Suppressed Non-small Cell Lung Cancer Cell Research Articles

Fluvastatin Inhibits HMG-CoA Reductase and Prevents Non-Small Cell Lung Carcinogenesis.

Lung cancer is the leading cause of cancer-related death worldwide. However, promising agents for lung cancer prevention are still very limited. Identification of preventive targets and novel effective preventive agents is urgently needed for clinical applications. In this study, we found that fluvastatin targeted 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGCR), which a rate-limiting enzyme in the mevalonate pathway, and inhibited non-small cell lung cancer (NSCLC) tumorigenesis. Initially, we demonstrated 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 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.

MicroRNA‑718 serves a tumor‑suppressive role innon‑smallcell lung cancer by directly targeting CCNB1.

MicroRNA-718 (miR-718) serves crucial roles in tumorigenesis and in the progression of a number of cancers. However, the expression profile, specific functions and mechanisms of action of miR-718 in non-small cell lung cancer (NSCLC) are still elusive. The aims of the present study were to quantify the expression of miR-718, determine its biological roles and elucidate the molecular mechanisms responsible for its activities in NSCLC cells. Reverse transcription-quantitative PCR was carried out to assess miR-718 expression in NSCLC tissue samples and cell lines. The Cell Counting Kit-8 assay, flow cytometry, cell migration and invasion assays, and a tumor xenograft experiment were performed to evaluate the effects of miR-718 overexpression on the malignant biological behaviors of NSCLC cells. miR-718 expression was demonstrated to be significantly decreased in NSCLC tissue samples and cell lines. This reduced expression was significantly associated with tumor, node, metastasis stage, tumor size, lymph node metastasis and poor overall survival among patients with NSCLC. Exogenous miR-718 expression suppressed NSCLC cell proliferation, migration and invasion, and promoted apoptosis in vitro; whereas it hindered tumor growth in vivo. Experiments to elucidate the mechanisms involved revealed that miR-718 functions by directly targeting cyclin B1 (CCNB1) mRNA. CCNB1 expression was found to be upregulated in NSCLC and inversely correlated with miR-718 levels. CCNB1 depletion had effects similar to those of miR-718 overexpression in NSCLC cells. Furthermore, restoration of CCNB1 expression attenuated the tumor-suppressive effects of miR-718 overexpression in NSCLC cells. These results indicated that miR-718 suppressed NSCLC progression in vitro and in vivo by directly targeting CCNB1 mRNA, which may indicate a potential target for the diagnosis and treatment of this fatal disease.

In silico identification of thiostrepton as an inhibitor of cancer stem cell growth and an enhancer for chemotherapy in non-small-cell lung cancer.

Cancer stem cells (CSCs) play an important role in cancer treatment resistance and disease progression. Identifying an effective anti‐CSC agent may lead to improved disease control. We used CSC‐associated gene signatures to identify drug candidates that may inhibit CSC growth by reversing the CSC gene signature. Thiostrepton, a natural cyclic oligopeptide antibiotic, was the top‐ranked candidate. In non–small‐cell lung cancer (NSCLC) cells, thiostrepton inhibited CSC growth in vitro and reduced protein expression of cancer stemness markers, including CD133, Nanog and Oct4A. In addition, metastasis‐associated Src tyrosine kinase signalling, cell migration and epithelial‐to‐mesenchymal transition (EMT) were all inhibited by thiostrepton. Mechanistically, thiostrepton treatment led to elevated levels of tumour suppressor miR‐98. Thiostrepton combined with gemcitabine synergistically suppressed NSCLC cell growth and induced apoptosis. The inhibition of NSCLC tumours and CSC growth by thiostrepton was also demonstrated in vivo. Our findings indicate that thiostrepton, an established drug identified in silico, is an inhibitor of CSC growth and a potential enhancer of chemotherapy in NSCLC.

