Resistant Non-small Cell Lung Cancer Cell Lines Research Articles

Identification of Key Genes and Signaling Pathways in Entrectinibresistant Non-small Cell Lung Cancer Using Bioinformatic Analysis and Experimental Verification.

Entrectinib, a ROS1 inhibitor, is effective in patients with ROS1-positive non-small-cell lung cancer (NSCLC). However, entrectinib resistance remains a challenge worldwide. The biomarkers of entrectinib resistance and molecular mechanisms have not been clarified based on the Gene Expression Omnibus (GEO) database. The aim of this study is to identify key genes and signaling pathways involved in the development of entrectinib-resistant NSCLC through bioinformatics analysis and experimental validation. Differentially expressed genes (DEGs) were screened between entrectinib resistant and parental human NSCLC cell lines of the GSE214715 dataset, lung adenocarcinoma (LUAD) and non-tumor adjacent tissues of the GSE75037 dataset, and NSCLC and non-tumor adjacent tissues of the GSE18842 dataset. Functional enrichment analyses were performed, including Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Overlapped DEGs among those three datasets were identified using the Venn diagram package. The transcriptional levels of key genes were investigated using the University of ALabama at Birmingham CANcer data analysis Portal (UALCAN). The association between transcriptional levels of key genes and survival was analyzed using Kaplan-Meier Plotter (https://www.kmplot.com/analysis/). The correlations between hub genes and immune cell infiltration were investigated using the Tumor Immune Estimation Resource (TIMER) database. Specific signaling pathway enrichment analysis was performed using Gene Set Enrichment Analysis (GSEA) of LinkedOmics. Competitive endogenous RNA (ceRNA) networks, genome-wide association studies (GWAS), and drug sensitivity analyses of key genes were further investigated. The expression of ZEB2 was subsequently confirmed in both parental HCC78 cells and entrectinib-resistant HCC78 cells using real-time quantitative polymerase chain reaction (qRT-PCR). 708 DEGs were identified between entrectinib-resistant CUTO28 (CUTO28-ER) and parental CUTO28 cell lines in the GSE214715 dataset. One thousand three hundred and ninety-five DEGs were identified between entrectinib resistant (CUTO37-ER) and parental CUTO37 cell lines in the GSE214715 dataset. Eight hundred and forty-nine DEGs were identified between LUAD and non-tumor adjacent tissues in the GSE75037 dataset. Seven hundred and sevety-three DEGs were identified between NSCLC and non-tumor adjacent tissues in the GSE18842 dataset. Among these three datasets, seven overlapped DEGs were identified, including ZBED2, CHI3L2, CELF2, SEMA5A, ZEB2, S100A12, and PDK4. Among these seven overlapped DEGs, the expression levels of CHI3L2, ZEB2, and S100A12 were downregulated in those three datasets. The results of analysis using the UALCAN database showed that these three genes were significantly downregulated in LUAD and LUSC patients compared with the normal population. However, only the lower transcriptional level of ZEB2 was linked to worse survival in patients with lung cancer. GSEA analysis revealed that ZEB2 was significantly negatively correlated with nucleotide excision repair (NER) in LUAD, and homologous recombination (HR) and NER in LUSC, which were linked to drug resistance. A ceRNA network of THRB-AS1/ has-miR-1293/ ZEB2 in LUAD was established. We have identified core genes associated with non-small cell resistance to entrectinib, including CHI3L2, ZEB2, and S100A12. ZEB2 is a core gene associated with acquired resistance to entetinib in NSCLC.

Frequency-Dependent Ecological Interactions Increase the Prevalence, and Shape the Distribution, of Preexisting Drug Resistance

The evolution of resistance remains one of the primary challenges for modern medicine, from infectious diseases to cancers. Many of these resistance-conferring mutations often carry a substantial fitness cost in the absence of treatment. As a result, we would expect these mutants to undergo purifying selection and be rapidly driven to extinction. Nevertheless, preexisting resistance is frequently observed from drug-resistant malaria to targeted cancer therapies in non-small-cell lung cancer (NSCLC) and melanoma. Solutions to this apparent paradox have taken several forms, from spatial rescue to simple mutation supply arguments. Recently, in an evolved resistant NSCLC cell line, we found that frequency-dependent ecological interactions between ancestor and resistant mutant ameliorate the cost of resistance in the absence of treatment. Here, we hypothesize that frequency-dependent ecological interactions in general play a major role in the prevalence of preexisting resistance. We combine numerical simulations with robust analytical approximations to provide a rigorous mathematical framework for studying the effects of frequency-dependent ecological interactions on the evolutionary dynamics of preexisting resistance. First, we find that ecological interactions significantly expand the parameter regime under which we expect to observe preexisting resistance. Next, even when positive ecological interactions between mutants and ancestors are rare, these resistant clones provide the primary mode of evolved resistance because even weak positive interaction leads to significantly longer extinction times. We then find that even in the case where mutation supply alone is sufficient to predict preexisting resistance, frequency-dependent ecological forces still contribute a strong evolutionary pressure that selects for increasingly positive ecological effects (negative frequency-dependent selection). Finally, we genetically engineer several of the most common clinically observed resistance mechanisms to targeted therapies in NSCLC, a treatment notorious for preexisting resistance. We find that each engineered mutant displays a positive ecological interaction with their ancestor. As a whole, these results suggest that frequency-dependent ecological effects can play a crucial role in shaping the evolutionary dynamics of preexisting resistance. Published by the American Physical Society 2024

