Non-small Cell Lung Carcinoma Cells Research Articles

Coumarin Derivatives Exert Anti-Lung Cancer Activity by Inhibition of Epithelial-Mesenchymal Transition and Migration in A549 Cells.

A series of coumarin derivatives and isosteres were synthesized from the reaction of triflic intermediates with phenylboronic acids, terminal alkynes, and organozinc compounds through palladium-catalyzed cross-coupling reactions. The in vitro cytotoxic effect of the compounds was evaluated against two non-small cell lung carcinoma (NSCLC) cell lines (A-549 and H2170) and a normal cell line (NIH-3T3) using cisplatin as a reference drug. Additionally, the effects of the most promising coumarin derivative (9f) in reversing the epithelial-to-mesenchymal transition (EMT) in IL-1β-stimulated A549 cells and in inhibiting the EMT-associated migratory ability in A549 cells were also evaluated. 9f had the greatest cytotoxic effect (CC50 = 7.1 ± 0.8 and 3.3 ± 0.5 μM, respectively against A549 and H2170 cells) and CC50 value of 25.8 µM for NIH-3T3 cells. 9f inhibited the IL-1β-induced EMT in epithelial cells by inhibiting the F-actin reorganization, attenuating changes in the actin cytoskeleton reorganization, and downregulating vimentin in A549 cells stimulated by IL-1β. Treatment of A549 cells with 9f at 7 µM for 24 h significantly reduced the migration of IL-1β-stimulated cells, which is a phenomenon confirmed by qualitative assessment of the wound closure. Taken together, our findings suggest that coumarin derivatives, especially compound 9f, may become a promising candidate for lung cancer therapy, especially in lung cancer promoted by NSCLC cell lines.

FOXP3 activated-LINC01232 accelerates the stemness of non-small cell lung carcinoma by activating TGF-β signaling pathway and recruiting IGF2BP2 to stabilize TGFBR1

Non-small cell lung carcinoma (NSCLC) is one of the most common malignant tumors worldwide with high incidence and mortality. Long non-coding RNAs (lncRNAs) have been reported to affect human cancer progression. The present study aimed to investigate the regulatory role and mechanism of long intergenic non-protein coding RNA 1232 (LINC01232) in NSCLC cells. RT-qPCR results revealed that LINC01232 expression was high in NSCLC cells. Flow cytometry and sphere formation assays indicated that LINC01232 significantly promoted NSCLC cell stemness. Luciferase reporter assay and ChIP assay validated that forkhead box P3 (FOXP3) could bind to LINC01232 promoter and activate LINC01232 transcription. Further, LINC01232 was certified to activate TGF-β signaling pathway through regulating transforming growth factor beta receptor 1 (TGFBR1). After RIP and RNA pull down assays, insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) was proven as the RNA-binding protein (RBP) for LINC01232. LINC01232 promoted TGFBR1 mRNA stability via recruiting IGF2BP2. Subsequently, LINC01232 was verified to accelerate NSCLC cell stemness and induce macrophage M2 polarization via upregulating TGFBR1. Taken together, FOXP3 activated-LINC01232 accelerated NSCLC cell stemness by activating TGF-β signaling pathway and recruiting IGF2BP2 to stabilize TGFBR1, which might offer a rationale for lncRNA-based treatment to NSCLC.

CircFBXW8 Acts an Oncogenic Role in the Malignant Progression of Non-small Cell Lung Carcinoma by miR-370-3p-Dependent Regulation of TRIM44.

