Response Of Lung Cancer Cells Research Articles

Overcoming Nutrient Stress: Integrin αvβ3-Driven Metabolic Adaptation Supports Tumor Initiation.

Nutrient stress accompanies several stages of tumor progression, including metastasis formation. Metabolic reprogramming is a hallmark of cancer, and it has been associated with stress tolerance and anchorage-independent cell survival. Adaptive responses are required to support cancer cell survival under these conditions. In this issue of Cancer Research, Nam and colleagues showed that the extracellular matrix (ECM) receptor integrin β3 was upregulated in lung cancer cells in response to nutrient starvation, resulting in increased cell survival that was independent from ECM binding. Delving into the molecular mechanisms responsible for this, the authors found that integrin β3 promoted glutamine metabolism and oxidative phosphorylation (OXPHOS) by activating a Src/AMPK/PGC1α signaling pathway. Importantly, in vivo experiments confirmed that OXPHOS inhibition suppressed tumor initiation in an orthotopic model of lung cancer, while β3 knockout completely abrogated tumor initiation. These observations indicate that targeting signaling pathways downstream of αvβ3 could represent a promising therapeutic avenue to prevent lung cancer progression and metastasis. See related article by Nam et al., p. 1630.

Abstract 6459: Molecular profiling of advanced non-small cell lung cancer in response to first-line immune checkpoint inhibitors and/or chemotherapy using multimodal real-world data

Abstract Background: Up to 85% of lung cancer patients have non-small cell lung cancer (NSCLC). Standard of care for NSCLC includes immune checkpoint inhibitors (ICI) and/or platinum-based chemotherapy (CT). However, therapy success is limited with up to 70% of patients not responding to ICI. Mechanisms of resistance across treatments are not fully understood. In this study, we explore real-world data to characterize molecular and genomic changes in advanced NSCLC treated with ICI, ICI+CT, or CT. Methods: Stage III/IV NSCLC patients receiving first-line (1L) ICI, ICI+CT, or CT were identified from Tempus Labs, Inc.’s de-identified, multimodal real-world database. Patients’ solid tissue biopsies taken pre- or post-1L underwent next generation sequencing with the Tempus xT assays (DNAseq of 648 genes at 500x coverage and whole transcriptome RNAseq). Patients’ clinical and outcome data were abstracted from electronic health records. We analyzed changes in gene expression, pathway enrichment, estimated immune cell components, and somatic mutations in responders (complete/partial response) and non-responders (progression/stable disease) upon 1L treatment. Results: 1L non-responders (70%, 51%, and 47%) were identified across cohorts (143 ICI, 764 ICI+CT, and 228 CT, respectively). 58% of pre-1L biopsies were taken from distal metastatic tissues. Distinct pathways altered post-1L include the downregulated E2F target pathway in CT cohort, and upregulated TNFα/NFκB signaling and the hypoxia and protein secretion pathways in ICI cohort. Significant (FDR < 0.05) augmented expression was observed for 90, 23, and 65 genes in non-responders vs. responders post ICI, ICI+CT, and CT, respectively. RNAseq deconvolution reveals decreased plasma and CD4+ activated T cell fraction in metastatic biopsies, whereas increased CD4+ activated T cell and monocyte fraction was seen after treatment only in ICI cohort. Genes with oncogenic driver mutations reported in NSCLC were detected including TP53 (68%), KRAS (30%), STK11 (13%), EGFR (11%), CDKN2A (10%), KEAP1 (9%), and NF1 (7%). Of the 250 mutated genes, 72 (29%) were commonly not detected post-1L, whereas 38, 3, and 8 unique genes with loss of detectable mutations were identified post-1L of ICI, ICI+CT, and CT, respectively. Since loss of detectable mutations is a sign of loss of subpopulations of cancer cells hosting such mutations that succumb to therapy, these genetic mutations could be used to select patients who can benefit from such therapy. Conclusions: Distinct molecular characteristics including tumor immune components and genomic alterations are associated with 1L response in advanced NSCLC. Real-world data analyses provide opportunity to explore distinct mechanisms of therapy resistance and identify potential biomarkers of therapy response for further validation in clinical studies. Citation Format: Xiaoyong Fu, Jordan Kardos, Paola Correa, Min Wang, Li, Jackson Egen, Shahed Iqbal. Molecular profiling of advanced non-small cell lung cancer in response to first-line immune checkpoint inhibitors and/or chemotherapy using multimodal real-world data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6459.

PTEN-Akt/mTOR Expression Level in A549 Lung Cancer Cells in Response to Cisplatin, Carotenoids, and their Combination

PTEN-Akt/mTOR Expression Level in A549 Lung Cancer Cells in Response to Cisplatin, Carotenoids, and their Combination

Transcriptome profiling of A549 non-small cell lung cancer cells in response to Trichinella spiralis muscle larvae excretory/secretory products.

