H1299 Non-small Cell Lung Cancer Cells Research Articles

PMEPA1 facilitates non-small cell lung cancer progression via activating the JNK signaling pathway.

Prostate transmembrane protein androgen-induced 1 (PMEPA1), a critical checkpoint of multiple signaling pathways, has been demonstrated to play a crucial role in various types of cancers. However, little is known about its function in non-small cell lung cancer (NSCLC). Our objective is to explore the function of PMEPA1 and its potential mechanisms in NSCLC progression. PMEPA1 expression and prognostic significance in adenocarcinoma of lung cancer (LUAD) and squamous cell carcinoma of lung cancer (LUSC) were determined using Gene Expression Profiling Interactive Analysis (GEPIA). Next, a series of cell assays were performed to examine whether overexpression or depletion of PMEPA1 affected the malignant behaviors of NSCLC H1299 cells, such as proliferation and migration. Luciferase reporter gene assays and SP600125 (a JNK inhibitor) were employed to ascertain the regulatory relationship between PMEPA1 and JNK. PMEPA1 is overexpressed in LUAD and LUSC tissues and portends a worse prognosis for cancer patients. Gain and loss of function experiments demonstrated that PMEPA1 executes oncogenetic function in H1299 cells. Mechanism studies elucidated that PMEPA1 stimulated the transcriptional activity of the JNK pathway. PMEPA1 increased the H1299 cell viability, proliferation, and migration which works, at least partially, by triggering the JNK activity. Hence, our findings support that the PMEPA1/JNK axis might be a promising therapeutic target for this challenging disease.

Role of the epithelial-mesenchymal transition-related circular RNA, circ-10720, in non-small-cell lung cancer.

BackgroundCircular RNAs (circRNAs) are non-coding RNAs with a circular structure that have recently emerged as important regulators of tumorogenesis. Recently, several circRNAS, including circ-10720 have been related to epithelial-mesenchymal transition (EMT) process. In the present study, we have analyzed the role of circ-10720 in non-small-cell lung cancer (NSCLC) and studied its prognostic relevance in resected stage I–IIIa NSCLC patients.MethodsCirc-10720 expression was analyzed using a custom TaqMan assay in four NSCLC cell lines (HCC44, A549, H23 and H1299) and in the normal immortalized lung cell line BEAS2B. Silencing of circ-10720 was performed using two custom siRNAs which were transfected using lipofectamine 2000. Protein levels were evaluated by Western blot and immunofluorescence. Wound healing and invasion assays were performed to evaluate the impact the circRNA on cell motility. Apoptosis was analyzed by evaluation of Caspase 3–7 activity and proliferation by MTS assay. Moreover, the expression levels of the circRNA were studied in 119 resected NSCLC patients. The expression in tumor tissue was correlated with the main clinicopathological characteristics and with time to relapse (TTR).ResultsCirc-10720 was overexpressed in HCC44 and A549 and underexpressed in H23 and H1299 NSCLC cell lines in comparison to BEAS2B normal immortalized lung cell line. CircRNA knockdown in the two circ-10720 overexpressing cell lines was associated with a decrease of Vimentin (VIM) and an increase of E-cadherin (CDH1) protein levels, loss of mesenchymal phenotype, and a significant reduction of migration and invasion capacity. After silencing circ-10720, the apoptosis rate increased and the proliferation was significantly reduced. Furthermore, circ-10720 was upregulated in tumor vs. normal tissue from 119 resected NSCLC patients. In the group of patients not receiving adjuvant treatment, those with high levels of circ-10720 had a shorter TTR than those with low levels and emerged as an independent prognostic value in the multivariate analysis. In tumor tissue, circ-10720 levels positively correlated with the EMT gene Twist1 levels.ConclusionsCirc-10720 regulates EMT, apoptosis and proliferation and acts as a biomarker of relapse in NSCLC.

G9a Promotes Invasion and Metastasis of Non-Small Cell Lung Cancer through Enhancing Focal Adhesion Kinase Activation via NF-κB Signaling Pathway.

