Cultured Lung Cancer Cells Research Articles

Evaluation of a Platinum-Acridine Anticancer Agent and Its Liposomal Formulation in an in vivo Model of Lung Adenocarcinoma.

Liposomal formulations have been developed for a highly cytotoxic platinum-acridine agent, [PtCl(pn)(C18 H21 N4 )](NO3 )2 (PA, pn=propane-1,3-diamine), and fully characterized. Nanoliposomes consisting of hydrogenated soybean phosphatidylcholine (HSPC), 1,2-dihexadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), and polyethylene glycol-2000-distearoylphosphatidylethanolamine (DSPE-mPEG2k ) were able to stably encapsulate PA at payload-to-lipid ratios of 2-20 %. The fusogenic properties of the liposomes promote efficient cellular uptake of PA across the plasma membrane, which results in vesicular transport of payload to the nucleus in cultured lung cancer cells. Unencapsulated PA and one of the newly designed liposomal formulations show promising tumor growth inhibition in tumor xenografts derived from A549 lung adenocarcinoma cells of 76 % and 72 %, respectively. Cisplatin showed no significant efficacy at a 10-fold higher dose. These findings underscore the utility of platinum-acridine agents for treating aggressive, chemoresistant forms of cancer and validate nanoliposomes as a biocompatible, expandable platform for their intravenous delivery and other potential routes of administration.

Downregulation of microRNA-324-3p inhibits lung cancer by blocking the NCAM1-MAPK axis through ALX4.

Lung cancer remains the principal cause of cancer-related death worldwide. As microRNAs (miRNAs) are critically involved in lung cancer, we investigated the potential role of miR-324-3p in lung cancer via the ALX4/NCAM1/MAPK axis. The expression of miR-324-3p and ALX4 was detected in clinical samples, and their interaction confirmed by miRNA-targeted luciferase reporter assay. The mechanisms involved in the miR-324-3p-ALX4 interaction in lung cancer cell biological processes were analyzed through gain- and loss-of function approaches. In addition, cultured lung cancer cells were treated with the p38MAPK pathway activator P79350 in order to explore the role of this pathway in the abovementioned axis. Further, a tumor xenograft model in nude mice was constructed to confirm the in vitro findings. miR-324-3p was highly expressed in lung cancer tissues and cells, and inhibited the expression of ALX4 in A549 cells. After confirming the targeted inhibition of ALX4 by miR-324-3p, we showed that this interaction upregulated the expression of NCAM1 and activated the MAPK pathway. The inhibition of miR-324-3p could suppress lung cancer cell invasion, migration, and autophagy, and retarded the growth of subcutaneous tumors in nude mice. Downregulation of ALX4 or NCAM1 overexpression reversed these favorable effects of decreased miR-324-3p. Our study demonstrated the promotive effect of miR-324-3p on the development and progression of lung cancer, thus suggesting a new target for treatment of this devastating disease.

Degradation of Mutant Protein Aggregates within the Endoplasmic Reticulum of Vasopressin Neurons.

SummaryMisfolded or unfolded proteins in the ER are said to be degraded only after translocation or isolation from the ER. Here, we describe a mechanism by which mutant proteins are degraded within the ER. Aggregates of mutant arginine vasopressin (AVP) precursor were confined to ER-associated compartments (ERACs) connected to the ER in AVP neurons of a mouse model of familial neurohypophysial diabetes insipidus. The ERACs were enclosed by membranes, an ER chaperone and marker protein of phagophores and autophagosomes were expressed around the aggregates, and lysosomes fused with the ERACs. Moreover, lysosome-related molecules were present within the ERACs, and aggregate degradation within the ERACs was dependent on autophagic-lysosomal activity. Thus, we demonstrate that protein aggregates can be degraded by autophagic-lysosomal machinery within specialized compartments of the ER.

