Chemoresistance In Lung Cancer Research Articles

L-Theanine Inhibits Chemoresistance of Lung Cancer Cells to Cisplatin by Regulating STAT3/NOTCH1-BMAL1 Signaling.

L-Theanine, a nonproteinogenic amino acid derived from green tea, is being recognized as an anti-cancer candidate. However, it's roles in the development of cancer chemoresistance is still unknown and the molecular mechanism is urgently to be explored. The effects of L-Theanine on lung cancer chemoresistance were validated by Cell Counting Kit-8 (CCK-8) assay, transwell assay, and in vitro tumor spheroid formation assay; the expression of proteins was detected by using polymerase chain reaction (PCR) and western blotting. RNA-sequencing (RNA-seq) and bioinformatics analysis were used to identify differentially expressed genes induced by L-Theanine. BMAL1 knockdown and overexpression were constructed by using a lentivirus-mediated transfection system. L-Theanine improved the chemoresistance to cis-diamminedichloroplatinum (DDP) and inhibited stemness of DDP-resistant lung cancer cells but not non-resistant lung cancer cells. The results from RNA-seq analysis showed that STAT3/NOTCH1 pathway was a potential dominant signaling involved in L-Theanine improving the chemoresistance in DDP-resistant lung cancer. Mechanistically, L-Theanine impeded migration and stemness activation of DDP-resistant lung cancer cells via regulating the expression of STAT3/NOTCH1/BMAL1 signaling-induced stemness markers as well as inhibiting the expression levels of drug resistance-related genes. In addition, a combination of L-Theanine and Stat3 blockade synergistically improved the chemoresistance in DDP-resistant lung cancer. L-Theanine improves the chemoresistance by regulating STAT3/NOTCH1/BMAL1 signaling, reducing stemness, and inhibiting the migration of DDP-resistant lung cancer cells. The finding might provide some evidence for therapeutic options in overcoming the chemoresistance in cancers, including lung cancer.

Inducing ferroptosis by traditional medicines: a novel approach to reverse chemoresistance in lung cancer

Lung cancer is the leading cause of global cancer-related deaths. Platinum-based chemotherapy is the first-line treatment for the most common type of lung cancer, i.e., non-small-cell lung cancer (NSCLC), but its therapeutic efficiency is limited by chemotherapeutic resistance. Therefore, it is vital to develop effective therapeutic modalities that bypass the common molecular mechanisms associated with chemotherapeutic resistance. Ferroptosis is a form of non-apoptotic regulated cell death characterized by iron-dependent lipid peroxidation (LPO). Ferroptosis is crucial for the proper therapeutic efficacy of lung cancer-associated chemotherapies. If targeted as a novel therapeutic mechanism, ferroptosis modulators present new opportunities for increasing the therapeutic efficacy of lung cancer chemotherapy. Emerging studies have revealed that the pharmacological induction of ferroptosis using natural compounds boosts the efficacy of chemotherapy in lung cancer or drug-resistant cancer. In this review, we first discuss chemotherapeutic resistance (or chemoresistance) in lung cancer and introduce the core mechanisms behind ferroptosis. Then, we comprehensively summarize the small-molecule compounds sourced from traditional medicines that may boost the anti-tumor activity of current chemotherapeutic agents and overcome chemotherapeutic resistance in NSCLC. Cumulatively, we suggest that traditional medicines with ferroptosis-related anticancer activity could serve as a starting point to overcome chemotherapeutic resistance in NSCLC by inducing ferroptosis, highlighting new potential therapeutic regimens used to overcome chemoresistance in NSCLC.