Potential roles of euchromatic histone methyltransferase 2 (EHMT2 or G9a) in invasion and metastasis are not well understood in non-small cell lung cancer (NSCLC). Here, we investigated the effect and underlying mechanisms of G9a and therapeutic implications of targeting G9a in the invasion and metastasis of NSCLC. Overexpression of G9a significantly enhanced in vitro proliferation and invasion, while knockdown of G9a drastically suppressed in vivo growth and metastasis of A549 and H1299 NSCLC cells. Knockdown or inhibition of G9a significantly decreased the expression of focal adhesion kinase (FAK) protein and activation of FAK pathway. In addition, defactinib, a potent FAK inhibitor, partially abolished the G9a-enhanced invasion in these NSCLC cells. Furthermore, targeting G9a was found to suppress NF-κB transcriptional activity in NSCLC cells through stabilizing NF-κB inhibitor alpha (IκBα), while an NF-κB inhibitor Parthenilide partially abolished the G9a-enhanced FAK activation, which suggests that G9a-enhanced invasion and activation of FAK is mediated by elevated NF-κB activity. Notably, a strong positive correlation between the IHC staining of G9a and phosphorylated FAK proteins was identified in H1299 xenografts and 159 cases of NSCLC tissues (R = 0.408). IMPLICATIONS: The findings of this study strongly demonstrate that G9a may promote invasion and metastasis of NSCLC cells by enhancing FAK signaling pathway via elevating NF-κB transcriptional activity, indicating potential significance and therapeutic implications of these pathways in the invasion and metastasis of NSCLCs that overexpress G9a protein.

Long non‑coding RNA MEG3 inhibits cell migration and invasion of non‑small cell lung cancer cells by regulating the miR‑21‑5p/PTEN axis.

Long non-coding RNAs (lncRNAs) are involved in the occurrence and progression of numerous types of cancer. The aim of the present study was to evaluate the effect of the lncRNA maternally expressed gene 3 (MEG3) on the migration and invasion of non-small cell lung cancer (NSCLC) H1299 and PC9 cells. Reverse transcription-quantitative (RT-q)PCR analysis showed that MEG3 was downregulated in NSCLC PC9 and H1299 cells. Additionally, bioinformatics analysis indicated that MEG3 sponges microRNA (miR)-21-5p; miR-21-5p was predicted to target the phosphatase and tensin homolog (PTEN) 3′-untranslated region sequence. MEG3 overexpression led to miR-21-5p suppression and PTEN upregulation in PC9 and H1299 cells, as detected by RT-qPCR. Subsequently, western blot analysis confirmed that MEG3 overexpression enhanced PTEN expression levels and inhibited the PI3K/AKT signaling pathway in NSCLC cells. These effects were attenuated by miR-21-5p. Dual luciferase assay supported the sponging effect of MEG3 on miR-21-5p and validated the direct interaction between miR-21-5p and PTEN. Furthermore, Transwell assay demonstrated that MEG3 overexpression had an inhibitory effect on cell migration and invasion. MEG3 overexpression also mediated epithelial-to-mesenchymal transition by significantly enhancing E-cadherin and decreasing N-cadherin, Vimentin and matrix metalloprotein 9 expression levels in NSCLC cells, as indicated by western blot analysis. These changes were partially reversed by an miR-21-5p mimic. These results indicated that MEG3 acted as a tumor suppressor that inhibited NSCLC cell migration and invasion via sponging miR-21-5p, which, in turn, enhanced the expression levels of PTEN, in part via the PI3K/AKT signaling pathway. The results of the present study have suggested the potential of MEG3 as a novel therapeutic target for NSCLC treatment.

Effect of EG00229 on Radiation Resistance of Lung Adenocarcinoma Cells.

Background: Neuropilin 1 (NRP1) is a pleiotropic receptor that interacts with multiple ligands and their receptors and plays a critical role in the process of tumor metastasis and radiation resistance in endothelial cells and tumor cells. In this study, we sought to investigate the mechanistic role of NRP1 in the radiation resistance of non-small cell lung cancer (NSCLC) cells and the role of EG00229 (an inhibitor of NRP1) on reversing radiation resistance.Materials and Methods: A549 and H1299 NSCLC cells were used to construct radiation resistance models. Western blot, ELISA, and qRT-PCR were used to detect protein and mRNA levels of NRP1, epithelial-mesenchymal transition (EMT) markers, and molecules in signaling pathways. Immunofluorescence was used to measure changes in co-expression of NRP1 and VEGF-165 in radiation-resistant model cells. An immunoprecipitation assay was used to detect the binding capacity of NRP1 and VEGF-165.Results: We successfully created two radiation resistant models (A549RR and H1299-RR). The expression levels of NRP1, EMT-related proteins, and proteins in metastasis-related pathways were increased in NSCLC cells with radiation resistance. After adding EG00229, the expression levels and binding capacity of NRP1 and VEGF-165 proteins were significantly reduced. The expression of EMT-related proteins and proteins in metastasis-related pathways were reduced in NSCLC cells with radiation resistance.Conclusion: Our data provide an insight into the molecular mechanisms of radiation resistance and suggest that EG00229 may contribute to reversing the radiation resistance of NSCLC cells by inhibiting the binding of NRP1 and VEGF-165. Our findings could provide a novel theoretical and experimental foundation for improving the efficacy of lung cancer radiotherapy.