P1.01-29 MLPH Promotes Epithelial–Mesenchymal Transition and Brain Metastasis via Cdc42/PAK1 Signaling in Non-Small Cell Lung Cancer

Brain metastasis (BM) is associated with poor prognosis, recurrence, and death in patients with non-small cell lung cancer (NSCLC). Therefore, a better understanding of molecular mechanisms underlying NSCLC development and progression could provide helpful insights for NSCLC prevention and effective treatment. Melanophilin (MLPH), an actin-based transport binding partner, involves in cancer progression. However, the role of MLPH in NSCLC remains unclear. Here we elucidated the functional significance, molecular mechanisms and clinical impact of MLPH in NSCLC. RNA-Sequencing was performed to identify differentially expressed genes in lung tissues of NSCLC patients with (BM+) and without BM (BM-). The expression of MLPH was examined in the serum of BM+ and BM- patients by PCR. Integrative database analysis was used to examine MLPH levels in NSCLC tissues and analyze the relationship between MLPH levels and patient survival. Lentivirus containing small hairpin (sh) RNA targeting MLPH or empty vector was designed to explore its role in NSCLC. The cell counting kit-8 assay, wound healing assay, transwell assay, flow cytometry analysis, Phalloidin staining, xenografted tumor model and brain metastasis model were used to determine the effects of MLPH on the proliferation, migration, invasion, EMT, tumorigenesis and brain metastasis of NSCLC. Western blot analysis was used to explore the underlying mechanism. High-throughput sequencing showed that MLPH mRNA was significantly differentially expressed in lung tumors between BM+ and BM- NSCLC patients. MLPH was frequently overexpressed in NSCLC tissues and cells, and high levels of MLPH correlated with poor prognosis of NSCLC patients. Silencing MLPH by shRNA suppressed NSCLC cell proliferation, migration, invasion and TGF-β-induced EMT, and triggered cell cycle arrest and apoptosis. Being in consistent with the in vitro findings, the in vivo experiment exhibited that knockdown of MLPH inhibited xenograft tumorigenesis and brain metastasis in nude mice. Mechanically, we identified TGF-β as a key downstream effector of MLPH. MLPH silencing attenuated Cdc42/PAK1 signaling activation at least in part through the downregulation of TGF-β. Furthermore, EMT phenotypes changes caused by MLPH knockdown were partially dependent on TGF-β inhibition. Our findings uncovered the role of MLPH in NSCLC progression and provided evidence for MLPH positively modulating the Cdc42/PAK1 signaling pathway to promote EMT and metastasis via TGF-β in NSCLC cells. MLPH may have the potential as a therapeutic target against metastatic NSCLC.

Downregulated MCOLN1 Attenuates The Progression Of Non-Small-Cell Lung Cancer By Inhibiting Lysosome-Autophagy.

ObjectivesAutophagy plays various roles in non-small-cell lung cancer (NSCLC). MCOLN1, a reactive oxygen species sensor, can regulate autophagy via lysosomal Ca(2+); however, the role of MCOLN1 in NSCLC is largely unknown. This study aimed to explore the effects of MCOLN1 on proliferation, invasion and migration in NSCLC and the underling mechanisms.Materials and methodsThe tissues of NSCLC patients were collected, then MCOLN1 expression in tumor and adjacent tissues was measured and its relationship with pathological staging was analyzed. The Cell Counting Kit-8 (CCK-8) assay, wound healing assay and transwell migration assay were used to evaluate the proliferation, migration and invasion ability, respectively. Live-cell imaging and transmission electron microscopy (TEM) were used to observe autophagic flux and autolysosomes.ResultsIt was found that MCOLN1 expression was significantly decreased in human NSCLC tissues compared with normal lung tissues while more MCOLN1 in stage III–IV was shown than stage I–II, indicating that MCOLN1 increased along with the progression of NSCLC. Furthermore, CCK-8 assay, wound healing assay and transwell migration assay confirmed that the inhibition of MCOLN1 suppressed NSCLC cells proliferation migration and invasion. Overexpression of MCOLN1 promoted autophagy in A549 and H1299 cells with increased LC3-II/I, lamp1 expression and autolysosomes as well as autophagic flux shown by live-cell imaging and TEM.ConclusionOur study shows that downregulated MCOLN1 reduced lysosome-autophagy activity contributing to inhibited tumor progression, which reveals a novel role of MCOLN1 in NSCLC, and targeting MCOLN1 may be a therapeutic potential for NSCLC.