CircMYBL1 suppressed acquired resistance to osimertinib in non-small-cell lung cancer

Circular RNAs (circRNAs) play an important role in the development of acquired resistance to many anticancer drugs. We developed the Non-Small-Cell Lung Cancer (NSCLC) cell lines with acquired resistance to osimertinib, a third-generation of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), and evaluated the different expression profiles of circRNAs in osimertinib-sensitive and -resistant NSCLC cell lines using RNA sequencing (RNA-Seq). The expression of selected differentially expressed circRNAs was verified using quantitative real-time PCR (qRT-PCR) in paired osimertinib-sensitive and -resistant NSCLC cell lines, and in plasma samples of osimertinib-sensitive and -resistant NSCLC patients. We found that circMYBL1(has_circ_0136924) was downregulated after acquired resistance to osimertinib, inhibiting circMYBL1 expression facilitated the proliferation, migration, and invasion in osimertinib-sensitive NSCLC cells. CircMYBL1 may be a novel molecular biomarker and therapeutic target for osimertinib-resistant NSCLC.

Meta-analysis of microarray data to determine gene indicators involved in cisplatin resistance in non-small cell lung cancer.

Lung cancer is a major cause of cancer-related mortality worldwide, with a 5-year survival rate of approximately 22%. Cisplatin is one of the standard first-line chemotherapeutic agents for non-small cell lung cancer (NSCLC), but its efficacy is often limited by the development of resistance. Despite extensive research on the molecular mechanisms of chemoresistance, the underlying causes remain elusive and complex. We analyzed three microarray datasets to find the gene signature and key pathways related to cisplatin resistance in NSCLC. We compared the gene expression of sensitive and resistant NSCLC cell lines treated with cisplatin. We found 274 DEGs, including 111 upregulated and 163 downregulated genes, in the resistant group. Gene set enrichment analysis showed the potential roles of several DEGs, such as TUBB2B, MAPK7, TUBAL3, MAP2K5, SMUG1, NTHL1, PARP3, NTRK1, G6PD, PDK1, HEY1, YTHDF2, CD274, and MAGEA1, in cisplatin resistance. Functional analysis revealed the involvement of pathways, such as gap junction, base excision repair, central carbon metabolism, and Notch signaling in the resistant cell lines. We identified several molecular factors that contribute to cisplatin resistance in NSCLC cell lines, involving genes and pathways that regulate gap junction communication, DNA damage repair, ROS balance, EMT induction, and stemness maintenance. These genes and pathways could be targets for future studies to overcome cisplatin resistance in NSCLC.

Impact of Folic Acid on the Resistance of Non-small Cell Lung Cancer Cells to Osimertinib by Regulating Methylation of DUSP1

Drug resistance is the main cause of high mortality of lung cancer. This study was conducted to investigate the effect of folic acid (FA) on the resistance of non-small cell lung cancer (NSCLC) cells to Osimertinib (OSM) by regulating the methylation of dual specificity phosphatase 1 (DUSP1). The OSM resistant NSCLC cell line PC9R was establishd by gradually escalation of OSM concentration in PC9 cells. PC9R cells were randomly grouped into Control group, OSM group (5 μmol/L OSM), FA group (600 nmol/L FA), methylation inhibitor decitabine (DAC) group (10 μmol/L DAC), FA+OSM group (600 nmol/L FA+5 μmol/L OSM), and FA+OSM+DAC group (600 nmol/L FA+5 μmol/L OSM+10 μmol/L DAC). CCK-8 method was applied to detect cell proliferation ability. Scratch test was applied to test the ability of cell migration. Transwell assay was applied to detect cell invasion ability. Flow cytometry was applied to measure and analyze the apoptosis rate of cells in each group. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) method was applied to detect the expression level of DUSP1 mRNA in cells. Methylation specific PCR (MSP) was applied to detect the methylation status of the DUSP1 promoter region in each group. Western blot was applied to analyze the expression levels of DUSP1 protein and key proteins in the DUSP1 downstream mitogen-activated protein kinase (MAPK) signaling pathway in each group. Compared with the Control group, the cell OD450 values (48 h, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the OSM group were obviously decreased (P<0.05); the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of extracellular regulated protein kinases (ERK) were obviously increased (P<0.05); the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the DAC group were obviously increased (P<0.05); the apoptosis rate, the expression of p38 MAPK protein, the phosphorylation level of ERK, and the methylation level of DUSP1 were obviously reduced (P<0.05). Compared with the OSM group, the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the FA+OSM group were obviously decreased (P<0.05); the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of ERK were obviously increased (P<0.05). Compared with the FA+OSM group, the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the FA+OSM+DAC group were obviously increased; the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of ERK were obviously reduced (P<0.05). FA may inhibit DUSP1 expression by enhancing DUSP1 methylation, regulate downstream MAPK signal pathway, then promote apoptosis, inhibit cell invasion and metastasis, and ultimately reduce OSM resistance in NSCLC cells.