The long non-coding RNA SNHG4/microRNA-let-7e/KDM3A/p21 pathway is involved in the development of non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is a foremost cause of malignancy-associated mortality globally. Recent studies have emphasized long non-coding RNAs (lncRNAs) as important biomarkers with diagnostic and therapeutic potential in regard to NSCLC. This study aimed to elucidate the functional role of lncRNA small nucleolar RNA host gene 4 (SNHG4) in NSCLC. Initially, 50 paired cancerous and noncancerous tissues were obtained from NSCLC patients. Human NSCLC H1299 cells were assayed to evaluate viability, colony formation, invasion, migration, cycle arrest, and apoptosis via Cell Counting Kit-8 (CCK-8), plate clone formation, and transwell invasion assays, as well as a scratch test and flow cytometry. A dual-luciferase reporter gene assay was used to examine lncRNA SNHG4 binding with miR-let-7e and miR-let-7e binding with lysine demethylase 3A (KDM3A). H1299 cells were xenografted into nude mice. lncRNAs SNHG4 and KDM3A were both upregulated in NSCLC tissues. The knockdown of lncRNA SNHG4 or KDM3A inhibited H1299 cell viability, colony formation, invasion, migration, and cycle progression while inducing apoptosis. lncRNA SNHG4 was found to bind to miR-let-7e that negatively targeted KDM3A. KDM3A inhibited p53-K372me1, thus reducing p21 expression. The NSCLC development was inhibited by downregulating lncRNA SNHG4 in nude mice. Taken together, the key findings of the current study demonstrate a novel lncRNA SNHG4/let-7e/KDM3A/p21 axis in NSCLC, highlighting a promising therapeutic target for NSCLC.

MicroRNA-let-7e in serum-derived exosomes inhibits the metastasis of non-small-cell lung cancer in a SUV39H2/LSD1/CDH1-dependent manner.

Non-small-cell lung cancer (NSCLC) remains the leading cause of cancer-related death worldwide. Accumulating research has highlighted the ability of exosome-encapsulated microRNAs (miRNAs or miRs) as potential circulating biomarkers for lung cancer. The current study aimed to evaluate the clinical significance of serum-derived exosomal miR-let-7e as a biomarker in the metastasis of NSCLC. Initially, the expression of miR-let-7e, SUV39H2, and CDH1 in human NSCLC tissues and exosomes isolated from the serum of NSCLC patients was determined by RT-qPCR, demonstrating that miR-let-7e was downregulated in NSCLC tissues and serum-derived exosomes, while SUV39H2 was upregulated in NSCLC tissues. Kaplan-Meier method revealed that both lower miR-let-7e expression and higher SUV39H2 expression were correlated with a lower survival rate of NSCLC patients. Next, SUV39H2 was predicted and validated to be a target of miR-let-7e using dual-luciferase reporter assay. NSCLC H1299 cells following ectopic expression and depletion experiments of miR-let-7e and SUV39H2 were treated with serum-derived exosomes, after which the viability, migration, and invasion of H1299 cells were detected using CCK-8 and Transwell assays. Further, in vivo experiments were conducted to elucidate the effect of exosomal miR-let-7e on tumorigenesis. Results revealed that miR-let-7e overexpression in serum-derived exosomes inhibited SUV39H2, resulting in impaired cell viability, migration, and invasion in vitro as well as delayed tumor growth in vivo. In conclusion, the key findings of the current study demonstrate that exosomal miR-let-7e from serum possesses anticarcinogenic properties against NSCLC via the SUV39H2/LSD1/CDH1 axis.

Beyond Blocking: Engineering RNAi-Mediated Targeted Immune Checkpoint Nanoblocker Enables T-Cell-Independent Cancer Treatment.