Suppression of MGAT3 expression and the epithelial–mesenchymal transition of lung cancer cells by miR-188-5p

BackgroundTo investigate the effect of miR-188-5p overexpression on the invasion and migration of cultured lung cancer cells, and on related cellular mechanisms that underlie epithelial mesenchymal transition (EMT). MethodsHuman lung cancer cell line 95D was transfected with miR-188-5p mimic. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were performed to quantify the expression levels of genes including E-cadherin, Snail, α-SMA, and MGAT3. Changes in cell motility, invasion and proliferation were studied using scratch migration assay, transwell invasion assay, and colony formation assay, respectively. The expression levels of EMT-related proteins and MGAT3 protein were also determined via immunofluorescent staining. The ability of miR-188-5p to regulate its target gene, MGAT3, was assessed using dual luciferase activity assay. ResultsLung cancer cell line 95D showed the lowest miR-188-5p expression level thus was used in this study. Transfection with miR-188-5p mimic significantly suppressed migration, invasion and clonal formation potency of 95D cells. Dual luciferase activity assay implicated that miR-188-5p exerts its negative regulatory effect on MGAT3 expression through recognizing the 3′ untranslated region (3′UTR) of the MGAT3 gene. Over-expression of miR-188-5p in 95D cells also remarkably increased E-cadherin protein expression and decreased the expression levels of Snail and α-SMA, which suppressed the EMT process. ConclusionMiR-188-5p reduces the expression of MGAT3 and inhibits the metastatic properties of a highly invasive lung cancer cell line, probably via targeted regulation of EMT process. Further research to explore the potential therapeutic value of miR-188-5p, both as a biomarker and as a drug candidate for the management of metastatic lung cancer may be warranted.

Development and validation of a rapid, robust and sensitive UPLC-QQQ-MS/MS method for simultaneous quantification of GSH metabolism in lung cancer cells

Changes in cellular metabolism accompany tumor therapeutic resistance. Metabolite concentrations specifically reflect the cellular state. Glutathione (GSH) metabolism maintains the redox homeostasis while also confers therapeutic resistance to cancer cells. However, analytical methods for studying GSH metabolism rely on high-resolution-based untargeted metabolomics. Since the aim of untargeted metabolomics studies is covering as much metabolites as possible, these methods lack sensitivity for simultaneous analysis of intracellular GSH-related metabolites with different polarities and structures. In this study, based on cultured lung cancer cells, we described a rapid, robust and sensitive ultra-performance liquid chromatography-triple quadrupole tandem mass spectrographic method (UPLC-QQQ-MS/MS) to simultaneously quantify a repertoire of GSH-related metabolites, including GSH, GSSG, glycine, cysteine, glutamine, glutamate, cystine, γ-glutamyl-cysteine and cysteinyl-glycine. This method avoided the use of derivatization and/or ion-pairing reagents and was validated according to United States Food and Drug Administration (US FDA) criteria. The lower limit of quantification was determined to be 0.5–100 ng/mL with lower limits of detection at 0.14–10.07 ng/mL. The intra- and inter-day precision values for all the analytes were <15% CV, and the accuracy ranged from 85.4% to 114% at three levels of quality control. This method combined simple preparation with rapid analytical procedure (8 min), allowed for high-throughput analysis of GSH metabolism in basic and therapeutic treatment conditions within cultured cells. Our data showed a significant alteration of GSH metabolism in two independent resistant cells compared to sensitive cells. This method monitored the impact of molecularly targeted drugs on GSH metabolism within lung cancer cells and therefore helped identifying potential metabolic vulnerability for the therapeutic resistance in lung cancer.

CXCL1 as an Unfavorable Prognosis Factor Negatively Regulated by DACH1 in Non-small Cell Lung Cancer.