LncRNA FEZF1-AS1 facilitates cisplatin resistance in non-small cell lung cancer through modulating the miR-32-5p-glutaminase axis.

Non-small cell lung cancer (NSCLC) is one of the prevalent malignancies. Cisplatin (CDDP) is a conventional chemotherapeutic agent against NSCLC. However, inherent and acquired chemoresistance limited the effectiveness of cisplatin in treatment of NSCLC. This study aimed to investigate the roles and underlying mechanisms of lncRNA-FEZF1-AS1 in mediating cisplatin sensitivity in NSCLC. We found that FEZF1-AS1 levels were significantly higher in lung cancer patients and cell lines. Blocking FEZF1-AS1 sensitized lung cancer cells to cisplatin. Additionally, both glutamine metabolism and FEZF1-AS1 were significantly elevated in cisplatin resistant NSCLC cell lines, A549/CDDP R and SK-MES-1 CDDP/R. Analysis using bioinformatics, RNA pull-down assay and luciferase assay demonstrated that FEZF1-AS1 sponged miR-32-5p, which acted as a tumor suppressor in NSCLC. Glutaminase (GLS), a key enzyme in the glutamine metabolism, was predicted and validated as the direct target of miR-32-5p in NSCLC cells. Inhibiting glutamine metabolism or reducing glutamine supply effectively resensitized cisplatin-resistant cells. Furthermore, restoring miR-32-5p in FEZF1-AS1-overexpressing cisplatin resistant cells successfully overcame FEZF1-AS1-mediated cisplatin resistance by targeting GLS. These findings were further supported by in vivo xenograft mice experiments. This study uncovered the roles and molecular mechanisms of lncRNA FEZF1-AS1 in mediating cisplatin resistance in NSCLC, specifically through modulating the miR-32-5p-GLS axis, providing support for the development of new therapeutic approaches against chemoresistant lung cancer.

Abstract B099: Targeting Non Small Cell Lung Cancer EGFR-Mutant and EGFR-Inhibitor resistant cell lines by ferroptosis induction: A potential therapeutic approach

Abstract The epidermal growth factor receptor (EGFR) is a crucial signaling protein involved in regulating cellular growth, survival, and proliferation. EGFR mutations, particularly in non-small cell lung cancer (NSCLC), have been extensively studied due to their role in driving tumor progression and resistance to conventional therapies. While EGFR-mutant tumors initially respond well to EGFR inhibitors, emergence of resistance remains a significant challenge. Ferroptosis is a recently discovered form of regulated cell death characterized by accumulation of lipid peroxides and oxidative stress. It has gained attention as a potential therapeutic strategy in combating drug resistance. However, the vulnerability of various EGFR-mutant and resistant NSCLC cells to ferroptosis induction remains poorly understood. To bridge the existing knowledge gap, we investigated the responsiveness of both EGFR-mutant and their resistant counterpart NSCLC cell lines to ferroptosis induction. Interestingly, NSCLC cell lines and patients’ tumors which harbor EGFR mutations are distinct from those carrying STK11 and KEAP1 mutations, which are known to contribute to resistance to ferroptosis. This observation prompted us to test whether ferroptosis inducers could effectively target this specific subset of NSCLC. In our experimental approach, we first utilized a panel of commercially available EGFR mutant cell lines (n=3), including one harboring T790M resistance mutation, which is a common mechanism of acquired resistance to first-generation inhibitors. We subjected the EGFR-mutant cell lines in vitro to a process of acquiring resistance against three generations of EGFR inhibitors (n=4). Subsequently, upon confirmation of resistance, we evaluated their susceptibility to our proprietary ferroptosis inducer. Exome sequencing data of EGFR-inhibitor resistant cell lines and their age-matched control cell lines revealed that the former acquired genetic alterations that have previously been found to confer resistance in EGFR-inhibitor resistant patients’ tumors, suggesting that our approach is valid for modeling relevant resistance mechanisms. Our results indicate that both EGFR-mutant and resistant cell lines exhibit sensitivity to ferroptosis induction. Furthermore, we observed that EGFR-mutant patient-derived cells, which are refractory to EGFR inhibitors of several generations, were strongly cytostatic to our ferroptosis inducer. These findings provide valuable insights into the interplay between EGFR mutations, drug resistance, and ferroptosis. Exploiting the vulnerability of EGFR-inhibitor resistant NSCLC cells to ferroptosis could offer novel therapeutic options for overcoming drug resistance in EGFR-driven cancers. Further investigations into the underlying molecular mechanisms of ferroptosis inducers in NSCLC are warranted for the development of effective therapies. Citation Format: Taronish Dubash, Maria Cristina Munteanu, Cristian Nunez, Shrouq Farah, Laurence Jadin, Branko Radetich, Francesco M Marincola, Darby Schmidt, Nadia Gurvich. Targeting Non Small Cell Lung Cancer EGFR-Mutant and EGFR-Inhibitor resistant cell lines by ferroptosis induction: A potential therapeutic approach [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B099.