FAM83A signaling induces epithelial-mesenchymal transition by the PI3K/AKT/Snail pathway in NSCLC.

Family with sequence similarity 83, member A (FAM83A), as a potential tumor promoter, was reported to contribute to the progression of several malignant tumors. However, the significance of FAM83A in invasion and metastasis of non-small cell lung cancer (NSCLC) remains largely unknown. In this study, we found that FAM83A expression was significantly increased in NSCLC tissues. High expression of FAM83A was positively associated with tumor metastasis and poor survival of NSCLC patients. Functional experiments revealed that FAM83A knockdown could suppress NSCLC cell migration and invasion both in vivo and in vitro. While opposite results were observed in FAM83A-transfected cells. Mechanically, we found that FAM83A promoted NSCLC cell migration and invasion by inducing epithelial-mesenchymal transition (EMT) via PI3K/ATK/Snail signaling. Rescue experiment demonstrated that inhibition of either AKT or Snail could partially counteract the promoting effect of FAM83A overexpression in NSCLC metastasis. Taken together, our findings are the first time to demonstrate that increased expression of FAM83A in NSCLC was correlated with EMT and tumor metastasis, which may provide a novel therapeutic target in NSCLC treatment.

RBAK is upregulated in non-small cell lung cancer and promotes cell migration and invasion.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-associated mortality worldwide, NCSCLC includes lung adenocarcinoma and lung squamous cell carcinoma. Tumor metastasis is the major cause of mortality of patients with NSCLC. However, the mechanisms underlying NSCLC metastasis remain largely elusive. In present study, the authors focused on exploring the roles of RBAK in NSCLC. The present study demonstrated that RB-associated KRAB zinc finger (RBAK) was upregulated in NSCLC compared with non-tumorous tissues by analyzing Gene Expression Omnibus (GEO) datasets and The Cancer Genome Atlas (TCGA) dataset. High expression of RBAK was associated with poor disease-free survival of patients with NSCLC by analyzing TCGA dataset. Furthermore, an RBAK-mediated protein-protein interaction network was constructed to reveal the potential underlying mechanisms by which RBAK drives NSCLC progression. The authors found that RBAK was involved in regulating a number of transcription factors, including androgen receptor, forkhead box A1, tumor protein 53, and E2F transcription factor 1, 2 and 4, suggesting that RBAK may have a role in regulating gene transcription. GO and KEGG enrichment analyses of the genes co-expressed with RBAK revealed that RBAK is involved in regulating a number of biological functions, including the Wnt signaling pathway, mRNA splicing, protein polyubiquitination, cell-cell adhesion and focal adhesion. Transwell and wound healing assays demonstrated that knockdown of RBAK suppressed NSCLC cell migration and invasion. The present study enhances the current understanding of the important roles of RBAK in NSCLC metastasis and may provide useful information for the development of novel treatment approaches.

Downregulation of CPA4 inhibits non small-cell lung cancer growth by suppressing the AKT/c-MYC pathway.

Carboxypeptidase A4 (CPA4) is a member of the metallocarboxypeptidase family. A previous study indicated that CPA4 may participate in the modulation of peptide hormone activity and hormone‐regulated tissue growth and differentiation. However, the role of CPA4 in lung tumorigenesis remains unclear. Our study revealed that CPA4 expression was higher in both lung cancer cells and tumor tissues. We performed 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assays, colony‐formation assays, and Cellomics ArrayScan Infinity analysis to demonstrate that CPA4 knockdown inhibited non small–cell lung cancer (NSCLC) cell proliferation. Conversely, ectopic expression of CPA4 enhanced lung cancer cell proliferation. Consistent with these observations, we generated xenograft tumor models to confirm that CPA4 downregulation suppressed NSCLC cell growth. Mechanistically, we revealed that CPA4 downregulation may induce apoptosis and G1‐S arrest by suppressing the protein kinase B/c‐MYC pathway. These results suggest that CPA4 has an oncogenic effect on lung cancer growth. Taken together, we identified a novel gene in lung cancer that might provide a basis for new therapeutic targets.