Trichinella spiralis (T. spiralis) muscle-larva excretory/secretory products (ML-ESPs) is a complex array of proteins with antitumor activity. We previously demonstrated that ML-ESPs inhibit the proliferation of A549 non-small cell lung cancer (NSCLC) cell line. However, the mechanism of ML-ESPs against A549 cells, especially on the transcriptional level, remains unknow. In this study, we systematically investigated a global profile bioinformatics analysis of transcriptional response of A549 cells treated with ML-ESPs. And then, we further explored the transcriptional regulation of genes related to glucose metabolism in A549 cells by ML-ESPs. The results showed that ML-ESPs altered the expression of 2,860 genes (1,634 upregulated and 1,226 downregulated). GO and KEGG analysis demonstrated that differentially expressed genes (DEGs) were mainly associated with pathway in cancer and metabolic process. The downregulated genes interaction network of metabolic process is mainly associated with glucose metabolism. Furthermore, the expression of phosphofructokinase muscle (PFKM), phosphofructokinase liver (PFKL), enolase 2 (ENO2), lactate dehydrogenase B (LDHB), 6-phosphogluconolactonase (6PGL), ribulose-phosphate-3-epimerase (PRE), transketolase (TKT), transaldolase 1 (TALDO1), which genes mainly regulate glycolysis and pentose phosphate pathway (PPP), were suppressed by ML-ESPs. Interestingly, tricarboxylic acid cycle (TCA)-related genes, such as pyruvate dehydrogenase phosphatase 1 (PDP1), PDP2, aconitate hydratase 1 (ACO1) and oxoglutarate dehydrogenase (OGDH) were upregulated by ML-ESPs. In summary, the transcriptome profiling of A549 cells were significantly altered by ML-ESPs. And we also provide new insight into how ML-ESPs induced a transcriptional reprogramming of glucose metabolism-related genes in A549 cells.

Network models of protein phosphorylation, acetylation, and ubiquitination connect metabolic and cell signaling pathways in lung cancer

We analyzed large-scale post-translational modification (PTM) data to outline cell signaling pathways affected by tyrosine kinase inhibitors (TKIs) in ten lung cancer cell lines. Tyrosine phosphorylated, lysine ubiquitinated, and lysine acetylated proteins were concomitantly identified using sequential enrichment of post translational modification (SEPTM) proteomics. Machine learning was used to identify PTM clusters that represent functional modules that respond to TKIs. To model lung cancer signaling at the protein level, PTM clusters were used to create a co-cluster correlation network (CCCN) and select protein-protein interactions (PPIs) from a large network of curated PPIs to create a cluster-filtered network (CFN). Next, we constructed a Pathway Crosstalk Network (PCN) by connecting pathways from NCATS BioPlanet whose member proteins have PTMs that co-cluster. Interrogating the CCCN, CFN, and PCN individually and in combination yields insights into the response of lung cancer cells to TKIs. We highlight examples where cell signaling pathways involving EGFR and ALK exhibit crosstalk with BioPlanet pathways: Transmembrane transport of small molecules; and Glycolysis and gluconeogenesis. These data identify known and previously unappreciated connections between receptor tyrosine kinase (RTK) signal transduction and oncogenic metabolic reprogramming in lung cancer. Comparison to a CFN generated from a previous multi-PTM analysis of lung cancer cell lines reveals a common core of PPIs involving heat shock/chaperone proteins, metabolic enzymes, cytoskeletal components, and RNA-binding proteins. Elucidation of points of crosstalk among signaling pathways employing different PTMs reveals new potential drug targets and candidates for synergistic attack through combination drug therapy.

Mechanism of miR-760 Reversing Lung Cancer Immune Escape by Downregulating IDO1 and Eliminating Regulatory T Cells Based on Mathematical Biology

In cancer biology, mathematical models have become indispensable. They are useful for gaining a mechanistic grasp of cancer's dynamic processes. In cancer research, mathematical modelling approaches are becoming more common. The complexity of cancer is well suited to quantitative approaches as it provides challenges and opportunities for new developments (Altrock et al., 2015). Background. MicroRNA-760 (miR-760), as an early discovered tumor suppressor gene, is poorly expressed in lung cancer (LC). Indoleamine 2,3-dioxygenase 1 (IDO1), as an important regulator of T cell function, is active in immune tolerance. We discovered that miR-760 has a targeted relationship with IDO1, but the regulatory mechanism between miR-760 and IDO1 is still unclear. Method. The miR-760 and IDO1 levels in NSCLC were tested via real-time quantitative polymerase chain reaction (qRT-PCR) and western blotting (WB). Cell growth was tested by CCK8, and NSCLC cell migration and invasion were analyzed through Transwell analysis. The binding conditions and target gene of miR-451 in NSCLC cells were determined via double luciferase reporter gene. The CD8+ T and CD4+ T cell ratio in CD45+cells was assessed by flow cytometry. Results. qRT-PCR revealed that miR-760 was low-expressed and IDO2 was highly expressed in LC. miR-760 mimics suppressed cell growth, invasiveness, and migration. We also observed that miR-760 could downregulate the IDO1 protein level. Significantly, we revealed that miR-760 could inhibit CD8+ T cell apoptosis by controlling IDO1 enzyme function. Conclusion. Our findings show that miR-760 inhibits CD8+ T cell responses in LC through regulating IDO1, laying the groundwork for the development of novel vaccination therapies for the treatment of LC.