Abstract 3751: Characterizing the role of the y chromosome-expressed long non-coding RNA linc-SPRY3 family in lung cancer radiation resistance

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide. A major issue for clinicians and patients alike is inherent resistance to radiation therapy. Recently, certain long non-coding RNAs (lnc-RNAs) have been shown to regulate pathways involved in resistances to lung cancer treatments. Our group recently published that a group of long non-coding RNAs, named the linc-SPRY3 family, play an important role in radiation therapy sensitivity. Originating from the Y-Chromosome, these long non-coding RNAs have been shown in vitro and in vivo to decrease the tumor burden after radiation. Interestingly, certain male Non-Small Cell Lung Cancer (NSCLC) cell lines have been shown to have complete loss of the Y-chromosome (LOY), showing a radiation resistant phenotype. To test this, the DYZ1 region of the Y-chromosome, where the linc-SPRY3 family is expressed, was artificially introduced into radiation resistant male NSCLC cell lines with LOY. Remarkably, by adding the DYZ1 region of the Y-chromosome into these cells, they became more sensitive to radiation and had increased apoptosis. Based on this preliminary data, we hypothesize the linc-SPRY3 family interacts with important pathways in the cell to increase sensitivity to DNA double strand breaks. Through RNA-Sequencing and other preliminary experiments, we found that the linc-SPRY3 family regulates the expression of Cell Division Cycle 6 (CDC6) and Cell Division Cycle 25A (CDC25A) genes. Preliminary data suggests these interactions play a role in influencing NSCLC ability to invade and metastasize. We are currently characterizing this family of linc-RNAs by flow cytometry, in vivo metastasis models, and fluorescent in situ hybridization. We will characterize the mechanism of the linc-SPRY3 family in radiation resistance through genetic modifications and further downstream analysis via the over-expression and knockdown of these linc-RNAs. The characterization of these linc-RNAs will be critical for the development of potential diagnostic or therapeutic therapies. Citation Format: Emily S. Westemeier-Rice. Characterizing the role of the y chromosome-expressed long non-coding RNA linc-SPRY3 family in lung cancer radiation resistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3751.

Activation of the HSP27-AKT axis contributes to gefitinib resistance in non-small cell lung cancer cells independent of EGFR mutations

PurposeAlthough epidermal growth factor receptor (EGFR)-activating mutations in non-small cell lung cancer (NSCLC) usually show sensitivity to first-generation EGFR-tyrosine kinase inhibitors (TKIs), most patients relapse because of drug resistance. Heat shock protein 27 (HSP27) has been reported to be involved in the resistance of EGFR-TKIs, although the underlying mechanism is unclear. Here, we explore the mechanisms of HSP27-mediated EGFR TKI resistance and propose novel therapeutic strategies.MethodsTo determine the mechanism of HSP27 associated gefitinib resistance, differences were assessed using gefitinib-sensitive and -resistant NSCLC cell lines. In vivo xenograft experiments were conducted to elucidate the combinatorial effects of J2, a small molecule HSP27 inhibitor, and gefitinib. Analyses of human NSCLC tissues and PDX tissues were also used for comparison of HSP27 and phosphorylated AKT expression.ResultsLarge-scale cohort analysis of NSCLC cases revealed that HSP27 expression correlated well with the incidence of EGFR mutations and affected patient survival. Increased pAKT and HSP27 was observed in gefitinib-resistant cells compared with gefitinib-sensitive cells. Moreover, increased phosphorylation of HSP27 by gefitinib augmented its protein stability and potentiated its binding activity with pAKT, which resulted in increased gefitinib resistance. However, in gefitinib-sensitive cells, stronger binding activity between EGFR and HSP27 was observed. Moreover, these phenomena occurred regardless of EGFR mutation including secondary mutations, such as T790M. AKT knockdown switched HSP27-pAKT binding to HSP27-EGFR, which promoted gefitinib sensitivity in gefitinib-resistant cells. Functional inhibition of HSP27 yielded sensitization to gefitinib in gefitinib-resistant cells by inhibiting the interaction between HSP27 and pAKT.ConclusionsOur results indicate that combination of EGFR-TKIs with HSP27 inhibitors may represent a good strategy to overcome resistance to EGFR-TKIs, especially in cancers exhibiting AKT pathway activation.

Identification of lncRNA-miRNA-mRNA Networks Linked to Non-small Lung Cancer Resistance to Inhibitors of Epidermal Growth Factor Receptor.