MicroRNA-510 Plays Oncogenic Roles in Non-Small Cell Lung Cancer by Directly Targeting SRC Kinase Signaling Inhibitor 1.

An increasing number of studies have demonstrated that microRNAs (miRNAs) may play key roles in various cancer carcinogenesis and progression, including non-small cell lung cancer (NSCLC). However, the expressions, roles, and mechanisms of miR-510 in NSCLC have, up to now, been largely undefined. In vivo assay showed that miR-510 was upregulated in NSCLC tissues compared with that in adjacent nontumor lung tissues. miR-510 expression was significantly correlated with TNM stage and lymph node metastasis. In vitro assay indicated that expressions of miR-510 were also increased in NSCLC cell lines. Downregulation of miR-510 suppressed NSCLC cell proliferation and invasion in vitro. We identified SRC kinase signaling inhibitor 1 (SRCIN1) as a direct target gene of miR-510 in NSCLC. Expression of SRCIN1 was downregulated in lung cancer cells and negatively correlated with miR-510 expression in tumor tissues. Downregulation of SRCIN1, leading to inhibition of miR-510 expression, reversed cell proliferation and invasion in NSCLC cells. These results showed that miR-510 acted as an oncogenic miRNA in NSCLC, partly by targeting SRCIN1, suggesting that miR-510 can be a potential approach for the treatment of patients with malignant lung cancer.

Abstract 4371: To investigate the role of heme flux and function in growth and progression of NSCLC in vivo

Abstract Lung cancer is the leading cause of cancer-related mortality and about 85% of the cases are non-small cell lung cancer (NSCLC). Previous research from our lab has shown that the levels of rate limiting heme biosynthetic enzyme ALAS1, heme uptake proteins HCP1 and HRG1 and oxygen utilizing hemoproteins are strongly elevated in NSCLC cells. Furthermore, specific inhibition of heme synthesis using a well-known inhibitor suppresses NSCLC cell proliferation, colony formation and migration compared to normal cells. These results suggest that heme flux and function is important for NSCLC growth and progression. Lentiviral particles overexpressing eGFP, Alas1, HRG1, and HCP1 were used to transfect NSCLC cell lines. Tumorigenic properties of these cells were analyzed in vitro using invasion, migration and colony formation assay. These cell lines were used to implant subcutaneous xenografts in NOD/SCID mice (n=6). Tumor growth was monitored using Bioluminescence imaging (BLI) and Vernier caliper measurements over a period of five weeks. Mice were sacrificed, tumor tissues were harvested and processed and embedded into FFPE blocks. The tissue sections were then used for histopathological analysis (Hematoxylin and Eosin staining) and immunohistochemistry experiments. Our data show a significant increase in invasion, migration and colony formation properties of cells lines overexpressing Alas1, HRG1 and HCP1 compared to eGFP overexpressing cell lines (Control) in vitro. At week 5, BLI radiance (total photons/seconds), tumor volumes and masses of Alas1, HCP1 and HRG1 overexpression subcutaneous tumor xenografts were significantly higher compared to eGFP overexpression controls. This was further corroborated by H and E staining. Our results demonstrate that overexpression of heme synthesis and uptake proteins causes an increase in tumorigenic properties of NSCLC cells in vivo. Thus, heme flux and function plays an important role in growth and progression of NSCLC. Further studies are underway to discern the molecular mechanisms underlying the role of heme in lung tumor growth. Citation Format: Adnin Ashrafi, Poorva Ghosh, Sagar Sohoni, Li Zhang. To investigate the role of heme flux and function in growth and progression of NSCLC in vivo [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 4371.

LncRNA DGCR5 regulates the non-small cell lung cancer cell growth, migration, and invasion through regulating miR-211-5p/EPHB6 axis.