Transcriptomic atlas of GNAT family members in pulmonary epithelia under pathological conditions using single‐cell and bulk cell sequencing

General control non-repressible 5-related N-acetyltransferases (GNAT) family members (n = 24) are primarily involved in the histone acetylation and translation process, to promote gene expression. Dysfunction of GNAT family genes was found to contribute to cancer cell migration and invasion. Of those, NAT10 and ELP3 were proposed to promote epithelial-to-mesenchymal transition, immune cell infiltration and cell circle regulation.1 KAT2A, KAT2B, SAT1 and NAA40 were considered as biomarkers of cancer prognosis. NAT1, NAT2, NAT8L, NAA10 and GNPNAT1 were altered in lung cancer, while little has been known about changes of GNAT family members in epithelia of inflammatory lung diseases and in response to challenges. Epithelial cells play important roles in the maintenance of pulmonary defence and mechanic function and act as primary receptors to pathogens and secondary initiators for systemic inflammation.2, 3 Cancer cells have the capacity of producing chemoattractive factors to recruit the infiltration of immune cells, while immune cell interactions within the microenvironment alter the biological behaviours of cancer cells. The present study aims at understanding transcriptomic phenomes of GNAT family genes in human lung epithelia of lung diseases using single-cell RNA sequencing (scRNA-seq), compare GNAT gene heterogeneity of lung cancer with other cancers using bulk RNA-seq,and exploring GNAT gene changes in normal airway epithelial cells and lung cancer cells in response to external challenges, for example, lipopolysaccharide (LPS), cigarette smoking extract (CSE), cholesterol, lysophosphatidylcholine (LPC) or drugs. To figure out the role of GNAT in lung epithelial under different pathophysiological conditions including chronic systematic inflammation status, immune system disorders and tumour microenvironment, we analyzed epithelial scRNA-seq data of 84 lung tissues from 20 healthy controls, 15 patients with chronic obstructive pulmonary disease (COPD), 15 with idiopathic pulmonary fibrosis (IPF), 8 with systemic sclerosis (SSC), 15 with lung adenocarcinoma (LUAD) and 11 corresponding para-cancer tissues acquired from the databases (GSE128169, GSE131907, GSE136831, GSE128033, E-MTAB-6653 and E-MTAB-6149).4-8 Eight epithelia were labelled and defined, as detailed in Supplemental Materials (Part 1). We found that expression of more GNAT genes (>8) was higher in alveolar type I (Figure 1A) of SSC, alveolar type II of LUAD and SSC (Figure 1B), basal cells of IPF and LUAD (Figure 1C), ciliated epithelia of LUAD (Figure 1D), club epithelia of LUAD and SSC (Figure 1E), goblet cells of LUAD (Figure 1F), mucous epithelia of IPF and LUAD (Figure 1G) and neuroendocrine cells of LUAD (Figure 1H). Of those, KAT2B and NAT10 mainly over-expressed in COPD, KAT2B, SAT1 and ELP3 in IPF and GNPNAT1, KAT2A, NAA20 and NAT9 in LUAD (p-values in Table S1). mRNA expression of AANAT, NAA11, NAT16, NAT8, NAT8L and SALT1 was hardly detected in most various human lung epithelia or human tumour tissues, of which mean values and SEM were listed in Tables S2 and S3. It seems that the imbalance of histone acetylation and deacetylation plays a critical role in epithelial carcinogenesis and progression of LUAD through altering microtubule stability, chromatin structure, telomerase activity and DNA damage repair.9 Some GNAT genes may contribute to the common pathway in the course of LUAD and COPD by mediating the synthesis of CoA-SH, biological behaviours of epithelia (differentiation, apoptosis and proliferation) and metabolism of amino acids-related pathways (Figure S1). To define the specificity of GNAT gene changes in epithelia of lung cancer, we compared GNAT gene expressions in LUAD with normal lung tissues, para-cancer tissues and 11 other cancers from the UCSC database (https://xenabroeser.net). The read count value of each GNAT family gene was converted to fragments per kilobase of exon model per million mapped fragments (FPKM) value. Expression folds above the normal of NAT9 were higher in LUAD, NAA20 and NAA50 in LUSC, SAT1 in THCA, NAGS and SAT2 in LIHC, KAT2A in CHOL, NAT8 in KIRP, NAA11 in BLCA or KAT2B, NAT16, NAT8L and SATL1 in GBM (Figure 2A). Expressions of GNAT genes elevated or reduced in various cancers, as compared with normal tissues or LUAD tissues, were presented in Figure 2B and summarized in Table S3. Of