The emergence of immune checkpoint blockade to activate host T cells to attack tumor cells has revolutionized the cancer treatment landscape over the past decade. However, sustained response has only been achieved in a small proportion of patients. This can be attributed to physiological barriers, such as T-cell heterogeneity and immunosuppressive tumor microenvironments. To this can be added obstacles intrinsic to traditional antibody-driven blockade methods, including the inability to inhibit checkpoint translocation from cytoplasm, systemic immune toxicity, and "bite back" effect on T cells. Using non-small cell lung cancer (NSCLC) as the cancer model, here we report an unconventional, yet powerful, tumor-targeted checkpoint blocking strategy by RNAi nanoengineering for T-cell-independent cancer therapy. Unlike antibodies, such nanoblocker silences both membranous and cytoplasmic PD-L1 in cancer cells, thus eliminating the binding step. Moreover, it is demonstrated that silencing of PD-L1 by the nanoblocker can cause the direct programmed cell death of NSCLC H460 cells, without the need of T-cell intervention. In vivo results from xenograft tumor models further demonstrate that tumor-homing peptide modification enables the nanoblocker to accumulate in the tumor tissue, downregulate the PD-L1 expression, and inhibit the tumor growth more efficiently than the nontargeted group. These findings may offer an effective means toward overcoming barriers against traditional checkpoint blockade and provide different insights into the molecular mechanism(s) underlying immunotherapy.

In Silico and In Vitro Analysis of lncRNA XIST Reveals a Panel of Possible Lung Cancer Regulators and a Five-Gene Diagnostic Signature.

Simple SummaryLong non-coding RNAs (lncRNA) have been associated with a number of diseases including cancer. A well-studied lncRNA called XIST (X-inactive specific transcript) acts as a major effector of the X-inactivation process. It is expressed on the inactive X chromosome providing a dosage equivalence between males and females. Recently XIST has been implicated in the development of lung cancer. Using a bioinformatics approach, we demonstrate the XIST is over-expressed in female patients compared to males. When XIST gene was silenced in two different cell lines (of male and female origin), a number of genes were differentially expressed; playing a role in signal transduction pathways, energy balance and metabolism, thus providing a better insight of the role of this lncRNA in cancer. Finally, we showed that expression of XIST with another 4 genes provided a strong diagnostic potential to discriminate lung cancer from healthy controls.Long non-coding RNAs (lncRNAs) perform a wide functional repertoire of roles in cell biology, ranging from RNA editing to gene regulation, as well as tumour genesis and tumour progression. The lncRNA X-inactive specific transcript (XIST) is involved in the aetiopathogenesis of non-small cell lung cancer (NSCLC). However, its role at the molecular level is not fully elucidated. The expression of XIST and co-regulated genes TSIX, hnRNPu, Bcl-2, and BRCA1 analyses in lung cancer (LC) and controls were performed in silico. Differentially expressed genes (DEGs) were determined using RNA-seq in H1975 and A549 NSCLC cell lines following siRNA for XIST. XIST exhibited sexual dimorphism, being up-regulated in females compared to males in both control and LC patient cohorts. RNA-seq revealed 944 and 751 DEGs for A549 and H1975 cell lines, respectively. These DEGs are involved in signal transduction, cell communication, energy pathways, and nucleic acid metabolism. XIST expression associated with TSIX, hnRNPu, Bcl-2, and BRCA1 provided a strong collective feature to discriminate between controls and LC, implying a diagnostic potential. There is a much more complex role for XIST in lung cancer. Further studies should concentrate on sex-specific changes and investigate the signalling pathways of the DEGs following silencing of this lncRNA.

TOPK mediates hypoxia-induced epithelial-mesenchymal transition and the invasion of nonsmall-cell lung cancer cells via the HIF-1α/snail axis