Non-small cell lung carcinoma (NSCLC) is an aggressive malignant tumor. Growing evidences have revealed that circular RNA (circRNA) is involved in NSCLC progression. This study aims to investigate the role of circular RNA F-box and WD repeat domain containing 8 (circFBXW8) in NSCLC progression and the underlying mechanism. The expression of circFBXW8, microRNA-370-3p (miR-370-3p) and tripartite motif containing 44 (TRIM44) mRNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Protein expression was detected by western blot analysis or immunohistochemistry assay. Additionally, cell viability, colony-forming ability, proliferation and apoptosis were investigated by 3-(4,5-Dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide, cell colony formation, 5-Ethynyl-29-deoxyuridine and flow cytometry analysis assays, respectively. Cell migratory and invasive abilities were examined by wound-healing and transwell assays. The regulatory relationship between miR-370-3p and circFBXW8 or TRIM44 was identified by dual-luciferase reporter and RNA pull-down assays. Furthermore, xenograft experiment was employed to explain the effect of circFBXW8 silencing on tumor formation. CircFBXW8 and TRIM44 expression were upregulated, while miR-370-3p was downregulated in NSCLC tissues, cells and the exosomes from NSCLC cells compared with respective controls. CircFBXW8 depletion repressed NSCLC cell proliferation, migration and invasion, but promoted cell apoptosis. CircFBXW8 acted as a sponge of miR-370-3p and regulated NSCLC cell malignancy by binding to miR-370-3p. Additionally, miR-370-3p repressed NSCLC cell processes by regulating TRIM44. CircFBXW8 knockdown inhibited tumor formation in vivo. Further, circFBXW8 secretion was mediated by exosomes. CircFBXW8 modulated NSCLC progression by increasing TRIM44 expression through sponging miR-370-3p, which provided a new direction for studying the therapy of NSCLC.

Circ_0006220 promotes non-small cell lung cancer progression via sponging miR-203-3p and regulating RGS17 expression.

Lung cancer is the most common malignancy, and its mortality ranks first among malignancies. Non-small cell lung carcinoma (NSCLC) is the most common pathological subtype of lung cancer. It is reported that circular RNAs (circRNAs) feature prominently in the occurrence and metastasis of NSCLC. This study aims to decipher the biological functions of circ_0006220 in NSCLC and the underlying mechanism. The microarray data (GSE101586) were downloaded from the Gene Expression Omnibus database, and differentially expressed circRNAs in NSCLC tissues were screened using the GEO2R tool. Quantitative real-time polymerase chain reaction was used for detecting the expression of circ_0006220, miR-203-3p, and regulator of G-protein signaling 17 (RGS17) mRNA in NSCLC tissues and cells. The connection between circ_0006220 expression and clinicopathological indicators was analyzed through the chi-square test. EdU and cell counting kit-8 assays were carried out to detect cell growth. Cell migration and invasion were detected by transwell assays. Bioinformatics was used to predict, and RNA immunoprecipitation assay and dual-luciferase reporter gene assay were conducted for verifying, the targeted relationship among circ_0006220, miR-203-3p, and RGS17. The expression of circ_0006220 was elevated in NSCLC cells and tissues, and high circ_0006220 expression was significantly associated with unfavorable clinicopathological indicators. In addition, it was revealed that circ_0006220 overexpression facilitated NSCLC cell growth, migration, and invasion, whereas knocking down circ_0006220 had contrary effects. Furthermore, miR-203-3p was identified as a downstream target of circ_0006220, and circ_0006220 could sponge miR-203-3p; RGS17 was identified as a downstream target of miR-203-3p and was positively modulated by circ_0006220. Circ_0006220 up-regulates RGS17 expression by adsorbing miR-203-3p to promote NSCLC development.

Oncogenic circRNA C190 Promotes Non-Small Cell Lung Cancer via Modulation of the EGFR/ERK Pathway.

Lung cancers are the leading cause of cancer-related mortality worldwide, and the majority of lung cancers are non-small cell lung carcinoma (NSCLC). Overexpressed or activated EGFR has been associated with a poor prognosis in NSCLC. We previously identified a circular noncoding RNA, hsa_circ_0000190 (C190), as a negative prognostic biomarker of lung cancer. Here, we attempted to dissect the mechanistic function of C190 and test the potential of C190 as a therapeutic target in NSCLC. C190 was upregulated in both NSCLC clinical samples and cell lines. Activation of the EGFR pathway increased C190 expression through a MAPK/ERK-dependent mechanism. Transient and stable overexpression of C190 induced ERK1/2 phosphorylation, proliferation, and migration in vitro and xenograft tumor growth in vivo. RNA sequencing and Expression2Kinases (X2K) analysis indicated that kinases associated with cell-cycle and global translation are involved in C190-activated networks, including CDKs and p70S6K, which were further validated by immunoblotting. CRISPR/Cas13a-mediated knockdown of C190 decreased proliferation and migration of NSCLC cells in vitro and suppressed tumor growth in vivo. TargetScan and CircInteractome databases predicted that C190 targets CDKs by sponging miR-142-5p. Analysis of clinical lung cancer samples showed that C190, CDK1, and CDK6 expressions were significantly higher in advanced-stage lung cancer than in early-stage lung cancer. In summary, C190 is directly involved in EGFR-MAPK-ERK signaling and may serve as a potential therapeutic target for the treatment of NSCLC. SIGNIFICANCE: The circRNA C190 is identified as a mediator of multiple pro-oncogenic signaling pathways in lung cancer and can be targeted to suppress tumor progression.

