ_0007385 suppressed NSCLC cell proliferation, migration, invasion, and DDP resistance in vitro, and tumor growth in vivo.circ_0007385 was upregulated in NSCLC tissues and cells, and was associated with poor overall survival. What this study adds miR‐519d‐3p could directly interact with circ_0007385 and HMGB1 in NSCLC cells.A promising circ_0007385/miR‐519d‐3p/HMGB1 regulatory pathway was determined in NSCLC cells.

Upregulated lncRNA CASC9 Contributes to Progression of Non-Small Cell Lung Cancer Through Inhibition of miR-335-3p and Activation S100A14 Expression.

IntroductionNon-small cell lung cancer (NSCLC) is a deadly cancer type worldwide and the main sub-type of lung cancer. Cancer susceptibility candidate-9 (CASC9) was reported to be a key player in cancer progression. However, its function and underlying mechanism in NSCLC remain unclear.Materials and MethodsExpression level of CASC9 in NSCLC tissues and cells was measured with RT-qPCR. Biological roles of CASC9 in NSCLC were analyzed with a series of in vitro experiments. Potential mechanisms of CASC9 in NSCLC were analyzed by predicting and validating the possible targets of CASC9 in NSCLC.ResultsIn this study, we found CASC9 expression was upregulated in NSCLC tissues and cell lines. High CASC9 expression was identified as a predictor for poorer overall survival of NSCLC patients. Furthermore, functional assays showed CASC9 knockdown suppressed NSCLC cell proliferation, migration, and invasion, while CASC9 overexpression caused opposite effects. We also found microRNA-335-3p (miR-335-3p) could act as a target of CASC9 in NSCLC and the inhibition effect of CASC9 knockdown on NSCLC progression required the activity of miR-335-3p. In addition, we identified S100 calcium-binding protein A14 (S100A14) acts as a target of miR-335-3p.DiscussionTaken together, our study suggested CASC9 could promote NSCLC progression via miR-335-3p/S100A14 axis. The CASC9/miR-335-3p/S100A14 regulatory triplets identified in this work might provide new therapeutic strategies for NSCLC treatment.

Combined inhibition of ACK1 and AKT shows potential toward targeted therapy against KRAS-mutant non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) with Kirsten RAt Sarcoma 2 viral oncogene homolog (KRAS) mutation has become a clinical challenge in cancer treatment as KRAS-mutant tumors are often resistant to conventional anti-tumor therapies. Activated CDC42-associated kinase 1 (ACK1), an activator of protein kinase B (AKT), is a promising target for KRAS-mutant tumor therapy, but the downstream ACK1 signaling remains poorly understood. The aim of this study was to evaluate the effectiveness of combined ACK1/AKT inhibition on the proliferation, migration, invasion, and apoptosis of KRAS-mutant NSCLC cell lines (NCI-H23, NCI-H358, and A549). The cells were treated with an inhibitor of either ACK1 (dasatinib or sunitinib) or AKT (MK-2206 or GDC-0068), and the optimal concentrations of the two yielding synergistic tumor-killing effects were determined by applying the Chou-Talalay equation for drug combinations. We showed that combined administration of ACK1 and AKT inhibitors at the optimal concentrations effectively suppressed NSCLC cell viability and promoted apoptosis while inducing cell cycle arrest at the G2 phase. Moreover, NSCLC cell migration and invasion were inhibited by combined ACK1/AKT inhibition. These phenomena were associated with the reduced phosphorylation levels of ACK1 and AKT (at Ser473 and Thr308), as well as alterations in caspase-dependent apoptotic signaling. Collectively, our results demonstrate the promising therapeutic potential of combined ACK1/AKT inhibition as a strategy against KRAS-mutant NSCLC. Our findings provide the basis for the clinical translation of biological targeted drugs (ACK1 and AKT inhibitors) and their rational combination in cancer treatment.