Hypoxia has been suggested to induce epithelial-mesenchymal transition (EMT) in various cancer types via the transcription factor hypoxia-inducible factor-1 alpha (HIF-1α). Here, we demonstrated that TOPK upregulates EMT and the invasion of H460 nonsmall-cell lung cancer cells through the induction of the HIF-1α/Snail axis and hypoxic signaling. The expression of endogenous TOPK, phosphorylated TOPK, HIF-1α and Snail was significantly increased upon hypoxia exposure, but TOPK depletion markedly abrogated the induced mRNA and protein levels of HIF-1α and Snail. Interestingly, TOPK knockdown restored the hypoxia-induced suppression of E-cadherin and diminished hypoxia-induced N-cadherin expression. In addition, Snail depletion suppressed hypoxia-induced N-cadherin expression, which was attenuated by TOPK knockdown. Moreover, knockdown of Snail decreased hypoxia-induced nonsmall-cell lung cancer cell migration and invasion, which were suppressed by TOPK depletion. In summary, we conclude that TOPK positively regulates HIF-1α expression through hypoxia signaling and thereby promotes Snail expression, leading to EMT and the invasion of nonsmall-cell lung cancer cells. These findings suggest that TOPK plays a critical role as a novel mediator of hypoxia signaling that regulates nonsmall-cell lung cancer development.

Exploitation of the chick embryo chorioallantoic membrane (CAM) as a platform for anti-metastatic drug testing

The establishment of clinically relevant models for tumor metastasis and drug testing is a major challenge in cancer research. Here we report a physiologically relevant assay enabling quantitative analysis of metastatic capacity of tumor cells following implantation into the chorioallantoic membrane (CAM). Engraftment of as few as 103 non-small cell lung cancer (NSCLC) and prostate cancer (PCa) cell lines was sufficient for both primary tumor and metastasis formation. Standard 2D-imaging as well as 3D optical tomography imaging were used for the detection of fluorescent metastatic foci in the chick embryo. H2228- and H1975-initiated metastases were confirmed by genomic analysis. We quantified the inhibitory effect of docetaxel on LNCaP, and that of cisplatin on A549- and H1299-initiated metastatic growths.The CAM assay also mimicked the sensitivity of ALK-rearranged H2228 and EGFR-mutated H1975 NSCLC cells to tyrosine kinase inhibitors crizotinib and gefitinib respectively, as well as sensitivity of LNCaP cells to androgen-dependent enzalutamide therapy. The assay was suggested to reconstitute the bone metastatic tropism of PCa cells. We show that the CAM chick embryo model may be a powerful preclinical platform for testing and targeting of the metastatic capacity of cancer cells.

Radiosensitization of NSCLC cells to X-rays and carbon ions by the CHK1/CHK2 inhibitor AZD7762, Honokiol and Tunicamycin.

Although radiotherapy, especially carbon-ion radiotherapy, is an effective treatmentmodality against non-small-cell lung cancer (NSCLC), studies using radiation combined with sensitizer for improving the efficacy of radiotherapy are still needed. In this work, we aimed to investigate in NSCLC A549 and H1299 cell lines the effects of different linear energy transfer (LET) radiations combined with diverse sensitizing compounds. Cells pretreated with the CHK1/CHK2 inhibitor AZD7762, Honokiol or Tunicamycin were irradiated with low-LET X-rays and high-LET carbon ions. Cell survival was assessed using the clonogenic cell survival assay. Cell cycle distribution and apoptosis were measured with flow cytometry, and DNA double strand break (DSB) and repair were detected using γ-H2AX immunofluorescence staining. Our results revealed that AZD7762, Honokiol and Tunicamycin demonstrated low cytotoxicity to NSCLC cells and a pronounced radiosensitizing effect on NSCLC cells exposed to carbon ions than X-rays. Unrepaired DNA DSB damages, the abrogation of G2/M arrest induced by irradiation, and finally apoptotic cell death were the main causes of the radiosensitizing effect. Thus, our data suggest that high-LET carbon ion combined with these compounds may be a potentially effective therapeutic strategy for locally advanced NSCLC.

Abstract 3842: Histone methyltransferase G9a promotes migration, invasion of non-small cell lung cancer through enhancing FAK and NF-kB signal pathways