Spherical Invasion Assay: A Novel Method to Measure Invasion of Cancer Cells.

The invasion of tumor cells into the neighboring blood vessels and lymph nodes is a vital step for distant metastasis. Traditionally, the invasive activity of growth factors (or the anti-invasive activity of drugs) is measured with the Boyden chamber assay. However, this assay has a few disadvantages like poor physiological relevance of transwell inserts and an inability to control chemokine gradients. The Boyden chamber assay is one of the most prevalent methods to measure the invasion of cancer cells. It would be advantageous to develop another assay that could validate the results of the Boyden chamber assay. With this in mind, our laboratory developed the spherical invasion assay (SIA) to measure the pro-invasive activity of human cancer cells. The SIA also circumvents some of the drawbacks of the Boyden chamber assay. The present manuscript measures the anti-invasive activity of the Src kinase inhibitor PP2 in A549 human non-small cell lung carcinoma (NSCLC) cells using the SIA. The SIA protocol is comprised of two steps. In the first step, A549 human NSCLC cells (treated or not with PP2) were mixed with Matrigel and seeded in the middle of an eight-well chamber slide. After 24 h, a second layer of Matrigel was overlaid over the first layer. Over the course of the next 24 h, the A549 cells invade from the primary to the secondary Matrigel layers. Subsequently, the cells are visualized by phase-contrast microscopy and the images obtained are quantified using ImageJ to calculate the anti-invasive activity of PP2 in A549 cells. The results of the SIA correlate well with Boyden chamber assays. The SIA may be adapted for multiple experimental designs, such as drug screening (to combat invasion and metastasis), measuring the pro-invasive activity of growth factors, and elucidating the signaling pathways underlying the pro-invasive/anti-invasive activity of biological modifiers. Graphic abstract: Diagrammatic illustration of the spherical invasion assay ( Hurley et al., 2017 ) . A. The first layer is comprised of human cancer cells mixed in a 1:1 suspension with Phenol Red containing Matrigel (represented as LAYER 1 in the figure). After 24 h, the cancer cells grow and extend up to the boundary of this first layer. B. A second layer of 1:1 solution Phenol Red-free Matrigel, in Phenol Red-free RPMI (represented as LAYER 2 in the figure) is added on top of the first Matrigel spot. The cells are incubated for 24 h at 37°C. C. Over these 24 h, the cancer cells invade from the primary layer into the secondary Matrigel layer. The chamber slides are observed by phase-contrast microscopy. D. A representative photograph of the images obtained by the SIA is shown. The black arrow indicates the cancer cells invading into the second layer of Matrigel. The dotted line represents the interface between the two layers. The distance to which the cells have traveled (into the secondary Matrigel layer) is measured at ten sites (for each photograph) in a randomized double-blind fashion by three independent observers, using NIH ImageJ Version 1.47. This process is repeated for three separate photographic fields per sample.

Non-small cell lung carcinoma spheroid models in agarose microwells for drug response studies.