MicroRNA-448/EPHA7 axis regulates cell proliferation, invasion and migration via regulation of PI3K/AKT signaling pathway and epithelial-to-mesenchymal transition in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a primary subtype of lung cancers which has a high morbidity and poor prognosis. Emerging evidence has demonstrated that aberrantly expressed microRNAs (miRNAs) were implicated in the regulatory functions of multiple processes during tumorigenesis. In the current study, we explored the functional roles and underlying mechanisms of miR-448 in NSCLC. Quantitative real-time polymerase chain reaction assays were conducted to measure miR-448 expressions in 51 pairs of NSCLC tissues and corresponding normal tissues. Moreover, the relationship between miR-448 expressions and clinicopathological characteristics of NSCLC patients was also determined. We then performed transwell assays to explore the functions of miR-448 in NSCLC cell invasion and migration. As we had identified EPHA7 as a functional target of miR-448 in NSCLC cells, the clinical significance of EPHA7 in NSCLC patients was further investigated. Finally, we detected the influence of miR-448 on tumor growth rate and tumor size of NSCLC using tumor xenografts. Underexpressed miR-448 was identified in NSCLC, and low miR-448 expression was confirmed to be associated with the poor prognosis and adverse clinicopathologic features of NSCLC patients. Moreover, functional assays demonstrated that miR-448 overexpression suppressed NSCLC cell proliferation, invasion and migration. EPHA7 was identified as a direct target of miR-448. Additionally, miR-448 restoration suppressed in vivo NSCLC cell growth. Finally, our studies also indicated that miR-448 exerted anti-NSCLC functions via regulating PI3K/AKT signaling pathway and EMT. These results showed that miR-448/EPHA7 axis maybe one of the useful diagnostic and prognostic biomarkers for NSCLC patients.

MiR-646 suppresses proliferation and metastasis of non-small cell lung cancer by repressing FGF2 and CCND2.

MicroRNA‐646 (miR‐646) has been implicated in several other cancers; however, its functional mechanism in non‐small cell lung cancer (NSCLC) remains unclear. In this study, we observed the downregulation of miR‐646 expression in NSCLC tissues and cell lines. Low‐level expression of miR‐646 was associated with metastasis and stage of NSCLCs. Functional assays showed that overexpression of miR‐646 could suppress NSCLC cell proliferation, clonogenicity, invasion, and inhibit epithelial‐mesenchymal transition (EMT), whereas decreased miR‐646 expression showed the opposite effects. Importantly, miR‐646 overexpression attenuated in vivo tumor growth and metastasis in nude mice models. Mechanically, miR‐646 directly targeted and suppressed fibroblast growth factor 2 (FGF2) and cyclin D2 (CCND2) expression. Reintroduction of FGF2 and CCND2 attenuated miR‐646‐mediated suppression of proliferation and invasion in NSCLC. Collectively, these results demonstrate that miR‐646 acts as a tumor suppressor in NSCLC by targeting FGF2 and CCND2, and may serve as a therapeutic target for patients with NSCLC.

Bioengineered microRNAs suppress lung cancer cell metabolism and proliferation through targeting of folate cycle