Background: Interaction between cancer cells with microenvironment is essential for cancer progression, therapeutic resistance and prognosis. Chemokine CXCL1 shows variable roles in the development of cancers. DACH1 has been considered as a tumor suppressor and represses the expressions of several chemokines. The relationship between CXCL1 and DACH1 in non-small cell lung cancer (SCLC) deserves further investigation.Methods: Immunohistochemistry staining was performed on tumor tissue microarrays from lung cancer patients to detect CXCL1 protein. The CXCL1 concentration in the serum of adenocarcinoma patients was measured by ELISA. The CXCL1 protein secreted by cancer cell lines was detected by SearchLight proteome array and human cytokine antibody array. The meta-analysis of CXCL1 expression form public databases was performed and correlation between CXCL1 and DACH1 was analyzed. Moreover, the association between clinicopathological features and prognosis with CXCL1 and DACH1 was analyzed by tissue array and KM-plotter from public database.Results: The protein abundance of CXCL1 in lung cancer tissues was significantly higher than that in adjacent normal tissues. CXCL1 was closely related to TNM stage, tumor size, and lymph node metastasis and predicted worse overall survival in adenocarcinoma. The level of CXCL1 in the peripheral blood of adenocarcinoma patients also significantly elevated and positively related with clinical stage. The meta-analysis demonstrated that CXCL1 mRNA level was increased in lung cancer tissues and high level of CXCL1 indicated tumor progression in lung adenocarcinoma. In addition, public database analyses showed that CXCL1 negatively correlated with DACH1. Stable overexpressing DACH1 in cultured lung cancer cells remarkably decreased CXCL1 protein. Moreover, ectopic expression of DACH1 significantly inhibited the expression of CXCL1, Ki67, and cyclin D1 in tumor tissues compared with A549 cells with empty vector. Survival analysis showed that high CXCL1 and low DACH1 indicated poor overall survival and progression-free survival.Conclusion: CXCL1 is closely associated with tumor progression and poor survival. DACH1 significantly inhibits the expression of CXCL1 and indicates good prognosis. Therefore, combined detection of CXCL1 and DACH1 could more precisely predict prognosis of lung adenocarcinoma.

A rapid liquid biopsy of lung cancer by separation and detection of exfoliated tumor cells from bronchoalveolar lavage fluid with a dual-layer "PERFECT" filter system.

Separation and detection of exfoliated tumor cells (ETCs) from bronchoalveolar lavage fluid (BALF), namely the liquid biopsy of BALF, has been proved to be a valuable tool for the diagnosis of lung cancer. Herein, we established a rapid liquid biopsy of BALF based on a dual-layer PERFECT (precise, efficient, rapid, flexible, easy-to-operate, controllable and thin) filter system for the first time.Methods: The dual-layer PERFECT filter system consists of an upper-layer filter with large micropores (feature size of 49.4 ± 0.5 μm) and a lower-layer filter with small micropores (9.1 ± 0.1 μm). The upper-layer filter contributes to the isolation of cell clusters and removal of mucus from BALF samples, meanwhile the lower-layer one targets for the separation of single ETCs. First, separation of 10000 spiked A549s (cultured lung cancer cells) from 10 mL clinical BALF samples (n=3) were performed to investigate the performance of the proposed system in rare cell separation. Furthermore, separation and detection of ETCs and ETC clusters from clinical BALF samples were performed with this system to test its efficacy and compared with the routine cytocentrifuge. The clinical BALF samples were collected from 33 lung cancer-suspected patients with visible lesions under bronchoscope. The final histopathological results showed that 20 samples were from lung cancer positive patients while the other 13 cases were from lung cancer negative patients.Results: The recovery rate of spiked A549 cells from clinical BALF samples with the developed system (89.8 ± 5.2%) is significantly higher than that with the cytocentrifuge (13.6 ± 7.8%). In the preliminary clinical trial, although 33 clinical BALF samples with volume ranging from 6 mL to 18 mL showed greatly varied turbidity, filtrations could be finished within 3 min for 54.6% of samples (18/33), and 10 min at most for the rest. The dual-layer PERFECT filter system is proved to have a much higher sensitivity (80.0%, 95% CI: 55.7%-93.4%) than the routine cytocentrifuge (45.0%, 95% CI: 23.8%-68.0%), p=0.016 (McNemar test, two-tail). Moreover, the sensitivity of this platform is neither interfered by the variations of turbidity of the BALF samples, nor associated with the types of lung cancer.Conclusions: The easy and rapid processing of BALF samples with varying volume and turbidity, competitive sensitivity and good versatility for different lung cancer types will make the established dual-layer PERFECT filter system a promising approach for the liquid biopsy of BALF. The high-performance BALF-based liquid biopsy will improve the cytopathological identification and diagnosis of lung cancer.