There is a growing interest in developing personalized treatment strategies for each cancer patient, especially those with non-small cell lung carcinoma (NSCLC) which annually accounts for the majority of cancer related deaths in the US. Yet identifying the optimal NSCLC treatment strategy for each cancer patient is critical due to a multitude of mutations, some of which develop following initial therapy and can result in drug resistance. A key difficulty in developing personalized therapies in NSCLC is the lack of clinically relevant assay systems that are suitable to evaluate drug sensitivity using a minuscule amount of patient-derived material available following biopsies. Herein we leverage 3D printing to demonstrate a platform based on miniature microwells in agarose to culture cancer cell spheroids. The agarose wells were shaped by 3D printing molds with 1000 microwells with a U-shaped bottom. Three NSCLC cell lines (HCC4006, H1975 and A549) were used to demonstrate size uniformity, spheroid viability, biomarker expressions and drug response in 3D agarose microwells. Results show that our approach yielded spheroids of uniform size (coefficient of variation <22%) and high viability (>83% after 1 week-culture). Studies using epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKIs) drugs gefitinib and osimertinib showed clinically relevant responses. Based on the physical features, cell phenotypes, and responses to therapy of our spheroid models, we conclude that our platform is suitable for in vitro culture and drug evaluation, especially in cases when tumor sample is limited.

P38 Mediates Resistance to FGFR Inhibition in Non-Small Cell Lung Cancer.

FGFR signalling is one of the most prominent pathways involved in cell growth and development as well as cancer progression. FGFR1 amplification occurs in approximately 20% of all squamous cell lung carcinomas (SCC), a predominant subtype of non-small cell lung carcinoma (NSCLC), indicating FGFR as a potential target for the new anti-cancer treatment. However, acquired resistance to this type of therapies remains a serious clinical challenge. Here, we investigated the NSCLC cell lines response and potential mechanism of acquired resistance to novel selective FGFR inhibitor CPL304110. We found that despite significant genomic differences between CPL304110-sensitive cell lines, their resistant variants were characterised by upregulated p38 expression/phosphorylation, as well as enhanced expression of genes involved in MAPK signalling. We revealed that p38 inhibition restored sensitivity to CPL304110 in these cells. Moreover, the overexpression of this kinase in parental cells led to impaired response to FGFR inhibition, thus confirming that p38 MAPK is a driver of resistance to a novel FGFR inhibitor. Taken together, our results provide an insight into the potential direction for NSCLC targeted therapy.

Botrychium schaffneri Underw. extract acts via DIABLO to induce apoptosis and inhibit proliferation of non-small cell lung carcinoma in vitro and in vivo.

BackgroundBotrychium schaffneri Underw. has been popularly consumed since ancient times as a traditional medicine in China to treat whooping cough, bronchial asthma, and febrile convulsive twitch disease. This led us to investigate whether Botrychium schaffneri Underw. extract (BSE) may be effective against lung cancer, especially non-small cell lung carcinoma (NSCLC).MethodsIn this study, we extracted the ethanolic root extract of the grass, Botrychium schaffneri Underw. In vitro study, the change of NCI-H1299 cell proliferation was observed with CCK8 and MTT assays. Cell apoptosis was assessed using a kit based on staining with FITC-conjugated annexin V. In vivo study, we establish a stable animal model of NSCLC in nude mice, tumor volume and weight was measured twice a week. We conduct gene microarray screened for differentially expressed genes (DEGs), between NCI-H1299 cells treated by BSE or not. Then the DEGs were functionally annotated and path enriched.ResultsIt was revealed that BSE significantly suppressed NSCLC cell proliferation (IC50 134 μg/mL) and induced apoptosis. It also slowed tumor growth without affecting body weight, and a dose of 25 g/kg led to significantly smaller tumors than in control animals (13.85±3.36 vs. 23.40±6.05, P=0.044). Apoptosis-related protein direct IAP Binding protein with low PI (DIABLO) expression was up-regulated by BSE, and DIABLO knockdown significantly attenuated the anti-tumor effects of the extract.ConclusionsIn conclusion, BSE reduces the viability of NSCLC cells and promotes apoptosis, and these effects may be mediated by DIABLO.

Transthyretin Suppressed Tumor Progression in Nonsmall Cell Lung Cancer by Inactivating MAPK/ERK Pathway.