Abstract Overexpression of euchromatic histone methyltransferase 2 (EHMT2 or G9a) is associated with carcinogenesis and poor prognosis in a number of cancer types. In this study, we investigated the effect of G9a on invasion of non-small cell lung cancer (NSCLC) cells and the mechanism underlying cancer cell invasion. Knockdown of G9a was shown to drastically suppress in vitro cellular proliferation, migration, and invasion of A549 and H1299 NSCLC cells, while overexpression of G9a significantly enhanced the proliferation and invasion of these NSCLC cells. Additionally, knockdown of G9a led to significant decrease in expression of focal adhesion kinase (FAK) gene and activation of FAK pathway. UNC0638, a selective inhibitor of G9a, significantly suppressed both the proliferation, migration and invasion of these NSCLC cells, and concurrently led to a dramatic drop of global demethylation of H3K9 (H3K9me2) and suppressed the activation of FAK pathway. In addition, Defactinib, a potent FAK inhibitor, partially abolished the enhanced invasion by overexpression of G9a in these NSCLC cells. Furthermore, G9a was proved to regulate nuclear NF-κB transcriptional activity in NSCLC cells, and NF-κB inhibitor partially abolished the activated FAK signaling pathway by overexpressed G9a, which indicates that G9a-enhanced invasion and activation of FAK pathway may be mediated by NF-κB transcriptional activity. Notably, immunohistochemistry analysis further revealed that activation of FAK pathway was associated with overexpressed G9a in about 150 cases of NSCLC tissues. Altogether, these data have strongly demonstrated that G9a may promote invasion of NSCLC cells by upregulating FAK protein and pathway via enhancing NF-κB transcriptional activity, indicating a therapeutic implication of targeting these two pathways in NSCLC with overexpressed G9a. Citation Format: Ting Sun, Keqiang Zhang, Rajendra P. Pangeni, Wendong Li, Dan J. Raz. Histone methyltransferase G9a promotes migration, invasion of non-small cell lung cancer through enhancing FAK and NF-kB signal pathways [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 3842.

Triptolide-induced apoptosis in non-small cell lung cancer via a novel miR204-5p/Caveolin-1/Akt-mediated pathway.

Lung cancer is one of the most prevalent malignancies world-wide with non-small cell lung cancer (NSCLC) comprising nearly 80% of all cases. Unfortunately, many lung cancer patients are diagnosed at advanced stages of the disease with an associated poor prognosis. Recently, the Chinese herb root extract Triptolide/Minnelide (TL) has shown significant promise as a therapeutic agent for NSCLC treatment both in vitro and in vivo. The aim of this study was to investigate the underlying mechanism(s) of action regarding TL-induced cytotoxicity in NSCLC. We demonstrate that triptolide treatment of A549 and H460 NSCLC cells decreases Caveolin-1 (CAV-1) mRNA/protein expression, resulting in activation of the Akt/Bcl-2-mediated mitochondrial apoptosis pathway. CAV-1 down-regulation was triggered by Micro-RNA 204-5p (miR204-5p) up-regulation and could be significantly blocked by pre-treatment with both Sirt-1/Sirt-3 specific siRNA and SIRT-1/SIRT-3 enzyme inhibitors, EX-527 and nicotinamide. Overall, our results provide evidence for a novel mechanism by which TL exerts its cytotoxic effects on NSCLC via CAV-1 down-regulation. Furthermore, these findings demonstrate a pivotal role for TL induction of the Akt/Bax pathway in apoptosis of human lung cancer.

LncRNA MEG8 promotes tumor progression of non-small cell lung cancer via regulating miR-107/CDK6 axis.

Mounting evidence has implicated the vital role of long noncoding RNAs (lncRNAs) in non-small cell lung cancer (NSCLC). This study aims to investigate the mechanism of lncRNA MEG8 on NSCLC progression. The mRNA expressions of MEG8 and miR-107 were examined in tumor and adjacent normal tissues from patients with NSCLC by qRT-PCR. Lung epithelial BEAS-2B cells were transfected with MEG8 overexpression plasmid, and NSCLC A549 and H1299 cells were transfected with MEG8 or miR-107 overexpression/knockdown plasmid to detect the function of MEG8 or miR-107 on cell activity. The function of MEG8 and miR-107 on cell proliferation, cell cycle changes, invasion and migration was separately determined by Cell counting kit-8 assay and 5-ethynyl-2'-deoxyuridine staining, flow cytometry, transwell and cell scratch test. Target sites for miR-107 and MEG8, miR-107 and CDK6 were determined and verified by a dual luciferase gene reporter assay. The expression levels of the Rb/E2F3 signal pathway related proteins (p21, p27, E2F3 and Rb) were inspected by Western blot. MEG8 was strongly expressed while miR-107 was lowly expressed in tumor tissues and cells. Overexpression of MEG8 potentiated cell proliferation, migration and invasion in BEAS-2B cells. Silencing MEG8 or overexpression of miR-107 clearly hindered cell progression in A549 and H1299 cells. Mechanistically, MEG8 and CDK6 can competitively bind to miR-107 and together regulate the progression of NSCLC. Additionally, silencing MEG8 or overexpression of miR-107 can inhibit the phosphorylation levels of Rb and E2F3. Evidence in this work indicated that MEG8 regulates miR-107/CDK6 axis to promote NSCLC progression by activating the Rb/E2F3 pathway.