those, ELP3, GNPNAT1, NAA20, NAA30, NAA50, NAT10 and NAT14 increased significantly in lung cancers, while AANAT, KAT2A, KAT2B, NAA60, NAT9, SAT1 and SAT2 were lower, of which p-values were shown in Table S3. Different from GNAT genes in LUSC, NAA10, NAA20, NAA40, NAA50 and NAT10 down-regulated and ELP3, GNPNAT1, KAT2B, NAGS, NAT14 and NAT9 up-regulated in LUAD tissues (Table S4). The results suggested that GNAT family genes could serve as novel biomarker candidates of epithelial pathologies distinguishing between LUAD and LUSC. Critical GNAT family genes (ELP3, SAT1 and SAT2) may participate in the tumorigenesis of LUAD as well as COPD by regulating chromatin structures and metabolism of polyamines for cellular functions (Figure S1). To uncover responses of GNAT genes to inflammatory stimuli (e.g., CSE, LPS and LysoPC) in normal lung epithelial cells (HBE) and lung cancer cells (A549, SPC-A1, H1299 and H460), we measured GNAT gene expression in different lung epithelia after exposure of pathogens using bulk RNA-seq, as detailed in Supplemental Materials. AANAT and KAT2A were down-expressed in A549 at 3 h and up-expressed at 48 h after CSE (Figure 3A,B), implying obvious time-dependent variations. The molecular mechanisms associated with GNAT expression in A549 challenged with CSE may promote the progression of chronic airway inflammation towards lung cancer, as indicated previously.10 NAA20 and NAT10 decreased and SAT1 increased in A549 at 6 h, while LPS induced more GNAT gene changes than CSE at 48 h in A549 and H1299 (Table S5). Cholesterol at 1μg/ml reduced NAT10 expression at 6 h and CSE-increased KAT2A, NAA20 and NAA40 expression at 24 h (Figure 4A, Table S6). KAT2B and SAT1 upregulated in HBE challenged with CSE for 6 h and showed time-dependence at 48 h (Figure 4B, Table S6). LysoPC at 100μM reduced the expression of NAA10, NAA20, NAT14 and SAT2 and increased NAA50 in SPC-A1 cells at 24 h (Figure 4C, Table S7). Effects of LysoPC on SPC-A1 were mainly focused on the different NAA catalytic submits. AR2 inhibitor (No306) reduced the expression of AANAT, KAT2, NAA20, NAA40, NAT14 and NAT8L and increased NAA60, NAT9, SAT1 and SAT2 in A549 cells at 24 h (Figure 4D, Table S7). We also noticed the difference in GNAT gene expression among various epithelial cell lines and among cultured times. Effects of cholesterol and LPC on the GNAT family genes in epithelial cell lines were relatively weak, as compared with CSE and LPS, which implied that the expression of the GNAT family was more prone to change in both chronic inflammation and tumour development. In order to elucidate the potential mechanism of GNAT family genes in lung diseases, we summarized and exampled critical pathways of GNAT family genes-associated regulations in gene expression of potential targets responsible for the development of lung diseases (Figure S1). It is possible that the dysregulation of GNAT family genes might mediate the dysfunction of lung epithelium through the synthesis of CoA-SH. We found that the critical gene NAA50 was associated with chromatin organization affecting transcription in COPD which was corresponding to the CSE exposure in normal airway epithelial cells (Figure 4B). In conclusion, the heterogeneity of transcriptomic profiles exists among GNAT family members, lung epithelia, pulmonary diseases, cancer types, cell-line types or responses to various challenges. We identified the part of GNAT family genes as specific hallmarks in epithelial cells of lung cancers and the shared features with LUAD and COPD. More obvious expression of GNAT genes was detected in goblet cells of COPD and LUAD, basal and mucous cells of IPF and AT2 and basal cells of SSC. Of those, NAA11, NAT16, NAT8, NAT8L and SATL1 were hardly detected in most human lung epithelia and some showed lung cancer specificificity. The detailed potential pathway and target molecular for GNAT family genes involved in lung epithelium injury of chronic inflammatory diseases need to be furthermore investigated. The current analysis may limit some interpretations. Overall, our data indicate that the heterogeneity and complexity of GNAT family members may contribute to the pathogenesis of lung diseases and can be an important panel for the discovery of lung disease-specific biomarkers and targets for therapy. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Long non-coding RNAs and exosomal lncRNAs: Potential functions in lung cancer progression, drug resistance and tumor microenvironment remodeling.