Background: Lung malignancy is a main source of disease passing all throughout the planet, whereas the transthyretin (TTR) is a specific biomarker for clinical diagnosis. However, its role in lung malignancy stays to be obscure. Materials and Methods: In the current examination, the authors made an endeavor to research impact of abnormal expression of TTR on nonsmall cell lung carcinoma (NSCLC) by overexpression or knockdown of TTR. To further explore the instruments' fundamental mechanism part of TTR in NSCLC, several signal pathways were searched and verified. To confirm the effect of TTR overexpression on tumors, in vivo experiments were conducted. Result: It was found that upregulated TTR clearly stifled cell proliferation, migration, invasion, and expanded apoptosis. Significant suppression of phosphor-extracellular signal-regulated kinase (ERK) was observed in TTR-treated NSCLC cells, implying that TTR was important for cellular progress by regulating mitogen-activated protein kinase/ERK signaling pathway. In in vivo experiment, overexpression of TTR promoted cell apoptosis and inhibited tumor growth. Conclusion: Overall, the results suggest that TTR has a potential antitumor effect in human NSCLC progression, which provides theoretical basis for the diagnosis and treatment of NSCLC. Above all, further understanding of TTR was useful for clinical care. Clinical Trial Registration Number: 2016-08.

HDAC/MIF dual inhibitor inhibits NSCLC cell survival and proliferation by blocking the AKT pathway

Non-small-cell lung carcinoma (NSCLC) is one of the most common forms of lung cancer, and a leading cause of cancer death among human beings. There is an urgent demand for novel therapeutics for the treatment of NSCLC to enhance the efficacy of the currently applied Tyrosine kinase inhibitors (TKIs) therapy and to overcome therapy-resistance. Here, we report a novel small-molecule inhibitor that simultaneously targets histone deacetylase (HDAC) and macrophage migration inhibitory factor (MIF). The HDAC/MIF dual inhibitor proved to be toxic for EGFR mutated (H1650, TKI-resistant) or knock out (A549 EGFR−/−) NSCLC cell lines. Further experiments showed that HDAC inhibition inhibits cell survival and proliferation, while MIF inhibition downregulates pAKT or AKT expression level, which both interfere with cell survival. Furthermore, the combination treatment of TKI and HDAC/MIF dual inhibitor showed that the dual inhibitor enhanced TKI inhibitory efficacy, highlighting the advantages of HDAC/MIF dual inhibitor for more effective treatment of NSCLC.

The Gut Microbiome and Cancer Immunotherapy: Can We Use the Gut Microbiome as a Predictive Biomarker for Clinical Response in Cancer Immunotherapy?

Simple SummaryThe current review assessed the effects of the gut microbiome on clinical outcomes of immunotherapy and related adverse events (AEs) in cancer patients. Studies (n = 10) consistently reported that the gut microbiome prior to administering immune checkpoint inhibitors (ICIs) was associated with enhanced efficacy of ICIs and reduced AEs. Recent fecal microbiome transplant (FMT) studies demonstrated the modulatory effects of FMT on the composition and diversity of the gut microbiome in patients with refractory cancers and the potential to improve the efficacy of ICIs.Background: Emerging evidence suggests that gut microbiota influences the clinical response to immunotherapy. This review of clinical studies examines the relationship between gut microbiota and immunotherapy outcomes. Method: A literature search was conducted in electronic databases Medline, PubMed and ScienceDirect, with searches for “cancer” and “immunotherapy/immune checkpoint inhibitor” and “microbiome/microbiota” and/or “fecal microbiome transplant FMT”. The relevant literature was selected for this article. Results: Ten studies examined patients diagnosed with advanced metastatic melanoma (n = 6), hepatocellular carcinoma (HCC) (n = 2), non-small cell lung carcinoma (NSCLC) (n = 1) and one study examined combination both NSCLC and renal cell carcinoma (RCC) (n = 1). These studies consistently reported that the gut microbiome profile prior to administering immune checkpoint inhibitors (ICIs) was related to clinical response as measured by progression-free survival (PFS) and overall survival (OS). Two studies reported that a low abundance of Bacteroidetes was associated with colitis. Two studies showed that patients with anti-PD-1 refractory metastatic melanoma experienced improved response rates and no added toxicity when receiving fecal microbiota transplant (FMT) from patients with anti-PD-1 responsive disease. Conclusions: Overall, significant differences in the diversity and composition of the gut microbiome were identified in ICIs responders and non-responders. Our findings provide new insights into the value of assessing the gut microbiome in immunotherapy. Further robust randomized controlled trials (RCTs) examining the modulatory effects of the gut microbiome and FMT on ICIs in patients not responding to immunotherapy are warranted.