Cetuximab-Triptolide Conjugate Suppresses the Growth of EGFR-Overexpressing Lung Cancers through Targeting RNA Polymerase II

To overcome poor pharmacokinetics and toxicity of triptolide (TPL), a natural compound that exhibits potent anticancer activities, we developed a novel antibody-drug conjugate (ADC) to specifically deliver TPL to epidermal growth factor receptor (EGFR)-overexpressing non-small cell lung cancer (NSCLC) and others. The ADC (Cet-TPL) is made by conjugation of TPL to lysine residues of cetuximab (Cet), a clinically available anti-EGFR monoclonal antibody. Studies of antitumor efficacy demonstrated that Cet-TPL drastically suppressed in vitro proliferation and in vivo growth of these EGFR-overexpressing cancers, including NSCLC A549 and H1299 cells and two patient-derived xenografts, and head and neck squamous carcinoma UM-SCC6 cell, while it did not inhibit the proliferation and growth of NSCLC H520 that rarely expresses EGFR. Furthermore, immunofluorescence analysis revealed that Cet-TPL was effectively internalized and transported into lysosomes of EGFR-overexpressing cells. Cet-TPL effectively led to degradation of RNA polymerase II (Pol II) and demethylation of histone H3 lysines, and significantly induced apoptosis in these EGFR-overexpressing cancers. Compared with TPL, Cet, or their combination, Cet-TPL displayed higher target-specific cytotoxicity against EGFR-expressing cancers and much lower in vivo toxicity. In addition, Cet-TPL efficiently suppressed the activated EGFR pathway in UM-SCC6 cancer cells. Taken together, Cet-TPL represents a potent targeting therapeutic agent against EGFR-overexpressing NSCLC and others.

The Role of Transcriptional Factor Brachyury on Cell Cycle Regulation in Non-small Cell Lung Cancer.

Lung cancer is the leading cause of cancer-related death, and non-small cell lung cancer (NSCLC) accounts for almost 80–85% of all lung cancer cases. The transcriptional factor brachyury has been verified to promote tumor cells migrate, invade, and metastasis in various types of tumors, whereas divergent roles of brachyury on cell proliferation have been reported in several types of tumor cells. In this study, we attempted to explore the effect of brachyury on the cell cycle progression and proliferation capability of NSCLC cells. Firstly, we performed RNA-sequence and ChIP-sequence to explore underlying downstream pathways regulated by brachyury. Cell proliferation and colony formation assays were utilized to detect the effect of brachyury on the proliferation ability of two types of lung NSCLC cells: H460 and Calu-1, which represent different brachyury expression levels. Following cell cycle and cell apoptosis assays were used to investigate the mechanism by which brachyury promotes NSCLC grow and progression. RNA-sequence and ChIP-sequence (ChIP-seq) showed that one of the vital downstream pathways regulated by brachyury involves in cell cycle progression. Through cell proliferation assays and colony formation assays, we found that inhibition of brachyury could decrease the capability of proliferation in H460 cells. We also found that brachyury overexpression could prevent the transition from G0/G1 to S phase in Calu-1 cells, and brachyury knockdown could decrease the transition of G2/M phase in H460 cells. The cell apoptosis assays showed that inhibition of brachyury could promote apoptosis in H460 cells. In this study we demonstrate that brachyury and downstream target genes together involve in tumor cell cycle regulation by inducing accelerated transition through G2/M, promote tumor cell proliferation and inhibit apoptosis in lung NSCLC H460 cells. Targeting brachyury expression could be developed into a promising avenue for the prevention of lung cancer progression.

Optimization of the Enrichment of Circulating Tumor Cells for Downstream Phenotypic Analysis in Patients with Non-Small Cell Lung Cancer Treated with Anti-PD-1 Immunotherapy.