Background: Tyrosine kinase inhibitors that act against epidermal growth factor receptor (EGFR) show strong efficacy against non-small cell lung cancer (NSCLC) involving mutated EGFRs. However, most such patients eventually develop resistance to EGFR-TKIs. Numerous researches have reported that messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs) may be involved in EGFR-TKI resistance, but the comprehensive expression profile and competitive endogenous RNA (ceRNA) regulatory network between mRNAs and ncRNAs in EGFR-TKI resistance of NSCLC are incompletely known. We aimed to define a ceRNA regulatory network linking mRNAs and non-coding RNAs that may mediate this resistance. Methods: Using datasets GSE83666, GSE75309 and GSE103352 from the Gene Expression Omnibus, we identified long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs differentially expressed between NSCLC cells that were sensitive or resistant to EGFR-TKIs. The potential biological functions of the corresponding differentially expressed genes were analyzed based KEGG pathways. We combined interactions among lncRNAs, miRNAs and mRNAs in the RNAInter database with KEGG pathways to generate transcriptional regulatory ceRNA networks associated with NSCLC resistance to EGFR-TKIs. Kaplan-Meier analysis was used to assess the ability of core ceRNA regulatory sub-networks to predict the progression-free interval and overall survival of NSCLC. The expression of two core ceRNA regulatory sub-networks in NSCLC was validated by quantitative real-time PCR. Results: We identified 8,989 lncRNAs, 1,083 miRNAs and 3,191 mRNAs that were differentially expressed between patients who were sensitive or resistant to the inhibitors. These DEGs were linked to 968 biological processes and 31 KEGG pathways. Pearson analysis of correlations among the DEGs of lncRNAs, miRNAs and mRNAs identified 12 core ceRNA regulatory sub-networks associated with resistance to EGFR-TKIs. The two lncRNAs ABTB1 and NPTN with the hsa-miR-150–5p and mRNA SERPINE1 were significantly associated with resistance to EGFR-TKIs and survival in NSCLC. These lncRNAs and the miRNA were found to be down-regulated, and the mRNA up-regulated, in a resistant NSCLC cell line relative to the corresponding sensitive cells. Conclusion: In this study, we provide new insights into the pathogenesis of NSCLC and the emergence of resistance to EGFR-TKIs, based on a lncRNA-miRNA-mRNA network.

Dihydroartemisinin overcomes the resistance to osimertinib in EGFR-mutant non-small-cell lung cancer

Osimertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI), is commonly used to treat EGFR-mutant non-small-cell lung cancer (NSCLC). However, acquired resistance to mutant EGFR (T790M) can evolve following osimertinib treatment. High reactive oxygen species (ROS) levels in lung cancer cells can influence heme levels and have an impact on osimertinib resistance. Here, we found that heme levels were increased in osimertinib resistant EGFR-mutant NSCLC cell lines and plasma heme levels were also elevated in osimertinib-treated EGFR-mutant NSCLC patients. The antimalarial drug dihydroartemisinin (DHA), which has anticancer effects and requires heme, was tested to determine its potential to revert osimertinib resistance. DHA downregulated the expression of heme oxygenase 1 and inhibited cell proliferation in osimertinib-resistant EGFR-mutant NSCLC cells (PC9-GR4-AZD1), which was further enhanced by addition of 5-aminolevulinic acid, protoporphyrin IX and hemin. DHA was synergistic with osimertinib in inhibiting cell proliferation and colony formation of all osimertinib-resistant cell lines tested. Combination treatment with osimertinib and DHA also increased the levels of ROS, downregulated the phosphorylation or protein levels of several RTKs that often are overexpressed in osimertinib-resistant EGFR-mutant NSCLC cells, and inhibited tumor growth without toxicity in a PC9-GR4-AZD1 xenograft mouse model. The results suggest that DHA is able to reverse the resistance to osimertinib in EGFR-mutant NSCLC by elevating ROS level and impair heme metabolism.

MicroRNA expression profiling and biomarker validation in treatment-naïve and drug resistant non-small cell lung cancer

BackgroundIn the absence of targetable mutations or immune checkpoints, cisplatin-doublet chemotherapy remains the standard of care in non-small cell lung cancer (NSCLC). Drug resistance has however become a significant clinical challenge. Exploring a role for small non-coding microRNAs (miRNA) as biomarker candidates in cisplatin resistant (CisR) lung cancer is lacking and warrants further investigation.MethodsmiRNA expression profiling was assessed in a panel of cisplatin sensitive and resistant NSCLC cell lines and validated by qPCR. Modulation of altered miRNAs was studied using antagomiRs and pre-miRs while functional assays were used to assess cisplatin response. The translational relevance of these miRNAs as potential biomarkers was assessed in serum and matched normal and tumour lung tissues from chemo-naïve NSCLC patients, in addition to xenograft formalin-fixed paraffin-embedded (FFPE) tumours derived from cisplatin sensitive and resistant cell lines.ResultsDifferential expression of a 5-miR signature (miR-30a-3p, miR-30b-5p, miR-30c-5p, miR-34a-5p, miR-4286) demonstrated their ability to distinguish between normal and tumour lung tissue and between NSCLC histologies. In squamous cell carcinoma (SqCC), tissue miRNA expression was associated with poor survival. miR-4286 showed promise as a blood-based diagnostic biomarker that could distinguish between adenocarcinoma and SqCC histologies. In a xenograft model of cisplatin resistance, using 7-9 week old female NOD/SCID mice (NOD.CB17-Prkdcscid/NCrCrl), a 5-miRNA panel showed altered expression between sensitive and resistant tumours.ConclusionsThis study identified a panel of miRNAs which may have diagnostic and prognostic potential as novel biomarkers in lung cancer and furthermore, may have a predictive role in monitoring the emergence of resistance to cisplatin.

A novel STAT3 inhibitor W2014-S regresses human non-small cell lung cancer xenografts and sensitizes EGFR-TKI acquired resistance.