Among the different kinds of tumors threatening human life, lung cancer is one that is commonly observed in both males and females. The aggressive behavior of lung cancer and interactions occurring in tumor microenvironment enhances the malignancy of this tumor. The lung tumor cells have demonstrated capacity in developing chemo- and radio-resistance. LncRNAs are a category of non-coding RNAs that do not encode proteins, but their aberrant expression is responsible for tumor development, especially lung cancer. In the present review, we focus on both lncRNAs and exosomal lncRNAs in lung cancer, and their ability in regulating proliferation and metastasis. Cell cycle progression and molecular mechanisms related to lung cancer metastasis such as EMT and MMPs are regulated by lncRNAs. LncRNAs interact with miRNAs, STAT, Wnt, EZH2, PTEN and PI3K/Akt signaling pathways to affect progression of lung cancer cells. LncRNAs demonstrate both tumor-suppressor and tumor-promoting functions in lung cancer. They can be considered as biomarkers in lung cancer and especially exosomal lncRNAs present in body fluids are potential tools for minimally invasive diagnosis. Furthermore, we discuss regulation of lncRNAs by anti-cancer drugs and genetic tools as well as the role of these factors in therapy response of lung cancer cells.

Lentinan triggers oxidative stress-mediated anti-inflammatory responses in lung cancer cells.

Inflammatory responses change several aspects of malignancies such as proliferation, survival, angiogenesis, and metastasis and lead to tumor progression. Lung cancer is the leading type of cancer worldwide and cancer-related inflammatory mediators challenge the successful treatments. Lentinan, a polysaccharide derived from Lentinula edodes, has shown anti-inflammatory characteristics in colitis and has been approved as an adjuvant therapy for cancer treatment. In the present study, we explored the mechanism underlying anti-inflammatory function of Lentinan in lung cancer cells. We showed that Lentinan reduced the inflammatory cytokines IL-6 and IL-1β in LPS-stimulated A549 cells at the concentrations much lower than the IC50. Lentinan failed to alter the NLRP3 expression profile at transcriptional and translational levels. However, it showed a huge inhibition of caspase-1 activity. Lentinan downregulated the expression of IL-6 and IL-1β at the mRNA level. We also showed that Lentinan altered the oxidative status of the cells by increasing the intracellular ROS content and attenuating the activity of GPx4, the key player in the anti-oxidative defense system. Lentinan-induced ROS generation was associated with caspase-3 activation and induction of DNA breaks. This alteration was also associated with mitochondrial membrane depolarization shown by TMRE staining. Using recombinant caspase-1, we showed that Lentinan did not directly target caspase-1 but it led to caspase-1 inhibition. In conclusion, cytotoxicity and anti-inflammatory functions are separated by the dose of Lentinan. Lentinan increased the ROS and mitochondrial dysfunction in a level that is insufficient to induce cell death, but is sufficient to regulate the NLRP3 activation.

Identification of potential target genes of non-small cell lung cancer in response to resveratrol treatment by bioinformatics analysis.

Non-small cell lung cancer (NSCLC) is the most common type in lung cancer in the world, and it severely threatens the life of patients. Resveratrol has been reported to inhibit cancer. However, mechanisms of resveratrol inhibiting NSCLC were unclear. The aim of this study was to identify differentially expressed genes (DEGs) of NSCLC treated with resveratrol and reveal the potential targets of resveratrol in NSCLC. We obtained mRNA expression profiles of two datasets from the National Center for Biotechnology Information Gene Expression Omnibus (NCBI-GEO) and 271 DEGs were selected for further analysis. Data from STRING shown that 177 nodes and 342 edges were in the protein-protein interaction (PPI) network, and 10 hub genes (ANPEP, CD69, ITGAL, PECAM1, PTPRC, CD34, ITGA1, CCL2, SOX2, and EGFR) were identified by Cytoscape plus-in cytoHubba. Survival analysis revealed that NSCLC patients showing low expression of PECAM1, ANPEP, CD69, ITGAL, and PTPRC were associated with worse overall survival (OS) (P < 0.05), and high expression of SOX2 and EGFR was associated with worse OS for NSCLC patients (P < 0.05). Overall, we identified ANPEP, CD69, ITGAL, and PTPRC as potential candidate genes which were main effects of resveratrol on the treatment of NSCLC. ANPEP, ITGAL, CD69, and PTPRC are all clusters of differentiation (CD) antigens, might be the targets of resveratrol. The bioinformatic results suggested that the inhibitory effect of resveratrol on lung cancer may be related to the immune signaling pathway. Further studies are needed to validate these findings and to explore their functional mechanisms.

Synergy of nanodiamond\u2013doxorubicin conjugates and PD-L1 blockade effectively turns tumor-associated macrophages against tumor cells

BackgroundTumor-associated macrophages (TAMs) are the most abundant stromal cells in the tumor microenvironment. Turning the TAMs against their host tumor cells is an intriguing therapeutic strategy particularly attractive for patients with immunologically “cold” tumors. This concept was mechanistically demonstrated on in vitro human and murine lung cancer cells and their corresponding TAM models through combinatorial use of nanodiamond-doxorubicin conjugates (Nano-DOX) and a PD-L1 blocking agent BMS-1. Nano-DOX are an agent previously proved to be able to stimulate tumor cells’ immunogenicity and thereby reactivate the TAMs into the anti-tumor M1 phenotype.ResultsNano-DOX were first shown to stimulate the tumor cells and the TAMs to release the cytokine HMGB1 which, regardless of its source, acted through the RAGE/NF-κB pathway to induce PD-L1 in the tumor cells and PD-L1/PD-1 in the TAMs. Interestingly, Nano-DOX also induced NF-κB-dependent RAGE expression in the tumor cells and thus reinforced HMGB1’s action thereon. Then, BMS-1 was shown to enhance Nano-DOX-stimulated M1-type activation of TAMs both by blocking Nano-DOX-induced PD-L1 in the TAMs and by blocking tumor cell PD-L1 ligation with TAM PD-1. The TAMs with enhanced M1-type repolarization both killed the tumor cells and suppressed their growth. BMS-1 could also potentiate Nano-DOX’s action to suppress tumor cell growth via blocking of Nano-DOX-induced PD-L1 therein. Finally, Nano-DOX and BMS-1 achieved synergistic therapeutic efficacy against in vivo tumor grafts in a TAM-dependent manner.ConclusionsPD-L1/PD-1 upregulation mediated by autocrine and paracrine activation of the HMGB1/RAGE/NF-κB signaling is a key response of lung cancer cells and their TAMs to stress, which can be induced by Nano-DOX. Blockade of Nano-DOX-induced PD-L1, both in the cancer cells and the TAMs, achieves enhanced activation of TAM-mediated anti-tumor response.Graphic abstract