Non-small cell lung cancer (NSCLC) accounts for about 80% of lung cancers worldwide. In recent years, importance of noncoding RNAs including long noncoding RNA and microRNA in regulating tumor progression has been appreciated. Abnormally expression of DiGeorge syndrome critical region gene 5 (DGCR5) was found in multiple human cancers but its function in NSCLC is largely unknown. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was conducted to explore DGCR5 expression level in NSCLC. Bioinformatic analyses were conducted to explore the targets of DGCR5. Cell counting kit-8 assay, wound-healing assay, and transwell invasion assay were performed to analyze functions of DGCR5. RT-qPCR revealed that DGCR5 expression in NSCLC cells was significantly lower than in normal cell. DGCR5 overexpression suppresses NSCLC cell growth, migration, and invasion. Online algorithms found EPH receptor B6 (EPHB6) and DGCR5 contains same miR-211-5p binding region. The predicted connections were further validated by luciferase activity reporter assay. Recue experiments showed DGCR5 regulates NSCLC cell behaviors via targeting miR-211-5p/EPHB6. These findings collectively identified DGCR5/miR-211-5p/EPHB6 triple axis in NSCLC, which may novel understanding regarding the tumorigenesis of NSCLC.

Circular RNA circPIP5K1A promotes non-small cell lung cancer proliferation and metastasis through miR-600/HIF-1α regulation.

Circular RNAs (circRNAs) have an important function in human diseases, especially in cancer. circRNA hsa_circ_0014130 (circPIP5K1A), a particularly abundant circRNA, participates in the tumorigenesis of non-small cell lung cancer (NSCLC), although the underlying regulatory mechanism remains unclear. Here, we investigated the circPIP5K1A role in NSCLC. Expression of circPIP5K1A in NSCLC cell lines was explored with quantitative real-time PCR. The effect of circPIP5K1A on NSCLC was evaluated with circPIP5K1A silencing, miR-600 mimic transfection, and hypoxia-inducible factor (HIF)-1α overexpression, followed by assessment of cell proliferation, metastasis, and tumorigenesis in nude mice. The subcellular localization of circPIP5K1A was evaluated via fluorescence in situ hybridization (FISH), and correlation between circPIP5K1A, miR-600, and HIF-1α was assessed by luciferase assay. The data demonstrated that circPIP5K1A expression was increased in NSCLC cells. FISH showed that circPIP5K1A localized to the cytoplasm. The circPIP5K1A knockdown suppressed NSCLC cell metastasis and proliferation by promoting expression of miR-600. Overexpression of miR-600 inhibited HIF-1α-mediated metastasis and proliferation of NSCLC cell by downregulating the endothelial mesenchymal transition-related proteins, Snail and vimentin, and upregulating E-cadherin. In vivo experiments illustrated that circPIP5K1A silence suppressed tumor growth and pulmonary metastasis. The circPIP5K1A may function as an miR-600 sponge to facilitate NSCLC proliferation and metastasis by promoting HIF-1α. A bifluorescein reporter experiment confirmed that miR-600 was the circPIP5K1A target, and miR-600 interacted with the 3' untranslated region of HIF-1α. These results show that circPIP5K1A acted as a tumor promoter through a novel circPIP5K1A/miR-600/HIF-1α axis, which provides candidate markers and therapeutic targets for NSCLC.

MiR-34b-3p represses cell proliferation, cell cycle progression and cell apoptosis in non-small-cell lung cancer (NSCLC) by targeting CDK4.