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) is a common feature in human non-small cell lung cancer (NSCLC). STAT3 plays an important role in cancer progression as a driver oncogene and acquired resistance of targeted therapies as an alternatively activated pathway. W2014-S with pharmacophore structure of imidazopyridine, which was firstly reported to be utilized in STAT3 inhibitor discovery, was screened out as a potent STAT3 inhibitor from a library of small molecules. The aim of this study is to investigate the antitumor activities and mechanisms of W2014-S in NSCLC and effect on epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) resistance in vitro and in vivo.Methods: SPR analysis, Co-immunoprecipitation, confocal microscope imaging, and luciferase report gene assays were utilized to determine the mechanisms. Cell viability, colonial survival, wound healing, cell invasion assay, human cancer cell xenografts and PDX tumor xenografts were used to determine antitumor activities.Results: W2014-S disrupted STAT3 dimerization and selectively inhibited aberrant STAT3 signaling in NSCLC cell line. W2014-S strongly suppressed proliferation, survival, migration and invasion of lung cancer cells with aberrant STAT3 activation and inhibited the growth of human NSCLC cell xenografts and PDX tumor xenografts in mouse model. Furthermore, W2014-S significantly sensitized resistant NSCLC cell line to gefitinib and erlotinib in vitro and enhances the anti-tumor effect of gefitinib in TKI-resistant lung cancer xenografts in vivo.Conclusions: Our study has provided a novel STAT3 inhibitor with significant anti-tumor activities in NSCLC and suggests that combination of STAT3 inhibitor such as W2014-S with gefitinib could serve as a promising strategy to overcome EGFR-TKIs acquired resistance in NSCLC patients.

Potential monoamine oxidase A inhibitor suppressing paclitaxel-resistant non-small cell lung cancer metastasis and growth.

BackgroundHigh expression of monoamine oxidase A (MAOA) in non‐small cell lung cancer (NSCLC) is related to epithelial‐mesenchymal transition (EMT) and the development of clinicopathological features of NSCLC. Nevertheless, the role of MAOA in drug resistance still remains unclear. Hence, the aim of this article was to evaluate a previously synthesized MAOA inhibitor (G11) on inhibiting paclitaxel‐resistant NSCLC metastasis and growth.MethodsFirst, MAOA expression level was evaluated in several NSCLC cell lines. An MTT assay was used to validate the inhibitory effect of G11 on NSCLC cells in vitro. Second, gene expression in G11‐treated H460/PTX cells was analyzed by microarray gene expression. Third, transwell assay was performed to assess the invasion and metastasis of G11‐treated A549/PTX and H460/PTX cells and western blot assay used to analyze vital protein expression level in G11‐treated H460/PTX cells. Finally, the antimetastatic effect of G11 was tested in an NSCLC in vivo model.ResultsOur data revealed that G11 significantly inhibited the viability of paclitaxel (PTX)‐resistant NSCLC cell lines (A549/PTX and H460/PTX). G11 dramatically reduced the expression of MAOA in A549/PTX and H460/PTX cells, which exhibited relatively high MAOA expression levels. Additionally, G11 was found to hinder A549/PTX and H460/PTX cell migration and invasion. Furthermore, the in vivo study indicated that the coadministration of G11 and paclitaxel significantly suppressed tumor metastasis in H460/PTX lung metastasis models.ConclusionsThese findings indicated G11 showed a moderate inhibitory effect on paclitaxel‐resistant NSCLC metastasis and growth, and support further investigation on MAOA potentially as a promising therapeutic target for paclitaxel‐resistant NSCLC treatment.Key pointsSignificant findings of the study Inhibition of MAOA might contribute to the suppression of metastasis and growth in PTX‐resistant NSCLC cells. What this study adds This study explored the potential function of MAOA in drug‐resistant NSCLC and might consider MAOA as a promising target for the treatment of drug‐resistant NSCLC.

Abstract 1874: Anti-VEGF therapy sensitizes EGFR/MET co-altered NSCLC cells to TKIs via inhibiting the VEGF/ERK/MET pathway

Abstract Mesenchymal epithelial transition factor (MET) activation has been considered to mediate primary and acquired resistance to EGFR tyrosine kinase inhibitors (TKI) in epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC), but the mechanisms are not wholly clear. Meanwhile, the effective therapeutic strategy for these patients remains controversial. We previously presented a lung adenocarcinoma patient with dual EGFR-MET alterations benefited from bevacizumab + erlotinib as first-line treatment, achieving 13 months partial response (PR). PFS of another phase reached to 9 months with the combination of osimertinib + crizotinib + bevacizumab when disease proceeded due to increased MET copy number. Persistent PFS benefit was confirmed when the patient was administrated with different TKIs treatment in the presence of bevacizumab. However, the underlying mechanism remains elusive. Thus, we assumed that VEGF might be involved in the regulation of the MET signaling pathway and the addition of anti-VEGF therapy might be promising. In this study, we firstly confirmed whether MET overexpression alone might causally responsible for resistance to gefitinib treatment. Thus, we conducted in vitro forward genetic experiments, we took advantage of the EGFR-mutant lung cancer cell line PC-9, which had been shown to be sensitive to gefitinib. Ectopic overexpression of MET in PC-9 parental cells (PC-9/MET) was able to confer resistance to gefitinib by activating AKT and ERK signaling and sustaining proliferation.Meanwhile we got a gefitinib resistant EGFR mutant NSCLC cell line, HCC827 (HCC827-GR), which showing acquired MET amplification. Interestingly, we found that VEGF promoted cell survival and elicited adaptive resistance to gefitinib and crizotinib alone or in combination in MET transduced PC9-MET cells. This phenomenon also existed in HCC827-GR which harboring MET amplification. Importantly, addition of the VEGF inhibitor bevacizumab was able to restore partial sensitivity to gefitinib in both PC-9/MET and HCC827-GR cells while triple inhibition of EGFR, MET, and VEGF could suppresses the cell proliferation more remarkable. Moreover, we observed that VEGF could facilitate MET phosphorylation and activate several intracellular signaling pathways, including ERK, AKT, and Stat3. Then we pretreated cells with several specific kinase inhibitors, followed by treatment with VEGF. We found that VEGF-mediated up-regulation of MET depended on the ERK pathway and was independent of AKT and Stat3 pathways. These findings were further confirmed in human NSCLC Xenograft models. Addition of bevacizumab to TKIs resulted in stronger inhibition of tumor growth than TKI alone or TKIs combination treatment, by blocking both EGFR and MET signal transduction. In conclusion, bevacizumab + TKIs enhanced antitumor activity in NSCLC with EGFR/MET co-alteration through inhibition of the VEGF/ ERK/MET signaling axis. Citation Format: Qian Chu, Shanshan Huang, Yuan Gao, Jie Lin, Yuan Chen, Bo Zhu, Kongming Wu. Anti-VEGF therapy sensitizes EGFR/MET co-altered NSCLC cells to TKIs via inhibiting the VEGF/ERK/MET pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1874.