Combination Treatment With Inhibitors of ERK and Autophagy Enhances Antitumor Activity of Betulinic Acid in Non-small-Cell Lung Cancer In Vivo and In Vitro.

Aberrant activation of the Ras–ERK signaling pathway drives many important cancer phenotypes, and several inhibitors targeting such pathways are under investigation and/or approved by the FDA as single- or multi-agent therapy for patients with melanoma and non–small-cell lung cancer (NSCLC). Here, we show that betulinic acid (BA), a natural pentacyclic triterpenoid, inhibits cell proliferation, and induces apoptosis and protective autophagy in NSCLC cells. Thus, the cancer cell killing activity of BA is enhanced by autophagy inhibition. Mitogen-activated protein kinases, and especially ERK that facilitates cancer cell survival, are also activated by BA treatment. As such, in the presence of ERK inhibitors (ERKi), lung cancer cells are much more sensitive to BA. However, the dual treatment of BA and ERKi results in increased protective autophagy and AKT phosphorylation. Accordingly, inhibition of AKT has a highly synergistic anticancer effect with co-treatment of BA and ERKi. Notably, autophagy inhibition by hydroxychloroquine (HCQ) increases the response of lung cancer cells to BA in combination with ERKi. In vivo, the three-drug combination (BA, ERKi, and HCQ), resulted in superior therapeutic efficacy than single or dual treatments in the xenograft mouse model. Thus, our study provides a combined therapy strategy that is a highly effective treatment for patients with NSCLC.

The G4 Resolvase DHX36 Possesses a Prognosis Significance and Exerts Tumour Suppressing Function Through Multiple Causal Regulations in Non-Small Cell Lung Cancer.

Lung cancer is one of the most prevalent cancers in both men and women worldwide. The nucleic acid G4 structures have been implicated in the transcriptional programmes of cancer-related genes in some cancers such as lung cancer. However, the role of the dominant G4 resolvase DHX36 in the progression of lung cancer remains unknown. In this study, by bioinformatic analysis of public datasets (TCGA and GEO), we find DHX36 is an independent prognosis indicator in non-small-cell lung carcinoma (NSCLC) with subtype dependence. The stable lentiviral knockdown of the DHX36 results in accelerated migration and aggregation of the S-phase subpopulation in lung cancer cells. The reduction of DHX36 level de-sensitises the proliferation response of lung cancer cells to chemotherapeutic drugs such as paclitaxel with cell dependence. The knockdown of this helicase leads to promoted tumour growth, demonstrated by a 3D fluorescence spheroid lung cancer model, and the stimulation of cell colony formation as shown by single-cell cultivation. High throughput proteomic array indicates that DHX36 functions in lung cancer cells through regulating multiple signalling pathways including activation of protein activity, protein autophosphorylation, Fc-receptor signalling pathway, response to peptide hormone and stress-activated protein kinase signalling cascade. A causal transcriptomic analysis suggests that DHX36 is significantly associated with mRNA surveillance, RNA degradation, DNA replication and Myc targets. Therefore, we unveil that DHX36 presents clinical significance and plays a role in tumour suppression in lung cancer, and propose a potentially new concept for an anti-cancer therapy based on helicase-specific targeting.

Differential Response of Lung Cancer Cells, with Various Driver Mutations, to Plant Polyphenol Resveratrol and Vitamin D Active Metabolite PRI-2191.

Plant polyphenols and vitamins D exhibit chemopreventive and therapeutic anticancer effects. We first evaluated the biological effects of the plant polyphenol resveratrol (RESV) and vitamin D active metabolite PRI-2191 on lung cancer cells having different genetic backgrounds. RESV and PRI-2191 showed divergent responses depending on the genetic profile of cells. Antiproliferative activity of PRI-2191 was noticeable in EGFRmut cells, while RESV showed the highest antiproliferative and caspase-3-inducing activity in KRASmut cells. RESV upregulated p53 expression in wtp53 cells, while downregulated it in mutp53 cells with simultaneous upregulation of p21 expression in both cases. The effect of PRI-2191 on the induction of CYP24A1 expression was enhanced by RESV in two KRASmut cell lines. The effect of RESV combined with PRI-2191 on cytokine production was pronounced and modulated. RESV cooperated with PRI-2191 in regulating the expression of IL-8 in EGFRmut cells, while OPN in KRASmut cells and PD-L1 in both cell subtypes. We hypothesize that the differences in response to RESV and PRI-2191 between EGFRmut and KRASmut cell lines result from the differences in epigenetic modifications since both cell subtypes are associated with the divergent smoking history that can induce epigenetic alterations.