Abstract C063: A novel Nrf2 inhibitor suppresses proliferation and enhances the sensitivity of cancer cells to chemotherapy

Abstract The Nrf2-Keap1-ARE pathway, a master defense mechanism against oxidative and electrophilic stresses, has emerged as a target for cancer therapy. Activation of Nrf2 has been a common strategy for cancer prevention, as Nrf2 deficiency enhances susceptibility to carcinogens in a wide variety of preclinical models. However, accumulating evidence suggests a tumor-promoting role for Nrf2 in established tumors. Constitutive activation of Nrf2 because of gain of functionmutations in NFE2L2 or loss of function mutations in its negative regulator Keap1, has been found in many cancers, especially lung cancer, and is associated with chemoresistance and poor survival in human patients. Nrf2 inhibitors, therefore, provide a potential novel strategy for treating cancer. Here, we report a novel Nrf2 inhibitor, MSU225, identified from an existing chemistry library. MSU225 dose-dependently downregulated tBHQ (tert-butyl hydroquinone)-induced Nrf2 transcriptional activity in a luciferase reporter assay without cytotoxicity. MSU225 also decreased the expression of Nrf2 downstream targets, including HO-1, NQO1, GST1A1, GCLC, GCLM, and UGT1A6 in A549 human lung cancer cells, in which Keap1 is mutated and Nrf2 activation is constitutive. Notably, MSU225 induced a striking and significant (p&amp;lt;0.05) decrease of Nrf2 protein in A549 cells. The effect of MSU225 on Nrf2 protein levels can be blocked with the proteasome inhibitor MG132, suggesting MSU225 enhances Nrf2 degradation through the proteasome system. Ubiquitination of Nrf2 was also increased after MSU225 treatment. Interestingly, MSU225 also decreased Nrf2 protein levels in Keap1 knockout and βTrCP knockout cells, which are the two major pathways inducing Nrf2 degradation. Nrf2 involvement in cancer cell proliferation is well known. MSU225 inhibited the growth of human lung cancer cells (A549, H460, A427) in both 2D cell culture and soft agar. Cells addicted to Nrf2 were more susceptible to MSU225 suppression of cell proliferation than cells not addicted to Nrf2. MSU225 also sensitized A549 cells to chemotherapies including doxorubicin, carboplatin, 5-fluorouracil and Topotecan. Our results suggest that MSU225 is a novel Nrf2 inhibitor that enhances the degradation of Nrf2 in a Keap1 and βTrCP independent manner. In vivo studies to assess the efficacy of MSU225 for treating experimental lung cancer are ongoing. Citation Format: Di Zhang, Kelly Aldrich, Thomas Dexheimer, Edmund Ellsworth, Aaron Odom, Karen Liby. A novel Nrf2 inhibitor suppresses proliferation and enhances the sensitivity of cancer cells to chemotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C063. doi:10.1158/1535-7163.TARG-19-C063

The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis.

Ferroptosis is a form of regulated cell death that is caused by the iron-dependent peroxidation of lipids1,2. The glutathione-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols3,4. Ferroptosis has been implicated in the cell death that underlies several degenerative conditions2, and induction of ferroptosis by inhibition of GPX4 has emerged as a therapeutic strategy to trigger cancer cell death5. However, sensitivity to GPX4 inhibitors varies greatly across cancer cell lines6, suggesting that additional factors govern resistance to ferroptosis. Here, employing a synthetic lethal CRISPR/Cas9 screen, we identify ferroptosis suppressor protein 1 (FSP1) (previously known as apoptosis-inducing factor mitochondrial 2 (AIFM2)) as a potent ferroptosis resistance factor. Our data indicate that myristoylation recruits FSP1 to the plasma membrane where it functions as an oxidoreductase that reduces coenzyme Q10 (CoQ), generating a lipophilic radical-trapping antioxidant (RTA) that halts the propagation of lipid peroxides. We further find that FSP1 expression positively correlates with ferroptosis resistance across hundreds of cancer cell lines, and that FSP1 mediates resistance to ferroptosis in lung cancer cells in culture and in mouse tumor xenografts. Thus, our data identify FSP1 as a key component of a non-mitochondrial CoQ antioxidant system that acts in parallel to the canonical glutathione-based GPX4 pathway. These findings define a new ferroptosis suppression pathway and indicate that pharmacological inhibition of FSP1 may provide an effective strategy to sensitize cancer cells to ferroptosis-inducing chemotherapeutics.