Lung cancer is the most common incident cancer, with a high mortality worldwide, and non‐small‐cell lung cancer (NSCLC) accounts for approximately 85% of cases. Numerous studies have shown that the aberrant expression of microRNAs (miRNAs) is associated with the development and progression of cancers. However, the clinical significance and biological roles of most miRNAs in NSCLC remain elusive. In this study, we identified a novel miRNA, miR‐34b‐3p, that suppressed NSCLC cell growth and investigated the underlying mechanism. miR‐34b‐3p was down‐regulated in both NSCLC tumour tissues and lung cancer cell lines (H1299 and A549). The overexpression of miR‐34b‐3p suppressed lung cancer cell (H1299 and A549) growth, including proliferation inhibition, cell cycle arrest and increased apoptosis. Furthermore, luciferase reporter assays confirmed that miR‐34b‐3p could bind to the cyclin‐dependent kinase 4 (CDK4) mRNA 3′‐untranslated region (3′‐UTR) to suppress the expression of CDK4 in NSCLC cells. H1299 and A549 cell proliferation inhibition is mediated by cell cycle arrest and apoptosis with CDK4 interference. Moreover, CDK4 overexpression effectively reversed miR‐34‐3p‐repressed NSCLC cell growth. In conclusion, our findings reveal that miR‐34b‐3p might function as a tumour suppressor in NSCLC by targeting CDK4 and that miR‐34b‐3p may, therefore, serve as a biomarker for the diagnosis and treatment of NSCLC.

Overexpression of LINC00261 inhibits non-small cell lung cancer cells progression by interacting with miR-522-3p and suppressing Wnt signaling.

Long noncoding RNA LINC00261 has been experimentally validated to function as a tumor suppressor in several cancers, but its pathological role and functional mechanism in non-small cell lung cancer (NSCLC) are largely unclear. In this study, LINC00261 was delineated in NSCLC to be significantly downregulated in cancer tissues compared with corresponding adjacent normal tissues. Low expression of LINC00261 predicted worse survival for patients withNSCLC. Overexpression of LINC00261 in NSCLC cell lines inhibited cell proliferation and invasion, meanwhile promoted apoptosis. Subcellular fractionation assay showed that LINC00261 existed mainly in the cytoplasm of NSCLC A549 cells and luciferase assay validated its direct interaction with miR-522-3p. Overexpression of miR-522-3p significantly ameliorated suppressive effects of LINC00261 on proliferation and invasion of NSCLC cells. Besides, miR-522-3p was found to be able to directly combine with the 3'-untranslated region of SFRP2, which was generally regarded as a suppressor of Wnt signaling. Further quantitative reverse transcription polymerase chain reaction and Western blot experiments showed that LINC00261 upregulation potentiated the expression of SFRP2 and inhibited Wnt signaling pathway, which could both be reversely modulated by miR-522-3p. Taken together, our study demonstrated that LINC00261 suppressed NSCLC cells progression via sponging miR-522-3p and inhibiting Wnt signaling. These results supported us to better understand the pathogenic mechanism of NSCLC and revealed a potential molecular target for this fatal disease.

MicroRNA-582-5p suppresses non-small cell lung cancer cells growth and invasion via downregulating NOTCH1.

Non-small cell lung cancer (NSCLC) is the most common cancer worldwide. MicroRNAs have been shown to be correlated with biological processes of various tumors. In this study, we observed that the expression of miR-582-5p was lower in NSCLC tissues than that in para-carcinoma tissues. Ectopic expression of miR-582-5p significantly inhibited NCI-H358 cell proliferation and invasion. Knockdown of miR-582-5p showed the opposite results, with cell growth rate and the invasive capacity of PC-9 cells enhanced. Furthermore, we elucidated that NOTCH1 is a target of miR-582-5p and there is an inverse correlation between miR-582-5p and NOTCH1 expression in NSCLC tissues. Overexpression of NOTCH1 in miR-582-5p-overexpressing NCI-H358 cells could partially reverse the inhibition of cell proliferation and invasion by miR-582-5p. Thus, our research demonstrated that miR-582-5p suppresses NSCLC cell lines’ growth and invasion via targeting oncoprotein NOTCH1 and restoration of miR-582-5p might be feasible therapeutic strategy for NSCLC.