Isorhamnetin Enhances the Radiosensitivity of A549 Cells Through Interleukin-13 and the NF-κB Signaling Pathway.

Isorhamnetin (ISO), a naturally occurring plant flavonoid, is widely used as a phytomedicine. The major treatment modality for non-small-cell lung carcinoma (NSCLC) is radiotherapy. However, radiotherapy can induce radioresistance in cancer cells, thereby resulting in a poor response rate. Our results demonstrated that pretreatment with ISO induced radiosensitizing effect in A549 cells using colony formation, micronucleus, and γH2AX foci assays. In addition, ISO pretreatment significantly enhanced the radiation-induced incidence of apoptosis, the collapse of mitochondrial membrane potential, and the expressions of proteins associated with cellular apoptosis and suppressed the upregulation of NF-κBp65 induced by irradiation in A549 cells. Interestingly, the expression of interleukin-13 (IL-13), an anti-inflammatory cytokine, was positively correlated with the ISO-mediated radiosensitization of A549 cells. The knockdown of IL-13 expression by RNA interference decreased the IL-13 level and thus reduced ISO-mediated radiosensitivity in cells. We also found that the IR-induced NF-κB signaling activation was inhibited by ISO pretreatment, and it was abrogated in IL-13 silenced cells. We speculated that ISO may confer radiosensitivity on A549 cells via increasing the expression of IL-13 and inhibiting the activation of NF-κB. To our knowledge, this is the first report demonstrating the effects of ISO treatment on the responsiveness of lung cancer cells to irradiation through IL-13 and the NF-κB signaling pathway. In summary, ISO is a naturally occurring radiosensitizer with a potential application in adjuvant radiotherapy.

MARCKS cooperates with NKAP to activate NF-kB signaling in smoke-related lung cancer.

Rationale: Cigarette smoking is a major risk factor for lung cancer development and progression; however, the mechanism of how cigarette smoke activates signaling pathways in promoting cancer malignancy remains to be established. Herein, we aimed to determine the contribution of a signaling protein, myristoylated alanine-rich C kinase substrate (MARCKS), in smoke-mediated lung cancer.Methods: We firstly examined the levels of phosphorylated MARCKS (phospho-MARCKS) in smoke-exposed human lung cancer cells and specimens as well as non-human primate airway epithelium. Next, the MARCKS-interactome and its gene networks were identified. We also used genetic and pharmacological approaches to verify the functionality and molecular mechanism of smoke-induced phospho-MARCKS.Results: We observed that MARCKS becomes activated in airway epithelium and lung cancer cells in response to cigarette smoke. Functional proteomics revealed MARCKS protein directly binds to NF-κB-activating protein (NKAP). Following MARCKS phosphorylation at ser159 and ser163, the MARCKS-NKAP interaction was inhibited, leading to the activation of NF-κB signaling. In a screen of two cohorts of lung cancer patients, we confirmed that phospho-MARCKS is positively correlated with phospho-NF-κB (phospho-p65), and poor survival. Surprisingly, smoke-induced phospho-MARCKS upregulated the expression of pro-inflammatory cytokines, epithelial-mesenchymal transition, and stem-like properties. Conversely, targeting of MARCKS phosphorylation with MPS peptide, a specific MARCKS phosphorylation inhibitor, suppressed smoke-mediated NF-κB signaling activity, pro-inflammatory cytokines expression, aggressiveness and stemness of lung cancer cells.Conclusion: Our results suggest that phospho-MARCKS is a novel NF-kB activator in smoke-mediated lung cancer progression and provide a promising molecular model for developing new anticancer strategies.

A Pilot Study: Changes of Intestinal Microbiota of Patients With Non-small Cell Lung Cancer in Response to Osimertinib Therapy.

Osimertinib contributes to the higher efficacy and few intestinal side effects in non-small cell lung cancer (NSCLC) patients with T790M mutation. Previous studies has reported that intestinal microbiota play important roles in drug efficacy and toxicity. However, we have known less about the changes of intestinal microbiota in response to osimertinib therapy. In this pilot study, we used longitudinal sampling with 6 weeks sampling collection intervals for about 1 year to model intestinal microbial changes based on the 16S rRNA genes sequencing in fecal samples from NSCLC patients in response to osimertinib therapy. The results showed that there was no significantly different on the intestinal microbial composition at the phylum, family, and genus level among NSCLC patients with different treatment cycles (P > 0.05). There were no significant differences in alpha diversity characterized by the richness, Shannon diversity, and phylogenetic diversity based on the Welch’s t-test among NSCLC patients in response to osimertinib therapy (P > 0.05). However, the dissimilarity test and principal coordination analysis showed a few differences among NSCLC patients. The intestinal microbial markers were changed in post-therapy (Sutterella, Peptoniphilus, and Anaeroglobus) compared to that in pre-therapy (Clostridium XIVa). Furthermore, the phylogenetic molecular ecological networks (MENs) were influenced by osimertinib therapy based on the module number, link number, and module taxa composition of the first six groups. Overall, it indicated that osimertinib therapy changed the intestinal microbiota to some extent, though not completely. In all, this pilot study provides an understanding of changes of intestinal microbiota from NSCLC patients in response to osimertinib therapy. No complete changes in intestinal microbiota seem to be closely linked with the few intestinal side effects and higher efficacy in response to osimertinib therapy.