Effects of reprogrammed splenic CD8+ T-cells in vitro and in mice with spontaneous metastatic Lewis lung carcinoma

BackgroundMetastatic disease is a major and difficult-to-treat complication of lung cancer. Considering insufficient effectiveness of existing therapies and taking into account the current problem of lung cancer chemoresistance, it is necessary to continue the development of new treatments.MethodsPreviously, we have demonstrated the antitumor effects of reprogrammed CD8+ T-cells (rCD8+ T-cells) from the spleen in mice with orthotopic lung carcinoma. Reprogramming was conducted by inhibiting the MAPK/ERK signalling pathway through MEKi and the immune checkpoint PD-1/PD-L1. Concurrently, CD8+ T-cells were trained in Lewis lung carcinoma (LLC) cells. We suggested that rCD8+ T-cells isolated from the spleen might impede the development of metastatic disease.ResultsThe present study has indicated that the reprogramming procedure enhances the survival and cytotoxicity of splenic CD8+ T-cells in LLC culture. In an LLC model of spontaneous metastasis, splenic rCD8 + T-cell therapy augmented the numbers of CD8+ T-cells and CD4+ T-cells in the lungs of mice. These changes can account for the partial reduction of tumors in the lungs and the mitigation of metastatic activity.ConclusionsOur proposed reprogramming method enhances the antitumor activity of CD8+ T-cells isolated from the spleen and could be valuable in formulating an approach to treating metastatic disease in patients with lung cancer.

Abstract LB020: Extracellular vesicle-mediated intrinsic induction of ferroptosis causes reversal of chemoresistance in lung cancer

Abstract Introduction: Resistance to chemotherapeutic drugs is the major cause of treatment failures and high mortality in lung cancer(LC). Thus, strategies to overcome chemoresistance is the major thrust area of cancer research. Ciplatin (CDDP) is the first line of chemotherapeutic interventions in lung cancer however, the treatment benefit diminishes due to develop resistance against the drug. Dysregulation of apoptosis is the major reason for developing chemoresistance. Ferroptosis is a recently discovered form of non-apoptotic regulated cell death (RCD) that can overcome the apoptosis mediated resistance. To exploit this our laboratory has developed a novel Extracellular Vesicle (EV)-iron oxide nanoparticle (IONP) hybrid system- 'ExoFeR,' that can induce ferroptosis. Experiments conducted on LC cells and patient derived tumoroids (PDTs) have shown reversal of CDDP resistance after ExoFeR treatment. Our study demonstrates the utility of ferroptosis as alternate treatment intervention for treating chemoresistant LC tumors. Materials and methods: EVs derived from normal lung fibroblast cells (MRC9) were loaded with IONPs. The complex was characterized by Transmission electron microscopy (TEM), Inductively coupled plasma mass spectrometry (ICP-MS) and through Zeta potential measurements. The induction ferroptosis by ExoFeR was confirmed in Non-Small Cell Lung Cancer (NSCLC) A549 cells and NSCLC PDTs by Prussian blue, FerroOrange, and Mito-FerroGreen staining. Further, ferroptosis markers like GPX4 and SCL7A11/xCT were evaluated to examine the induction of ferroptosis. Finally, A549 cells and resistant NSCLC PDTs were tested for sensitivity to chemo drugs. Results: Physicochemical characterization indicated the successful formulation of ExoFeR. Reduction in the expression level of SLC7A11/xCT- and GPX4 proteins in ExoFeR-treated NSCLC cells and PDTs suggested induction of ferroptosis which also showed sensitization of chemo drug (cisplatin). Summary/Conclusion: We report here the synthesis of a novel EV-IONP-based system that can intrinsically induce ferroptosis and overcome chemoresistance by circumventing the conventional apoptosis-based resistance. Our finding is expected to develop a potent cancer treatment strategy for efficiently treating resistant NSCLC tumors. Citation Format: Akhil Srivastava, Anjugam Paramananantham, Rahmat Asfiya. Extracellular vesicle-mediated intrinsic induction of ferroptosis causes reversal of chemoresistance in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB020.

Linc00673-V3 positively regulates autophagy by promoting Smad3-mediated LC3B transcription in NSCLC.