DIAPH3 promotes the tumorigenesis of lung adenocarcinoma

Aberrant activation of MEKK-MEK-ERK signaling is frequently observed in lung cancer. Several inhibitors, which target this pathway, have shown clinical potential for the lung cancer treatment. Better understanding the regulation of this pathway would help the development of treatment strategies. In this study, we have identified the DIAPH3 as an up-regulated gene in lung adenocarcinoma. DIAPH3 promoted the growth of lung cancer cells both in the liquid culture and in the soft agar, and knockdown DIAPH3 inhibited the tumorigenesis both in the nude mice and in the de novo mouse model. In the molecular mechanism study, DIAPH3 was identified as the binding protein of STK38, impaired the interaction between STK38 and MEKK, and activated ERK signaling. Taken together, this study demonstrated the oncogenic roles of DIAPH3 in the tumorigenesis of lung cancer by interacting with STK38.

Long non-coding RNA OECC promotes cell proliferation and metastasis through the PI3K/Akt/mTOR signaling pathway in human lung cancer.

Lung cancer is one of the most common malignancies worldwide; however, its detailed molecular mechanism remains largely unknown. Long non-coding RNAs (lncRNAs) have been identified to serve critical roles in tumorigenesis. The aim of the present study was to investigate the role of a newly identified lncRNA, overexpressed in colorectal cancer (OECC), in human lung cancer. It was initially revealed that the relative transcript level of OECC was highly upregulated in clinical human lung cancer tissues as well as in cultured lung cancer cells. Knockdown of OECC with specific short hairpin RNAs in lung cancer cell lines A549 and 95D inhibited colony formation and cell viability, as evidenced using colony formation assays and cell proliferation assays. Furthermore, depletion of OECC in A549 and 95D cells suppressed migration and invasion, which was verified using Transwell assays. RNA-sequence analysis suggested that the phosphoinositide 3-kinase/protein kinase B (Akt)/mammalian target of rapamycin signaling pathway was positively regulated by OECC in lung cancer cells A549. In addition, overexpression of Akt in OECC-depleted A549 and 95D cells reversed the suppression of proliferation and migration caused by OECC depletion. The results of the present study identified lncRNA OECC as a novel regulator of lung cancer progression and provided new clues for the clinical treatment of lung cancer.

Abstract 1418: Brother of regulator of imprinted sites (BORIS) inhibits cisplatin induced DNA damage in non-small-cell lung cancer

Abstract Cisplatin (DDP) treatment is primary modality for non-small-cell lung cancer (NSCLC) chemotherapy. However, patients benefited from DDP treatment limitedly due to the DDP resistance. Brother of Regulator of Imprinted Sites (BORIS, CTCFL) is the paralogue of CCCTC-binding factor (CTCF) and expressed abnormally in most type of cancers and is potential tumor target for breast, cervical carcinoma and lung cancer. However, the funciton of BORIS on carcinoma is unrevealed. In our previous study, we found that BORIS suppressed apoptosis and resisted 5-FU (Fluorouracil) treatment in colorectal cancer. As either 5-FU or DDP induces DNA damage and apoptosis of carcinoma cells, BORIS perhaps resists DDP treatment too. Based on the prevalent expression of BORIS and the high incidence of DDP resistance in NSCLC, we proposed that BORIS contributed to DDP resistance by suppressing DNA damage in NSCLC. We collected RNA sequencing data of 156 NSCLC patients who received DDP chemotherapy from The Cancer Genome Atlas and analyzed the expressions of BORIS. We found high BORIS expression correlated with short survival in NSCLC patients who received DDP chemotherapy. BORIS expression declined from prospective to retrospective collected tissue which responded to DDP chemotherapy. It suggested the probability function of BORIS on DNA damage repair. In cell cultured lung cancer cell lines, BORIS was silenced or over-expressed including DDP resistant cell line. It indicated that A549/DDP (DDP resistant A549 cell line) expressed relative higher BORIS than A549 parental cells and BORISexpression levels correlated with cell viability. H1299 and A549/DDP expressed comparative BORIS and growed comparativly. H460 and A549 expressed relative lower BORIS levels and grew slower than H1299 and A549/DDP. BORIS knockdown inhibited the proliferation of NSCLC cells, enhanced the sensitivity of A549/DDP and H1299 to DDP treatment and induced DNA damage. Reversely DDP induced DNA damage was suppressed by BORIS over-expression. The excision repairing gene ERCC1, which is regulated by BORIS, might be the assistant for DDP resistance. Either high expression of BORIS or ERCC1 indicated short survival rates of NSCLC patients. Our data suggested that BORIS suppressed DNA damage and promoted the progression of NSCLC and DDP resistance, which indicated its potential in NSCLC prognosis and therapy. Citation Format: Yongfei Song, Chao Li, Mengdie Fang, Juan Ren, Jianfei Fang, Xiaoju Wang, Yanmei Zhang. Brother of regulator of imprinted sites (BORIS) inhibits cisplatin induced DNA damage in non-small-cell lung cancer [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 1418.