Lung cancer remains as the leading cause of cancer deaths in the world. Ver recently, we have established a novel technology for the production of bioengineered miRNA agents for the study of cancer biology and new therapies. Using recombinant miRNA molecules produced and folded in living cells, we identified a number of the most effective miRNAs for the inhibition of non‐small cell lung cancer (NSCLC) cell proliferation, among which miR‐22‐3p, miR‐9‐5p and miR‐218‐5p are all predicted to interfere with folate cycle and one‐carbon metabolism. The serine hydroxymethyltransferase‐1 (SHMT‐1), a tumor biomarker and important enzyme in folate biotransformation, was verified as a new target for both miR‐9‐5p and miR‐218‐5p. The methylenetetrahydrofolate dehydrogenase 1 like (MTHFD1L) was proven to be regulated by miR‐9‐5p directly. Furthermore, both methylenetetrahydrofolate reductase (MTHFR) and methylenetetrahydrofolate dehydrogenase‐2 (MTHFD2) were significantly suppressed by miR‐22‐3p in NSCLC cells. As determined by accurate LC‐MS/MS methods, folate metabolites and amino acid metabolome were altered remarkably by individual miRNAs in human NSCLC cells. Consequently, glycolysis rate and mitochondrial function were significantly inhibited in NSCLC cells treated with these miRNAs, accompanied by cell cycle arrest, ROS accumulation, NADP+/NADPH imbalance and other metabolic dysfunctions. In conclusion, our results demonstrated that bioengineered miR‐22‐3p, miR‐9‐5p, and miR‐218‐5p act on folate cycle to suppress NSCLC cell metabolic capacity and exhibit strong antiproliferation activities. These findings provide new insights into mechanistic actions of tumor suppressive miRNAs and development of novel therapeutic strategies.Support or Funding InformationThis study was supported by National Cancer Institute (grant No. R01CA225958) and National Institute of General Medical Sciences (R01GM113888), National Institutes of Health.

KCP10043F Represses the Proliferation of Human Non-Small Cell Lung Cancer Cells by Caspase-Mediated Apoptosis via STAT3 Inactivation.

We previously reported that 4-(4-fluorobenzylcarbamoylmethyl)-3-(4-cyclohexylphenyl)-2-[3-(N,N-dimethylureido)-N′-methylpropylamino]-3,4-dihydroquinazoline (KCP10043F) can induce G1-phase arrest and synergistic cell death in combination with etoposide in lung cancer cells. Here, we investigated the underlying mechanism by which KCP10043F induces cell death in non-small cell lung cancer (NSCLC). Propidium iodide (PI) and annexin V staining revealed that KCP10043F-induced cytotoxicity was caused by apoptosis. KCP10043F induced a series of intracellular events: (1) downregulation of Bcl-2 and Bcl-xL and upregulation of Bax and cleaved Bid; (2) loss of mitochondrial membrane potential; (3) increase of cytochrome c release; (4) cleavage of procaspase-8, procaspase-9, procaspase-3, and poly (ADP-ribose) polymerase (PARP). In addition, KCP10043F exhibited potent inhibitory effects on constitutive or interleukin-6 (IL-6)-induced signal transducer and activator of transcription (STAT3) phosphorylation and STAT3-regulated genes including survivin, Mcl-1, and cyclin D1. Furthermore, STAT3 overexpression attenuated KCP10043F-induced apoptosis and the cleavage of caspase-9, caspase-3, and PARP. Docking analysis disclosed that KCP10043F could bind to a pocket in the SH2 domain of STAT3 and prevent STAT3 phosphorylation. The oral administration of KCP10043F decreased tumor growth in an A549 xenograft mouse model, as associated with the reduced phosphorylated STAT3, survivin, Mcl-1, and Bcl-2 expression and increased TUNEL staining and PARP cleavage in tumor tissues. Collectively, our data suggest that KCP10043F suppresses NSCLC cell growth through apoptosis induction via STAT3 inactivation.

Overexpression of miR-425-5p is associated with poor prognosis and tumor progression in non-small cell lung cancer.

MicroRNA-425-5p (miR-425-5p) has been investigated in some human cancers, but the understanding of its clinical and functional roles in non-small cell lung cancer (NSCLC) remains poor. This study sought to measure the expression of miR-425-5p in NSCLC samples, assess its prognostic significance in cancer patients, and explore its functional role during tumor progression. Expression of miR-425-5p was examined using quantitative Real-time polymerase chain reaction (qRT-PCR). Kaplan-Meier survival analysis and Cox analysis were conducted to evaluate the prognostic value of miR-425-5p. The effects of miR-425-5p on NSCLC cell proliferation, migration and invasion were assessed using cell experiments. Expression of miR-425-5p was upregulated in NSCLC tissues and cells compared with the normal controls (all P< 0.05). The increased miR-425-5p expression was associated with positive lymph node metastasis and TNM advanced stage of the patients (all P< 0.05). The survival curves and Cox analysis indicated that high miR-425-5p was correlated with poor overall survival and acted as an independent prognostic factor in NSCLC patients. Cell experiments suggested that miR-425-5p overexpression could enhance, whereas its reduction could suppress NSCLC cell proliferation, migration and invasion (all P< 0.05). Forhead box J3 (FOXJ3) was proved to be a direct target of miR-425-5p in NSCLC. Overexpression of miR-425-5p predicts poor prognosis of NSCLC and promotes cancer cell proliferation, migration and invasion by targeting FOXJ3.