Tumor-derived exosomal miR-3157-3p promotes angiogenesis, vascular permeability and metastasis by targeting TIMP/KLF2 in non-small cell lung cancer

Metastasis is the main cause of death in patients with advanced lung cancer. The exosomes released by cancer cells create tumor microenvironment, and then accelerate tumor metastasis. Cancer-derived exosomes are considered to be the main driving force for metastasis niche formation at foreign sites, but the mechanism in Non-small cell lung carcinoma (NSCLC) is unclear. In metastatic NSCLC patients, the expression level of miR-3157-3p in circulating exosomes was significantly higher than that of non-metastatic NSCLC patients. Here, we found that miR-3157-3p can be transferred from NSCLC cells to vascular endothelial cells through exosomes. Our work indicates that exosome miR-3157-3p is involved in the formation of pre-metastatic niche formation before tumor metastasis and may be used as a blood-based biomarker for NSCLC metastasis. Exosome miR-3157-3p has regulated the expression of VEGF/MMP2/MMP9 and occludin in endothelial cells by targeting TIMP/KLF2, thereby promoted angiogenesis and increased vascular permeability. In addition, exosome miR-3157-3p promoted the metastasis of NSCLC in vivo.

Quercetin inhibits proliferation of and induces apoptosis in non-small-cell lung carcinoma via the lncRNA SNHG7/miR-34a-5p pathway

Objective To determine the role of quercetin in non-small cell lung carcinoma (NSCLC) and the biological outcomes using transfection experiments. Materials and methods Real-time reverse transcription-PCR and data collection were performed to determine lncRNA and miRNA levels. Transwell assay was performed to assess the invasion ability of cells. Apoptosis of cells digested with trypsin was determined using the Annexin V-FITC kit. Luciferase activity was determined using the luciferase reporter gene system. Cell viability was tested using the Cell Counting Kit-8 assay. A xenograft mouse model was established to investigate the effects of quercetin on tumor growth. Results The expression levels of the long non-coding RNA (lncRNA) small nucleolar RNA host gene 7 (SNHG7) were elevated in NSCLC cells, and the expression levels of the microRNA miR-34a-5p were decreased compared with those in normal cells. Further investigation revealed that quercetin decreased SNHG7 and elevated miR-34a-5p levels in NSCLC cells (p < .05). The luciferase reporter gene assay, RNA-binding protein immunoprecipitation assay, and transfection experiments revealed target-binding sequences between SNHG7 and miR-34a-5p. Overexpression of SNHG7 or miR-34a-5p inhibitor promoted NSCLC cell proliferation and accelerated tumor cell growth and metastasis. The therapeutic effect of quercetin on NSCLC cells was counteracted by co-transfection of SNHG7 mimic or miR-34a-5p inhibitor. Quercetin inhibited the survival, proliferation, migration, and invasion of NSCLC cells and enhanced their apoptosis. Using the mouse model, quercetin was shown to inhibit tumor growth. Conclusions Quercetin inhibits the proliferation and induces apoptosis of NSCLC cells by mediating signaling via the lncRNA SNHG7/miR-34a-5p pathway.

Flubendazole Elicits Antitumor Effects by Inhibiting STAT3 and Activating Autophagy in Non-small Cell Lung Cancer.

Non-small cell lung carcinoma (NSCLC) is a major neoplastic disease with a high mortality worldwide; however, effective treatment of this disease remains a challenge. Flubendazole, a traditional anthelmintic drug, possesses potent antitumor properties; however, the detailed molecular mechanism of flubendazole activity in NSCLC needs to be further explored. In the present study, flubendazole was found to exhibit valid antitumor activity in vitro as well as in vivo. Flubendazole blocked phosphorylation of STAT3 in a dose- and time-dependent manner and regulated the transcription of STAT3 target genes encoding apoptotic proteins. Further, flubendazole inhibited STAT3 activation by inhibiting its phosphorylation and nuclear localization induced by interleukin-6 (IL-6). Notably, the autophagic flux of NSCLC cell lines was increased after flubendazole treatment. Furthermore, flubendazole downregulated the expression of BCL2, P62, and phosphorylated-mTOR, but it upregulated LC3-I/II and Beclin-1 expression, which are the main genes associated with autophagy. Collectively, these data contribute to elucidating the efficacy of flubendazole as an anticancer drug, demonstrating its potential as a therapeutic agent via its suppression of STAT3 activity and the activation of autophagy in NSCLC.