Tumor microenvironment determines drug efficacy in vitro - apoptotic and anti-inflammatory effects of 15-lipoxygenase metabolite, 13-HpOTrE

Recent studies using 3D scaffolds have emphasized the importance of the surrounding stroma on chemoresistance in drug efficacy screenings. Since 15-lipoxygenase (15-LOX) metabolites reduced growth of breast, colon, prostate, lung and leukemia cancer cells in 2D cell culture, we were intrigued by the direct comparison of 15-LOX metabolite efficacy in 2D and 3D culture including a stroma equivalent. Herein, we studied the effects of 15-LOX metabolites 13-HpOTrE, 13-HpODE, and 15-HpETE on cutaneous squamous cell carcinoma cells.All metabolites reduced the viability of cancer cells in 2D culture below 10% at 100 µM of each substance. 13-HpOTrE, being the most active agent with respect to cytotoxicity and apoptosis was selected for further experiments. Other than with the 2D culture, we did not obverse cell death, neither from lactate dehydrogenase release, nor from morphology when applying 13-HpOTrE onto the surface of the 3D tumor constructs for one week. Next, we investigated the protein expression of peroxisome proliferator activated receptor gamma, for which the ligand is 13-HpOTrE, and Bcl-2 protein, an apoptosis regulator, but did not find any change following 13-HpOTrE administration. However, 13-HpOTrE treatment reduced the release of interleukin-6, bringing it closer to the level of tumor-free constructs.In conclusion, 13-HpOTrE reduces viability of skin cancer cells in 2D cultures only but modulates inflammatory cytokine levels in the corresponding 3D tumor constructs, too. These studies highlight the need for screening of anticancer drugs employing 3D tumors and including tumor microenvironment in the screening process to increase the low success rate of clinical trials in oncology.

Long non‑coding RNA FER1L4 inhibits cell proliferation and metastasis through regulation of the PI3K/AKT signaling pathway in lung cancer cells.

Lung cancer is among the most common malignancies worldwide; however, the current understanding of its detailed mechanism remains limited. Long non-coding RNAs (lncRNAs) were previously identified to serve significant roles in tumorigenesis. The present study aimed to investigate the role of a novel lncRNA, Fer-1-like family member 4 (FER1L4), in lung tumorigenesis. In the present study, it was demonstrated that the expression level of FER1L4 was significantly decreased in clinical lung cancer tissues and in cultured lung cancer cells, as evidenced by reverse transcription-quantitative polymerase chain reaction analysis. Overexpression of FER1L4 in lung cancer cell lines A549 and 95D inhibited colony formation, cell proliferation and cell migration capacity, measured by colony formation assays, cell proliferation assays and Transwell assays, respectively. Overexpression of FER1L4 led to a reduction in the expression levels of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) in A549 and 95D cells, whereas, activation of PI3K/Akt signaling using a small molecular inhibitor of phosphatase and tensin homolog, reversed the inhibitory effects of FER1L4 on cell proliferation and metastasis. All of these results suggested that the lncRNA FER1L4 suppressed cell proliferation and metastasis by inhibiting the PI3K/Akt signaling pathway in lung cancer.