ELF1 activated long non-coding RNA CASC2 inhibits cisplatin resistance of non-small cell lung cancer via the miR-18a/IRF-2 signaling pathway.

Chemoresistance is the leading cause of recurrence in non-small cell lung cancer (NSCLC). The long non-coding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2) inhibits the tumorigenesis of various cancers. However, the regulatory function of CASC2 on the chemoresistance of NSCLC remains unclear. The levels of CASC2 and miR-18a in cisplatin (DDP)-resistant NSCLC tissues and cell lines were evaluated by quantitative Polymerase Chain Reaction (qPCR). The role of low CASC2 levels on overall survival in patients with NSCLC was tested using the log-rank test. The Chi-squared test was used to assess the relation between CASC2 expression and clinicopathological features of NSCLC patients. Cell Counting Kit-8 (CCK-8) assays tested the cell proliferation of cisplatin-resistant NSCLC cells (H226/DDP and A549/DDP). The underlying regulatory mechanism between CASC2 and miR-18a or miR-18a and interferon regulatory factor 2 (IRF-2) was predicted by bioinformatics and verified by a Dual-Luciferase reporter assay, RNA transfection, qPCR, and Western blotting. Chromatin immunoprecipitation (ChIP) assay was done to exam the relation between E74 like factor 1 (ELF1) and CASC2 gene. Mice xenografts were applied to exam the function of CASC2 on chemosensitivity of cisplatin of NSCLC cells in vivo. Low CASC2 expression is more likely to present in patients with advanced TNM stage (IV), cisplatin-resistance, and poor overall survival. The expression of CASC2 sharply decreased in cisplatin-resistant NSCLC tissues and cell lines (H226/DDP and A549/DDP). CASC2 overexpression strongly inhibited proliferation, migration, and invasion of cisplatin-resistant NSCLC cells (H226/DDP and A549/DDP) in vitro and inhibited tumor growth in vivo. Besides, CASC2 repressed miR-18a function by binding to the complementary sites of miR-18a as competing endogenous RNAs (ceRNAs). MiR-18a released by the declining expression of CASC2 reduced the protein concentration of IRF-2 in NSCLC cells. Furthermore, the transcription factor ELF1 was found to be promotor of CASC2 and increased its levels in cisplatin-resistant NSCLC cells. IRF-2 expression mediated by the ELF1/CASC2/miR-18a axis is markedly associated with the proliferation, migration, and invasion of cisplatin-resistant NSCLC, resulting in inferior survival. These findings suggest that this regulatory axis may serve as a novel therapeutic target in NSCLC.

SLC43A3 Is a Biomarker of Sensitivity to the Telomeric DNA Damage Mediator 6-Thio-2'-Deoxyguanosine.

Cell membrane transporters facilitate the passage of nucleobases and nucleosides for nucleotide synthesis and metabolism, and are important for the delivery of nucleoside analogues used in anticancer drug therapy. Here, we investigated if cell membrane transporters are involved in the cellular uptake of the nucleoside analogue DNA damage mediator 6-thio-2'-deoxyguanosine (6-thio-dG). A large panel of non-small cell lung cancer (NSCLC) cell lines (73 of 77) were sensitive to 6-thio-dG; only four NSCLC lines were resistant to 6-thio-dG. When analyzed by microarray and RNA sequencing, the resistant NSCLC cell lines clustered together, providing a molecular signature for patients that may not respond to 6-thio-dG. Significant downregulation of solute carrier family 43 A3 (SLC43A3), an equilibrative nucleobase transporter, was identified as a candidate in this molecular resistance signature. High levels of SLC43A3 mRNA predicted sensitivity to 6-thio-dG and therefore SLC43A3 could serve as a promising biomarker for 6-thio-dG sensitivity in patients with NSCLC. SIGNIFICANCE: These findings identify a biomarker of resistance to the telomeric DNA damage mediator 6-thio-2'-deoxyguanosine.