Identification and characterization of non-small cell lung cancer associated sialoglycoproteins

Aberrantly sialylated cellular glycoconjugates were found to be involved in different processes during tumorigenesis. Such alteration was also noted in case of lung cancer, an important cause of cancer-related death throughout the world. Thus, study on lung cancer associated sialoglycoproteins is of paramount relevance to have a deeper insight into the mechanism of the disease pathogenesis. In the present study, sialic acid specific lectin (Maackia amurensis agglutinin and Sambcus nigra agglutinin)-based affinity chromatography followed by 2D-PAGE and MALDI-TOF/TOF mass spectrometric analysis were done to explore the disease-associated serum proteins of squamous cell carcinoma and adenocarcinoma [the major two subtypes of NSCLC (non-small cell lung carcinoma)] patients. Among seven identified proteins, α1-antitrypsin and haptoglobin-β were preferred for further studies. These two proteins were characterized as the disease associated serum-sialoglycoproteins of NSCLC-patients by western immunoblotting using each lectin specific inhibitor. The presence of these sialoglycoproteins was found on NSCLC cell lines (NCI-H520 & A549) by confocal microscopy. Both these proteins were also present in tissue samples of NSCLC origin and involved in proliferation, invasion and migration of NSCLC cells. Our findings suggest that α1-antitrypsin and haptoglobin-β may be the disease-associated sialoglycoproteins in NSCLC, which seem to be involved in disease progression. SignificanceOur contribution regarding the identification of the NSCLC associated sialoglycoproteins may provide a new vision towards the development of clinically useful newer strategies for the treatment of this disease.

PFKFB3 Inhibition Impairs Erlotinib-Induced Autophagy in NSCLCs.

Tyrosine kinase inhibitors (TKIs) targeting the kinase domain of the epidermal growth factor receptor (EGFR), such as erlotinib, have dramatically improved clinical outcomes of patients with EGFR-driven non-small cell lung carcinomas (NSCLCs). However, intrinsic or acquired resistance remains a clinical barrier to the success of FDA-approved EGFR TKIs. Multiple mechanisms of resistance have been identified, including the activation of prosurvival autophagy. We have previously shown that the expression and activity of PFKFB3—a known driver of glycolysis—is associated with resistance to erlotinib and that PFKFB3 inhibition improves the response of NSCLC cells to erlotinib. This study focuses on investigating the role of PFKFB3 in regulating erlotinib-driven autophagy to escape resistance to erlotinib. We evaluated the consequence of pharmacological inhibition of PFKFB3 on erlotinib-driven autophagy in NSCLC cells with different mutation statuses. Here, we identify PFKFB3 as a mediator of erlotinib-induced autophagy in NSCLCs. We demonstrate that PFKFB3 inhibition sensitizes NCSLCs to erlotinib via impairing autophagy flux. In summary, our studies uncovered a novel crosstalk between PFKFB3 and EGFR that regulates erlotinib-induced autophagy, thus contributing to erlotinib sensitivity in NSCLCs.

Bruton\u2019s tyrosine kinase (BTK) mediates resistance to EGFR inhibition in non-small-cell lung carcinoma