Therapeutic antibody discovery in lung tumors by B-cell receptor sequencing

Abstract Malignancies of the lung are expected to be responsible for over 25% of all cancer-associated mortalities in the United States in 2019. Clinical treatment of lung cancer is complicated by both poor detection of early disease activity and relapse or unresponsiveness to administered therapy. Taken together, these realities of patient outcome underscore the need for alternative therapeutic strategies. Research efforts in the cancer immunology field focus primarily on T cells in the tumor microenvironment, however, there is evidence that B cells may impart a clinical benefit in patients. The formation of tertiary lymphoid structures and antibody secretion in lung tumors associate with positive clinical outcomes yet remain understudied and poorly characterized. In an effort to expand upon these observations, we isolated B cells from cryopreserved human lung cancer tissue and recovered B-cell receptor (BCR) sequences by paired heavy and light chain single cell RNA sequencing. From these experiments, we identified clonally expanded B-cell populations and convergent BCR sequences shared between different patients. We further demonstrated that recombinant antibodies derived from lung cancer patients bind cultured lung cancer cell lines in a dose-dependent manner. This preliminary data supports the notion that tumor-resident B-cells secrete functional antibodies that may additionally be tumor-reactive. Future characterization of these antibodies includes antigen identification and the measurement of in vitro and in vivo cytotoxic activity. Human patient-derived antibodies not only could serve as a novel source for therapeutic and diagnostic agents but may also help inform vaccine design in lung cancer indications.

Snail1-dependent cancer-associated fibroblasts induce epithelial-mesenchymal transition in lung cancer cells via exosomes.

Epithelial-mesenchymal transition (EMT) is an essential component of metastasis. Our previous study demonstrated that cancer-associated fibroblasts (CAFs) induce EMT in lung cancer cells. In recent years, many studies have demonstrated that CAFs induce metastasis and drug resistance in cancer cells via exosomes. We sought to discover the mechanism underlying how CAFs induce EMT in lung cancer cells, unveiling the role of exosomes in lung cancer progression. We cultured lung cancer cell (i) with control medium, normal fibroblasts (NFs) or CAFs; (ii) with SNAI1-transfected or NC (negative control)-transfected CAFs; (iii) with exosomes extracted from NF- or CAF-conditioned medium; (iv) with exosomes released by SNAI1 or NC-transfected CAFs; (v) with CAF-conditioned medium or exosome-depleted CAF-conditioned medium. qRT-PCR was conducted to examine the expression of CDH1 (gene of E-cadherin) and VIM (gene of Vimentin), western blotting was conducted to examine E-cadherin and vimentin levels in lung cancer cells. Exosomes released by CAFs-promoted EMT in lung cancer cells. Interestingly, SNAI1 levels in exosomes secreted from CAFs were correlated with SNAI1 expression in CAFs. Furthermore, the level of SNAI1 in exosomes was crucial for inducing EMT in lung cancer cells. Finally, treatment of CAFs with GW4869, an inhibitor of exosome release, noticeably inhibited their EMT-inducing effect on recipient epithelial cells. The molecular mechanism underlying how CAFs induce EMT in cancer cells may be that CAFs deliver SNAI1 to recipient cancer cells via exosomes.

Tristetraprolin inhibits autophagy in cultured lung cancer cells via the nuclear factor-κB pathway

To explore the expression of the RNA-binding protein tristetraprolin in lung adenocarcinoma cells and its molecular mechanism for inhibiting autophagy. Quantitative real-time PCR and Western blotting were performed to detect the expression of autophagy-related genes (including Beclin1, LC3-Ⅱ/LC3-Ⅰ and SQSTM1/p62) in cultured lung adenocarcinoma cells at 24, 48 and 72 h after transient transfection with a tristetraprolin-overexpressing plasmid and the empty plasmid. The effects of transfection with the tristetraprolin-overexpressing plasmid and empty plasmids in the presence or absence of tumor necrosis factor-α (TNF-α) on the expressions of nuclear factor-κB (NF-κB) p65, c-rel, and p50 were examined in lung adenocarcinoma cells using immunofluorescence assay and Western blotting. The cells were also transfected with the IκBα-mut plasmid and the tristetraprolin-overexpressing plasmid, either alone or in combination, and the changes in the expressions of tristetraprolin and autophagy-related genes were detected using RT-qPCR and Western blotting. The expressions of tristetraprolin were significantly reduced at both the mRNA and protein levels in lung adenocarcinoma cells (P &lt; 0.001). Overexpression of tristetraprolin in the cells significantly lowered the expressions of autophagy-related genes Beclin1 and the ratio of LC3-Ⅱ/LC3-Ⅰ at the mRNA and protein levels (P &lt; 0.001), obviously lowered the expressions of NF-κB p65 and c-rel, and almost totally blocked the nuclear translocation of NF-κB p65 and c-rel (P &lt; 0.05); the expression of p50, however, did not undergo significant changes in response to tristetraprolin overexpression (P &gt; 0.05). The inhibitory effect of tristetraprolin overexpression on autophagy was abrogated by transfection of the cells with IκBα-mut plasmid, which blocked the NF-κB signaling pathway. Co-transfection of the cells with IκBα-mut also attenuated the inhibitory effect of tristetraprolin overexpression on Beclin1 and the LC3-Ⅱ/LC3-Ⅰ ratio at both the mRNA and protein levels (P &lt; 0.05). The expression of tristetraprolin is low in lung adenocarcinoma cells. Tristetraprolin overexpression causes inhibition of autophagy in lung adenocarcinoma cells possibly by blocking NF-κB p65 and c-rel nuclear translocation.