STAT3-induced long noncoding RNA LINC00668 promotes migration and invasion of non-small cell lung cancer via the miR-193a/KLF7 axis

Long noncoding RNAs (lncRNAs) have been demonstrated to play significant roles in non-small cell lung cancer (NSCLC) progression. Recently, a newly identified lncRNA, LncRNA LINC00668 (LINC00668), was reported to be involved in the regulation of progression of several tumors. However, the expression pattern and biological function of LINC00668 in NSCLC remains largely unclear. In this study, we found that LINC00668 expression was significantly up-regulated in both NSCLC tissues and cell lines. we also showed that LINC00668 upregulation was induced by transcription factor STAT3. Clinical investigation demonstrated that high expression level of LINC00668 was associated with advanced TNM stage, histological grade and lymph node metastasis. Moreover, multivariate analysis confirmed LINC00668 expression level to be an independent prognostic indicator for overall survival of NSCLC patients. Functional assays indicated that knockdown of LINC00668 suppressed NSCLC cells proliferation, migration and invasion, and promoted apoptosis. Mechanistic studies indicated that LINC00668 is a direct target of miR-193a, leading to down-regulation in the expression of its target gene KLF7. Our findings suggested that STAT3-induced LINC00668 contributed to NSCLC progression through upregulating KLF7 expression by sponging miR-193a, and may serve as a prognostic biomarker and a potential target for NSCLC.

Novel ROR1 inhibitor ARI-1 suppresses the development of non-small cell lung cancer.

Limited drug response and severe drug resistance confer the high mortality of non-small-cell lung cancer (NSCLC), a leading cause of cancer death worldwide. There is an urgent need for novel treatment against NSCLC. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is aberrantly overexpressed and participats in NSCLC development and EGFR-TKIs-induced drug resistance. Increasing evidences indicate that oncogenic ROR1 is a potential target for NSCLC therapy. However, nearly no ROR1 inhibitor was reported until now. Here, combining the computer-aided drug design and cell-based activity screening, we discover (R)-5,7-bis(methoxymethoxy)-2-(4-methoxyphenyl)chroman-4-one (ARI-1) as a novel ROR1 inhibitor. Biological evaluation demonstrates that ARI-1 specifically targets the extracellular frizzled domain of ROR1 and potently suppresses NSCLC cell proliferation and migration by regulating PI3K/AKT/mTOR signaling in a ROR1-dependent manner. Moreover, ARI-1 significantly inhibits tumor growth in vivo without obvious toxicity. Intriguingly, ARI-1 is effective to EGFR-TKIs-resistant NSCLC cells with high ROR1 expression. Therefore, our work suggests that the ROR1 inhibitor ARI-1 is a novel drug candidate for NSCLC treatment, especially for EGFR-TKIs-resisted NSCLC with high ROR1 expression.

SP1-mediated overexpression of lncRNA LINC01234 as a ceRNA facilitates non-small-cell lung cancer progression via regulating OTUB1.

Long noncoding RNAs (lncRNAs) have been confirmed to be strongly associated with the progression of various types of cancer. LncRNA LINC01234 (LINC01234) is a newly identified tumor-related lncRNA whose upregulation has been confirmed in some tumors. However, its potential expressions and possible functions in non-small-cell lung cancer (NSCLC) have not been explored. In this study, we first found that LINC01234 expressions were distinctly upregulated in both NSCLC samples and cell lines using RT-PCR. Our group also showed that LINC01234 upregulations were modulated by nuclear transcription factor SP1. The results form clinical investigations indicated that high LINC01234 expressions were associated with positively lymph node metastasis and advanced tumor-metastasis-node (TMN) stage. Kaplan-Meier assays indicated that patients with NSCLC having high LINC01234 expressions tend to have unfavorable clinical prognosis. Using multivariate assays, it was confirmed that LINC01234 was an independent prognostic factor for patients with NSCLC. In vitro assays showed that inhibition of LINC01234 suppressed NSCLC cell proliferation, cell colony formation and metastasis, and greatly promoted apoptosis. Mechanistic investigations revealed LINC01234 promotes the progression of NSCLC cells by the modulation of miR-140 to positively regulate OTUB1 expression. Taken together our findings, they provided an exhaustive assay of LINC01234 in NSCLC and imperative clues for insights into the potential effects of lncRNAs-miRNAs regulatory network in NSCLC.