Since its first discovery, long noncoding RNA Linc00673 has been linked to carcinogenesis and metastasis of various human cancers. Linc00673 had five transcriptional isoforms and their biological functions remained to be explored. Here we have reported that Linc00673-V3, one of the isoforms of Linc00673, promoted non-small cell lung cancer chemoresistance, and increased Linc00673-V3 expression level was associated with enhanced autophagy. Mechanistically, we discerned the existence of a stem-loop configuration engendered by the 1-100-nt and 2200-2275-nt fragments within Linc00673-V3. This structure inherently interacted with Smad3, thereby impeding its ubiquitination and subsequent degradation orchestrated by E3 ligase STUB1. The accumulation of Smad3 contributed to autophagy via up-regulation of LC3B transcription and ultimately conferred chemoresistance in NSCLC. Our results revealed a novel transcriptional regulation network between Linc00673-V3, Smad3, and LC3B, which provided an important insight into the interplay between autophagy regulation and non-canonical function of Smad3. Furthermore, the results from in vivo experiments suggested Linc00673-V3 targeted antisense oligonucleotide as a promising therapeutic strategy to overcome chemotherapy resistance in NSCLC.

YAP/TEAD involvement in resistance to paclitaxel chemotherapy in lung cancer.

The Yes-associated protein (YAP) oncoprotein has been linked to both metastases and resistance to targeted therapy of lung cancer cells. We aimed to investigate the effect of YAP pharmacological inhibition, using YAP/TEA domain (TEAD) transcription factor interaction inhibitors in chemo-resistant lung cancer cells. YAP subcellular localization, as a readout for YAP activation, cell migration, and TEAD transcription factor functional transcriptional activity were investigated in cancer cell lines with up-regulated YAP, with and without YAP/TEAD interaction inhibitors. Parental (A549) and paclitaxel-resistant (A549R) cell transcriptomes were analyzed. The half-maximal inhibitory concentration (IC50) of paclitaxel or trametinib, which are Mitogen-Activated protein kinase and Erk Kinase (MEK) inhibitors, combined with a YAP/TEAD inhibitor (IV#6), was determined. A three-dimensional (3D) microfluidic culture device enabled us to study the effect of IV#6/paclitaxel combination on cancer cells isolated from fresh resected lung cancer samples. YAP activity was significantly higher in paclitaxel-resistant cell lines. The YAP/TEAD inhibitor induced a decreased YAP activity in A549, PC9, and H2052 cells, with reduced YAP nuclear staining. Wound healing assays upon YAP inhibition revealed impaired cell motility of lung cancer A549 and mesothelioma H2052 cells. Combining YAP pharmacological inhibition with trametinib in K-Ras mutated A549 cells recapitulated synthetic lethality, thereby sensitizing these cells to MEK inhibition. The YAP/TEAD inhibitor lowered the IC50 of paclitaxel in A549R cells. Differential transcriptomic analysis of parental and A549R cells revealed an increased YAP/TEAD transcriptomic signature in resistant cells, downregulated upon YAP inhibition. The YAP/TEAD inhibitor restored paclitaxel sensitivity of A549R cells cultured in a 3D microfluidic system, with lung cancer cells from a fresh tumor efficiently killed by YAP/TEAD inhibitor/paclitaxel doublet. Evidence of the YAP/TEAD transcriptional program's role in chemotherapy resistance paves the way for YAP therapeutic targeting.

Blocking lncRNA HCG18 re-sensitizes Taxol resistant lung cancer cells to Taxol through modulating the miR-34a-5p/HDAC1 axis