LncRNA CASC2 inhibits proliferation and migration of adenocarcinoma cells via miR-4735-3p and mTOR.

Lung adenocarcinoma is a major form of non-small-cell lung cancer that frequently strikes nonsmokers. The disease is often diagnosed at a late stage and the 5-year survival rate is very low. Although previous studies found many somatic alterations associated with lung adenocarcinoma, the molecular basis of the development and progression of the disease is not well understood. We found that long noncoding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2), a putative tumor suppressor, was downregulated in both patient adenocarcinoma tissues and cultured lung cancer cells. Its tumor suppression function seemed to be dependent on its binding to miR-4735-5p. Changing the levels of CASC2 and miR-4735-3p in the cultured adenocarcinoma cells could affect the malignant phenotypes as well as growth of tumors derived from the cells injected into nude mice. Furthermore, the lncRNA and miR-4735-3p interplay likely the suppressed tumor growth through the downstream mammalian target of rapamycin signaling pathway. The results have revealed molecular details that may be critical for the development of lung adenocarcinoma, opening opportunities for the development of novel, and therapeutic tools.

Long non-coding RNA CRYBG3 regulates glycolysis of lung cancer cells by interacting with lactate dehydrogenase A.

Cancer cells usually utilize glucose as a carbon source for aerobic glycolysis, a phenomenon known as the Warburg effect. And a high rate of glycolysis has been observed in lung cancer cells. The growing evidence indicates that long non-coding RNAs (lncRNAs) are important players in lung cancer initiation and progression. However, the correlation between lncRNAs and glycolysis remains unclear. In this study, we recognized a lncRNA, LNC CRYBG3, which can interact with lactate dehydrogenase A (LDHA), a vital enzyme of glycolysis, is highly upregulated in both clinical lung cancer tissues and in vitro cultured lung cancer cell lines. A positive correlation between the expression level of LNC CRYBG3 and LDHA expression levels is observed. In another hand, LNC CRYBG3 is a regulator of glycolysis and its overexpression promoted the uptake of glucose and the production of lactate whereas the knockdown of LNC CRYBG3 led to opposite results and suppressed cell proliferation. These results indicated that LNC CRYBG3 might be a novel target for lung cancer treatment.

Imaging Extracellular Lactate In Vitro and In Vivo Using CEST MRI and a Paramagnetic Shift Reagent.

Overproduction of lactate is a hallmark of cancer, yet a method to quantitatively measure lactate production by cancer cells is not straight-forward. Chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) can potentially be used to image lactate but the small difference in chemical shift of the lactate -OH proton and water proton resonances make it challenging. Like other spectroscopic methods, CEST MRI cannot discriminate intracellular lactate from extracellular lactate. Herein, we demonstrate a relatively simple way to shift the lactate -OH proton resonance far away from water by addition of the paramagnetic shift reagent, EuDO3A, while retaining the CEST properties of lactate itself. The potential of the method was demonstrated by imaging extracellular lactate excreted from lung cancer cells in tissue culture without interference from other components in the culture media and by imaging excess lactate excreted into the bladder of a mouse.