MicroRNA-625 inhibits the progression of non‑small cell lung cancer by directly targeting HOXB5 and deactivating the Wnt/β-catenin pathway.

Numerous microRNAs (miRs) are dysregulated in non‑small cell lung cancer (NSCLC), serving pivotal roles in its formation and progression. miR‑625 is dysregulated in several types of human cancer, but its involvement in the formation and development of NSCLC remains poorly understood. In the present study, we aimed to investigate miR‑625 expression in NSCLC and its role in regulating NSCLC cell behavior. miR‑625 expression in NSCLC tissues and cell lines was detected using reverse transcription‑quantitative polymerase chain reaction. The effects of miR‑625 overexpression on NSCLC cell proliferation, apoptosis, migration and invasion in vitro were assessed using an MTT assay, flow cytometry, and cell migration and invasion assays, respectively. The effects of miR‑625 upregulation on NSCLC growth were evaluated in an in vivo xenograft model. The molecular mechanisms underlying the tumor‑suppressing roles of miR‑625 in NSCLC were explored in detail. miR‑625 expression was determined to be downregulated in NSCLC tissues and cell lines. This decreased expression was associated with advanced clinical features and poor overall survival of patients with NSCLC. Exogenous miR‑625 expression suppressed NSCLC cell proliferation, migration and invasion, and induced apoptosis in vitro. miR‑625 upregulation hindered NSCLC tumor growth in vivo. Homeobox B5 (HOXB5) was proposed to be the direct target gene of miR‑625 in NSCLC cells. The tumor‑suppressing effects of HOXB5 silencing were similar to those of miR‑625 overexpression in NSCLC cells. In rescue experiments, HOXB5 overexpression partially reversed the inhibitory effects of miR‑625 in NSCLC cells. miR‑625 upregulation directly targeted HOXB5 to deactivate the Wnt/β‑catenin signaling pathway in NSCLC cells in vitro and in vivo. miR‑625 was determined to be associated with HOXB5 suppression and Wnt/β‑catenin pathway deactivation, which in turn inhibited the aggressive behavior of NSCLC cells in vitro and in vivo.

Diosmetin induces apoptosis and enhances the chemotherapeutic efficacy of paclitaxel in non-small cell lung cancer cells via Nrf2 inhibition.

Non-small-cell lung cancer (NSCLC) accounts for up to 80-85% of all lung cancers and has a disappointing prognosis. Flavonoids exert anticancer properties, mostly involving stimulation of ROS production without significant toxicity to normal cells. This study was aimed to delineate the effect of diosmetin, a natural flavonoid, on NSCLC cells and its ability to enhance the antitumour activity of paclitaxel. NSCLC cells, normal cell lines HLF-1 and BEAS-2B, and immunodeficient mice were chosen as models to study the effects of diosmetin. Changes in cell viability, apoptosis, and ROS were analysed by MTT assay, flow cytometry assay, and fluorescent probe DCFH-DA. Expression of proteins and mRNA was determined by Western blotting and real-time RT-PCR. Growth of xenografted tumours was measured. Spleens and other vital organs were analysed with histological and immunohistochemical techniques. Diosmetin induced selective apoptotic death in NSCLC cells but spared normal cells, via ROS accumulation. Diosmetin induced ROS production in NSCLC cells probably via reducing Nrf2 stability through disruption of the PI3K/Akt/GSK-3β pathway. The in vitro and in vivo xenograft studies showed that combined treatment of diosmetin and paclitaxel synergistically suppressed NSCLC cells. Histological analysis of vital organs showed no obvious toxicity of diosmetin, which matched our in vitro findings. Diosmetin selectively induced apoptosis and enhanced the efficacy of paclitaxel in NSCLC cells via ROS accumulation through disruption of the PI3K/Akt/GSK-3β/Nrf2 pathway. Therefore, diosmetin may be a promising candidate for adjuvant treatment of NSCLC.