SPIB promotes anoikis resistance via elevated autolysosomal process in lung cancer cells.

Anoikis (detachment-induced cell death) is a specific type of programmed cell death which occurs in response to the loss of the correct extracellular matrix connections. Anoikis resistance is an important mechanism in cancer invasiveness and metastatic behavior. Autophagy, on the other hand, involves the degradation of damaged organelles and the recycling of misfolded proteins and intracellular components. However, the intersection of these two cellular responses in lung cancer cells has not been extensively studied. Here, we identified that upon matrix deprivation, the lymphocyte lineage-specific Ets transcription factor SPIB was activated and directly enhanced SNAP47 transcription in certain lung cancer cells. Loss of attachment-induced autophagy significantly increased anoikis resistance by SPIB activation. Consistent with this function, SPIB depletion by short hairpin RNA abrogated SNAP47 transcriptional activation upon matrix deprivation. Therefore, these data delineate an important role of SPIB in autophagy-mediated anoikis resistance in lung cancer cells. Accordingly, these findings suggest that manipulating SPIB-regulated pathways invivo and evaluating the impact of anoikis resistance warrant further investigation. DATABASE: RNA sequencing and ChIP sequencing data are available in Gene Expression Omnibus database under the accession numbers GSE106592 and GSE125561, respectively.

Cellular processes involved in lung cancer cells exposed to direct current electric field

With the rapid breakthrough of electrochemical treatment of tumors, electric field (EF)-sensitive genes, previously rarely exploited, have become an emerging field recently. Here, we reported our work for the identification of EF-sensitive genes in lung cancer cells. The gene expression profile (GSE33845), in which the human lung cancer CL1-0 cells were treated with a direct current electric field (dcEF) (300 mV/mm) for 2 h, was retrieved from GEO database. Differentially expressed genes (DEGs) were acquired, followed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) and protein-protein interaction (PPI) analysis. Hub genes were acquired and analyzed by various tools including the Human Protein Atlas, Kaplan-Meier analysis, Cytoscape, FunRich, Oncomine and cBioPortal. Subsequently, three-dimensional protein models of hub genes were modeled by Modeller 9.20 and Rosetta 3.9. Finally, a 100 ns molecular dynamics simulation for each hub protein was performed with GROMACS 2018.2. A total of 257 DEGs were acquired and analyzed by GO, KEGG and PPI. Then, 10 hub genes were obtained, and the signal pathway analysis showed that two inflammatory pathways were activated: the FoxO signaling pathway and the AGE-RAGE signaling pathway. The molecular dynamic analysis including RMSD and the radius of gyration hinted that the 3D structures of hub proteins were built. Overall, our work identified EF-sensitive genes in lung cancer cells and identified that the inflammatory state of tumor cells may be involved in the feedback mechanism of lung cancer cells in response to electric field stimulation. In addition, qualified three-dimensional protein models of hub genes were also constructed, which will be helpful in understanding the complex effects of dcEF on human lung cancer CL1-0 cells.

MiR-451a attenuates doxorubicin resistance in lung cancer via suppressing epithelialmesenchymal transition (EMT) through targeting c-Myc

Chemoresistance is still a major obstacle for lung cancer treatment. Increasing studies have demonstrated that microRNAs (miRNAs) are essential meditators of chemoresistance during cancer progression. MiR-451a is reported to be a tumor suppressor during cancer development. However, its effects on lung cancer and drug resistance in lung cancer are still unclear. In the study, the results showed that miR-451a exhibited a significant role in suppressing the drug resistance in lung cancer cells when treated with doxorubicin (DOX) through alleviating epithelialmesenchymal transition (EMT), as evidenced by the markedly reduced expression of N-cadherin and Vimentin, while the enhanced expression of E-cadherin. In addition, miR-451a over-expression markedly promoted the sensitivity of lung cancer cells to DOX treatments, and also disrupted the EMT of lung cancer cells. Mechanistically, miR-451a was found to directly target c-Myc to affect the EMT and drug resistance in lung cancer cells in response to DOX incubation. Furthermore, c-Myc knockdown markedly elevated the sensitivity of lung cancer cells to DOX, whereas over-expressing c-Myc markedly reversed the anti-tumor role of DOX, which was slightly diminished by miR-451a mimic. The in vivo experiments confirmed that miR-451a promoted the sensitivity of lung cancer cells-derived tumors to DOX treatment by reducing c-Myc. Therefore, our results revealed a new insight into DOX resistance of lung cancer cells and miR-451a could be considered as a potential therapeutic target to overcome drug resistance in lung cancer.