Overexpression of tripartite motif containing 26 inhibits non-small cell lung cancer cell growth by suppressing PI3K/AKT signaling.

It has been reported that tripartite motif containing 26 (TRIM26) is involved in the tumorigenesis of some cancers, but its function in non-small cell lung cancer (NSCLC) is still unclear. In this study, we found that TRIM26 was markedly down-regulated in both of NSCLC tumor tissues and cell lines. Additionally, high expression of TRIM26 in NSCLC patients predicted a positive index for patients' overall survival. What is more, overexpression of TRIM26 significantly suppressed NSCLC cell growth. Our further studies indicated that overexpression of TRIM26 inhibited the phosphorylation of PI3K p85 and AKT. And overexpressed TRIM26 regulated cell cycle-related genes' expression, including downregulating CDK4, Cyclin A, Cyclin D1, Cyclin D3, and Cyclin E, and upregulating p27 expression. Finally, we found that TRIM26 up-regulated PTEN expression by stabilizing PTEN protein in NSCLC cells. Collectively, our present study indicated that TRIM26 was decreased in NSCLC and overexpression of TRIM26 inhibited NSCLC cell growth by suppressing PI3K/AKT pathway, which suggested that TRIM26 could be as a potential target for the treatment of NSCLC in the future.

Sesamin suppresses NSCLC cell proliferation and induces apoptosis via Akt/p53 pathway

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer with a disappointing prognosis. The aim of this study was to investigate the anticancer effect of sesamin and the underlying mechanism. The MTT assay was used to detect the proliferation of NSCLC cells. The cell cycle and apoptosis were analyzed by flow cytometry. The protein levels of Akt, p-Akt (Ser473), p53, cyclin D1, CDK2, MDM2, p-MDM2 (Ser166) were detected by western blotting. The expression of p-Akt (Ser473), p53 and Ki67 in vivo was analyzed by IHC. Histopathologic analyses of major organs (heart, liver, spleen, lung and kidney) were performed by H&E staining. The results show that sesamin suppressed cell proliferation and induced apoptosis of NSCLC cells (A549 and H1792) in a dose-dependent manner. Treatment with sesamin caused cell cycle arrest at G1 phase and inhibited cyclin D1 and CDK2 expression. In addition, sesamin inhibited Akt activity and upregulated p53 expression both in vivo and in vitro. When Akt and p53 were suppressed by LY294002 and PFTα, respectively, sesamin exerted no additional effects. The in vivo results mostly matched the in vitro findings. Specifically, sesamin exerted little damage to major organs. Taken together, this study demonstrates that sesamin suppresses NSCLC cell proliferation by induction of G1 phase cell cycle arrest and apoptosis via Akt/p53 pathway. Therefore, sesamin may be a promising adjuvant treatment for NSCLC therapy.