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are current standard of care for patients with EGFR mutation and metastatic non-small-cell lung carcinoma (NSCLC), but most patients using EGFR TKIs acquire resistance later. So, overcoming resistance of EGFR TKIs has become an important issue in the treatment of NSCLC. Previously, therapeutics targeting Bruton’s tyrosine kinase (BTK) have been successful in treating several hematologic malignancies. However, the role of BTK in NSCLC is still unknown. In this study, by examining surgical specimens from 80 NSCLC patients and their clinicopathologic parameters, we found significant correlation between high BTK expression and tumor differentiation, p-stage, lymph node metastatic status, maximum tumor size, and poor prognosis of patients. Using two NSCLC cell lines A540 and PC9, we demonstrated that BTKpos cells exhibited more stemness (OCT4, SOX2) and EMT (E-Cadherin, Slug) markers than BTKneg cells. Knockdown of BTK sensitized the NSCLC cells to Gefitinib. Meanwhile, the second-generation BTK inhibitor Acalabrutinib effectively suppressed SOX2, STAT3/JAK2/Akt axis and potentiated the anti-proliferative effect of Gefitinib and Osimertinib in NSCLC cells, including the T790M H1975 cells. Furthermore, Acalabrutinib and Osimertinib combination exhibited significant tumor growth inhibition of H1975-derived tumors in vivo. Our findings suggested that BTK mediates stemness and EMT properties, and inhibition of BTK potentiates the effect of Gefitinib and Osimertinib in NSCLC cells resistant to TKI. This implies a new approach to treat the NSCLC patients with resistance to previous TKI treatment.

CircRNA_103615 contributes to tumor progression and cisplatin resistance in NSCLC by regulating ABCB1.

Lung cancer is one of the main causes of tumor lethality worldwide. Circular RNAs (circRNAs) have essential roles in tumor progression. However, in non-small cell lung carcinoma (NSCLC), the role of circRNAs remains unknown. In the present study, the expression, function and molecular mechanisms of a new circRNA, circRNA_103615, were investigated in NSCLC. Reverse transcription-quantitative PCR (RT-qPCR) was used to detect circRNA_103615 expression levels in NSCLC and normal tissues, as well as in NSCLC cell lines. MTT assay, flow cytometric assay, colony formation assay, and cell migration and invasion assays were used to examine the function of circRNA_103615 in NSCLC cells. MTT assay, colony formation assay, RT-qPCR, and western blotting were used to detect the effect of circRNA_103615 on cisplatin resistance. The results demonstrated that NSCLC cell lines and tissues had increased levels of circRNA_103615 compared with normal cells and normal tissues, respectively. Functionally, silencing of circRNA_103615 by small interfering RNA resulted in suppression of cell growth, migration, and invasion, but promotion of cell apoptosis. In addition, the cisplatin resistance of NSCLC was reversed by the silencing of circRNA_103615. Notably, ATP binding cassette subfamily Bmember 1 (ABCB1) expression was significantly decreased following circRNA_103615 knockdown, and ABCB1 overexpression reversed the effects of circRNA_103615 silencing on NSCLC cisplatin resistance. Thus, the present study indicated that circRNA_103615 may serve as a critical oncogene and potential novel biomarker in NSCLC, as well as a potential cisplatin resistance promoter, by regulating ABCB1 expression.

Dual targeting of MEK and PI3K effectively controls the proliferation of human EGFR-TKI resistant non-small cell lung carcinoma cell lines with different genetic backgrounds

BackgroundMolecular targeted therapy for non-small cell lung carcinoma (NSCLC) is restricted due to resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). This study evaluated the effects of dual targeting of MEK and PI3K in human EGFR-TKI resistant NSCLC cell lines.MethodsEGFR-TKI resistant NSCLC cell lines H1975, H460, and A549, with different mutation and amplification status in EGFR, K-RAS, PIK3CA, and MET genes, were treated with a MEK162 (MEK inhibitor) and BKM120 (PI3K inhibitor) combination or a BIBW2992 (EGFR inhibitor) and ARQ197 (MET inhibitor) combination and assayed for cell proliferation, apoptosis, and cell cycle distribution.ResultsDual targeting of MEK and PI3K efficiently inhibited the cell proliferation, induced apoptosis and the G0/G1 cell cycle, and decreased the phosphorylation of ERK1/2, AKT, S6, and 4E-BP1. H460 cells with K-RAS and PIK3CA mutation were most sensitive to MEK162 and BKM120 combinations. H1975 cells with EGFR and PIK3CA mutation and MET amplification were sensitive to BIBW2992 and ARQ197 combinations.ConclusionDual targeting regulated the proliferation of EGFR-TKI-resistant NSCLC cells, especially mutants in K-RAS and PIK3CA that are promising for EGFR-TKI-resistant NSCLC therapeutics.