The current study aimed at the optimization of circulating tumor cell (CTC) enrichment for downstream protein expression analyses in non-small cell lung cancer (NSCLC) to serve as a tool for the investigation of immune checkpoints in real time. Different enrichment approaches—ficoll density, erythrolysis, their combination with magnetic separation, ISET, and Parsortix—were compared in spiking experiments using the A549, H1975, and SKMES-1 NSCLC cell lines. The most efficient methods were tested in patients (n = 15) receiving immunotherapy targeting programmed cell death-1 (PD-1). Samples were immunofluorescently stained for a) cytokeratins (CK)/epithelial cell adhesion molecule (EpCAM)/leukocyte common antigen (CD45), and b) CK/programmed cell death ligand-1 (PD-L1)/ indoleamine-2,3-dioxygenase (IDO). Ficoll, ISET, and Parsortix presented the highest yields and compatibility with phenotypic analysis; however, at the patient level, they provided discordant CTC positivity (13%, 33%, and 60% of patients, respectively) and enriched for distinct CTC populations. IDO and PD-L1 were expressed in 44% and 33% and co-expressed in 19% of CTCs. CTC detection was associated with progressive disease (PD) (p = 0.006), reduced progression-free survival PFS (p = 0.007), and increased risk of relapse (hazard ratio; HR: 10.733; p = 0.026). IDO-positive CTCs were associated with shorter PFS (p = 0.039) and overall survival OS (p = 0.021) and increased risk of death (HR: 5.462; p = 0.039). The current study indicates that CTC analysis according to distinct immune checkpoints is feasible and may provide valuable biomarkers to monitor NSCLC patients treated with anti-PD-1 agents.

Kinesin superfamily protein 21B acts as an oncogene in non-small cell lung cancer

BackgroundKinesin superfamily proteins (KIFs) serve as microtubule-dependent molecular motors, and are involved in the progression of many malignant tumors. In this study, we aimed to investigate the expression pattern and precise role of kinesin family member 21B (KIF21B) in non-small cell lung cancer (NSCLC).MethodsKIF21B expression in 72 cases of NSCLC tissues was measured by immunohistochemical staining (IHC). We used shRNA-KIF21B interference to silence KIF21B in NSCLC H1299 and A549 cells and normal lung epithelial bronchus BEAS-2B cells. The biological roles of KIF21B in the growth and metastasis abilities of NSCLC cells were measured by Cell Counting Kit-8 (CCK8), colony formation and Hoechst 33342/PI, wound-healing, and Transwell assays, respectively. Expression of apoptosis-related proteins was determined using western blot. The effect of KIF21B on tumor growth in vivo was examined using nude mice model.ResultsKIF21B was up-regulated in NSCLC tissues, and correlated with pathological lymph node and pTNM stage, its high expression was predicted a poor prognosis of patients with NSCLC. Silencing of KIF21B mediated by lentivirus-delivered shRNA significantly inhibited the proliferation ability of H1299 and A549 cells. KIF21B knockdown increased apoptosis in H1299 and A549 cells, down-regulated the expression of Bcl-2 and up-regulated the expression of Bax and active Caspase 3. Moreover, KIF21B knockdown decreased the level of phosphorylated form of Akt (p-Akt) and Cyclin D1 expression in H1299 and A549 cells. In addition, silencing of KIF21B impeded the migration and invasion of H1299 and A549 cells. Further, silencing of KIF 21B dramatically inhibited xenograft growth in BALB/c nude mice. However, silencing of KIF21B did not affect the proliferation, migration and invasion of BEAS-2B cells.ConclusionsThese results reveal that KIF21B is up-regulated in NSCLC and acts as an oncogene in the growth and metastasis of NSCLC, which may function as a potential therapeutic target and a prognostic biomarker for NSCLC.

NCAPH Is Required for Proliferation, Migration and Invasion of Non-small-cell Lung Cancer Cells.

Non-structural maintenance of chromosomes condensin I complex subunit H (NCAPH) is implicated in correct chromosome condensation and segregation during mitosis. However, the functional role of NCAPH in the pathogenesis of non-small-cell lung cancer (NSCLC) remains unclear. The aim of this study was to elucidate the role of NCAPH in NSCLC cells. A549 and H1299 NSCLC cells were transfected with small-interfering RNA (siRNA) against NCAPH. Subsequently, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony-formation assay and flow cytometry analysis were performed to reveal the role of NCAPH in NSCLC cells. In addition, migration and invasion assay were also performed. NCAPH knockdown inhibited cell proliferation, induced cell-cycle arrest at G2/M phase, and prevented colony formation, migration and invasion by NSCLC cells. NCAPH is involved in NSCLC progression and development, and may be a potential therapeutic target for NSCLC treatment.