MiR-501-3p functions as a tumor suppressor in non-small cell lung cancer by downregulating RAP1A

MicroRNA-501-3p (miR-501-3p) has been reported to play tumor-suppressive roles in different cancers; however, its expression pattern and biological function in non–small cell lung cancer (NSCLC) remain unknown. In this study, we noted downregulation of miR-501-3p in NSCLC tissues and cell lines. Functional assays showed that overexpression of miR-501-3p suppressed NSCLC cell proliferation, clonogenicity, migration, and invasion. Moreover, miR-501-3p overexpression attenuated in vivo tumor growth in a nude mouse model. In terms of the mechanism, RAP1A was identified as a novel target of miR-501-3p. Overexpression of RAP1A strongly attenuated the inhibitory effects of miR-501-3p on the capacity of NSCLC cells for proliferation and motility. In the clinical samples of NSCLC, miR-501-3p levels negatively correlated with RAP1A expression, which was upregulated in NSCLC. Collectively, these results indicate that miR-501-3p acts as a tumor suppressor in NSCLC by directly targeting RAP1A mRNA and may serve as a theranostic biomarker for patients with NSCLC.

Long Non-Coding RNA HOXA11-AS Promotes Non-Small Cell Lung Cancer Tumorigenesis Through microRNA-148a-3p/DNMT1 Regulatory Axis.

ObjectiveOur present study aimed to further investigate the molecular basis of long non-coding RNA homeobox A11 antisense (HOXA11-AS) in the tumorigenesis of non-small cell lung cancer (NSCLC).MethodsHOXA11-AS, microRNA-148a-3p (miR-148a-3p), and DNA methyltransferase 1 (DNMT1) mRNA levels were measured by RT-qPCR assay. DNMT1 protein level was determined by Western blot assay. Cell proliferative capacity and apoptotic rate were determined by CCK-8 assay and flow cytometry analysis, respectively. The relationships of HOXA11-AS, miR-148a-3p, and DNMT1 were tested through bioinformatics analysis, luciferase assay, and RNA pull down assay. Mouse xenograft models of NSCLC were established to examine the biological function of HOXA11-AS in vivo.ResultsHOXA11-AS expression was notably upregulated and miR-148a-3p expression was conspicuously downregulated in NSCLC tissues and cells. HOXA11-AS knockdown curbed NSCLC cell proliferation and promoted cell apoptosis through directly increasing miR-148a-3p expression. Moreover, miR-148a-3p overexpression suppressed NSCLC cell proliferation and induced cell apoptosis. HOXA11-AS functioned as a competing endogenous RNA (ceRNA) of miR-148a-3p to increase DNMT1 expression in NSCLC cells. And, DNMT1 upregulation weakened the influence of HOXA11-AS1 loss on NSCLC cell proliferation and apoptosis. Additionally, HOXA11-AS knockdown suppressed NSCLC xenograft growth by upregulating miR-148a-3p and downregulating DNMT1 in vivo.ConclusionHOXA11-AS facilitated NSCLC tumorigenesis through miR-148a-3p/DNMT1 axis in vitro and in vivo, deepening our understanding of the molecular basis of HOXA11-AS in the development of NSCLC.

LncRNA LINC02418 regulates proliferation and apoptosis of non-small cell lung cancer cells by regulating miR-4677-3p/SEC61G.

Increasing studies indicated that long non-coding RNA (lncRNA) has crucial roles in cancer development, including non-small cell lung cancer (NSCLC). LINC02418 was reported to promote colorectal cancer development. However, whether LINC02418 has a role in NSCLC remains to be explored. First, expression level of LINC02418 in NSCLC tissues and normal tissues was analyzed at ENCORI. Moreover, expression level of LINC02418 in NSCLC cells and normal cell was analyzed with quantitative real-time PCR. Cell counting kit-8 assay, transwell invasion assay, and flow cytometry assay were used to analyze cell proliferation, cell invasion, and cell apoptosis. LINC02418 was found as upregulated expression in both NSCLC tissues and cells. Functional assays showed that LINC02418 knockdown suppressed NSCLC cell proliferation and invasion but promoted cell apoptosis, while the overexpression of LINC02418 exerts opposite effects. Mechanistically, we showed LINC02418 could interact with microRNA-4677-3p (miR-4677-3p) to regulate Sec61 gamma subunit (SEC61G) expression. These results indicated that LINC02418 functions as an oncogene, and regulated miR-4677-3p/SEC61G axis to accelerate NSCLC progression.