Dissecting the m6A methylation affection on afatinib resistance in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer deaths. Afatinib is the first-line anti-cancer agent for treatment of NSCLC. However, unexpected resistance has been a major obstacle for its clinical efficacy. In this study, we dissected afatinib resistance from the perspective of N6-Methyladenosine (m6A) modification. First, we depicted the m6A modification profiles for the afatinib resistant and sensitive NSCLC cell lines (H1299 and A549). We found that the sum enrichment scores of the resistant cell line (H1299) was much higher than that of the sensitive cell line (A549). Next, we identified the functionally m6A-modified genes, which were the intersection of the differentially m6A methylated genes and the differentially expressed genes between H1299 and A549, as well as negative correlation between m6A modification levels and gene expression levels. In addition, functional enrichment analysis of the functionally m6A-modified genes indicated that m6A methylation might modify cell cycle to affect afatinib response. Furthermore, the functionally m6A-modified genes were over-represented in the putative drug resistance-associated genes and the FDA-approved drug targets, and had significantly higher average degree and clustering coefficient than other genes in protein-protein interaction (PPI) network. We also identified five network modules, which were all related to drug resistance functions. Finally, survival analysis demonstrated that m6A modification could affect prognosis of NSCLC patients. In conclusion, we conducted a first attempt to dissect m6A methylation affection on afatinib resistance in NSCLC, and brought inspiration for the study of epigenetic roles in drug resistance.

Inhibitory effects of polyphyllins I and VII on human cisplatin-resistant NSCLC via p53 upregulation and CIP2A/AKT/mTOR signaling axis inhibition.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a human oncoprotein that is overexpressed in multiple kinds of cancers including non-small cell lung cancer (NSCLC). CIP2A plays an 'oncogenic nexus' to participate in the tumorigenesis and chemoresistance in several cancer types. AKT and mTORC1 overactivation are detected in NSCLC and many other cancers. Previous studies found that the CIP2A/AKT/mTOR pathway controls cell growth, apoptosis, autophagy process. Polyphyllin I (PPI) and polyphyllin VII (PPVII) are natural components extracted from Paris polyphylla that display anti-cancer properties. In the present study, we investigated whether PPI and PPVII can be used in the cisplatin (DDP)-resistant human NSCLC cell line A549/DDP. Results demonstrated that PPI and PPVII treatment significantly suppressed A549/DDP cell proliferation, migration, invasion and EMT, induced apoptosis and autophagy. Further examination of the mechanism revealed that the PPI and PPVII significantly upregulated the p53, induced caspase-dependent apoptosis and suppressed the CIP2A/AKT/mTOR pathway. The activation of autophagy was mediated through PPI and PPVII induced inhibition of mTOR. We propose that PPI and PPVII might be developed as candidate drugs for DDP-resistant NSCLC.

Abstract 3806: Overcoming drug-tolerant cancer cell subpopulations showing AXL activation and epithelial-mesenchymal transition is critical in conquering ALK-positive lung cancer

Abstract Anaplastic lymphoma kinase tyrosine kinase inhibitors (ALK-TKIs) induce a dramatic response in non-small cell lung cancer (NSCLC) patients with the ALK fusion gene. However, acquired resistance to ALK-TKIs remains an inevitable problem. In this study, we aimed to discover novel therapeutic targets to conquer ALK-positive lung cancer. We established three types of ALK-TKI (crizotinib, alectinib and ceritinib) -resistant H2228 NSCLC cell lines by high exposure and stepwise methods. We found these cells showed a loss of ALK signaling, overexpressed AXL with epithelial-mesenchymal transition (EMT), and had cancer stem cell-like (CSC) properties, suggesting drug-tolerant cancer cell subpopulations. Similarly, we demonstrated that TGF-β1 treated H2228 cells also showed AXL overexpression with EMT features and ALK-TKI resistance. The AXL inhibitor, R428, or HSP90 inhibitor, ganetespib, were effective in reversing ALK-TKI resistance and EMT changes in both ALK-TKI-resistant and TGF-β1-exposed H2228 cells. Tumor volumes of xenograft mice implanted with established H2228-ceritinib-resistant (H2228-CER) cells were significantly reduced after treatment with ganetespib, or ganetespib in combination with ceritinib. Some ALK-positive NSCLC patients with AXL overexpression showed a poorer response to crizotinib therapy than patients with a low expression of AXL. ALK signaling-independent AXL overexpressed in drug-tolerant cancer cell subpopulations with EMT and CSC features may be commonly involved commonly involved in intrinsic and acquired resistance to ALK-TKIs. This suggests AXL and HSP90 inhibitors may be promising therapeutic drugs to overcome drug-tolerant cancer cell subpopulations in ALK-positive NSCLC patients for the reason that ALK-positive NSCLC cells do not live through ALK-TKI therapy. Citation Format: Shinji Nakamichi, Seike Masahiro, Akihiko Miyanaga, Akiko Takahashi, Rintaro Noro, Kaoru Kubota, Akihiko Gemma. Overcoming drug-tolerant cancer cell subpopulations showing AXL activation and epithelial-mesenchymal transition is critical in conquering ALK-positive 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 3806.