Lung cancer is one of the most frequently diagnosed cancers worldwide, associated with a poor survival rate. Taxol (Paclitaxel) is commonly used as a chemotherapeutic treatment for advanced lung cancers. While Taxol has improved clinical outcomes for lung cancer patients, a significant number of them develop resistance to Taxol, resulting in treatment failure. The role of the long noncoding RNA HCG18 in lung cancer and Taxol resistance has not yet been fully understood. To investigate this, we examined the expression of HCG18 and miR-34a-5p in lung tumors and normal lung tissues using qRT-PCR. We also assessed Taxol resistance through cell viability and apoptosis assays. Through the starBase online service, we analyzed the interactions between lncRNA and mRNA as well as miRNA and mRNA. We further validated the association between lncRNA and miRNA through luciferase and RNA pull-down assays. Our findings demonstrated that HCG18 was significantly upregulated in lung cancer tissues compared to normal lung tissues. Silencing HCG18 increased the sensitivity of lung cancer cells to Taxol. Additionally, our study established a Taxol-resistant cell line and observed a substantial upregulation of HCG18 in Taxol-resistant lung cancer cells. Bioinformatic analysis predicted that HCG18 could bind to miR-34a-5p, forming a competing endogenous RNA network, which was confirmed through luciferase assay. We found that miR-34a-5p was downregulated in lung cancer tissues and negatively correlated with Taxol resistance, as it directly bound to the 3’UTR region of HDAC1. Further results showed that inhibition of HCG18 significantly increased miR-34a-5p expression and sensitized lung cancer cells to Taxol. This sensitization could be reversed by inhibiting miR-34a-5p. Finally, we demonstrated in a xenograft mouse model that inhibition of HCG18 sensitized Taxol-resistant lung cancer cells to Taxol treatment by modulating the miR-34a-5p-HDAC1 axis. In conclusion, our in vitro and in vivo results uncover a novel molecular mechanism by which HCG18 promotes Taxol resistance through modulation of the miR-34a-5p/HDAC1 axis. These findings contribute to the diagnosis and treatment of chemo-resistant lung cancer.

Attenuation of PI3K-Akt-mTOR Pathway to Reduce Cancer Stemness on Chemoresistant Lung Cancer Cells by Shikonin and Synergy with BEZ235 Inhibitor.

Lung cancer is considered the number one cause of cancer-related deaths worldwide. Although current treatments initially reduce the lung cancer burden, relapse occurs in most cases; the major causes of mortality are drug resistance and cancer stemness. Recent investigations have provided evidence that shikonin generates various bioactivities related to the treatment of cancer. We used shikonin to treat multi-resistant non-small lung cancer cells (DOC-resistant A549/D16, VCR-resistant A549/V16 cells) and defined the anti-cancer efficacy of shikonin. Our results showed shikonin induces apoptosis in these ABCB1-dependent and independent chemoresistance cancer sublines. Furthermore, we found that low doses of shikonin inhibit the proliferation of lung cancer stem-like cells by inhibiting spheroid formation. Concomitantly, the mRNA level and protein of stemness genes (Nanog and Oct4) were repressed significantly on both sublines. Shikonin reduces the phosphorylated Akt and p70s6k levels, indicating that the PI3K/Akt/mTOR signaling pathway is downregulated by shikonin. We further applied several signaling pathway inhibitors that have been used in anti-cancer clinical trials to test whether shikonin is suitable as a sensitizer for various signaling pathway inhibitors. In these experiments, we found that low doses shikonin and dual PI3K-mTOR inhibitor (BEZ235) have a synergistic effect that inhibits the spheroid formation from chemoresistant lung cancer sublines. Inhibiting the proliferation of lung cancer stem cells is believed to reduce the recurrence of lung cancer; therefore, shikonin's anti-drug resistance and anti-cancer stem cell activities make it a highly interesting molecule for future combined lung cancer therapy.

Inhibition of NNMT enhances drug sensitivity in lung cancer cells through mediation of autophagy.

This study aimed to investigate the role of Nicotinamide N-methyltransferase (NNMT) in the drug sensitivity of non-small cell lung cancer (NSCLC) cells, with a focus on its impact on autophagy and resistance to the chemotherapeutic agent osimertinib. The study hypothesized that NNMT knockdown would enhance drug sensitivity by modifying autophagic processes, providing a potential new therapeutic target for overcoming chemoresistance in lung cancer. Proteomic analysis was utilized to identify changes in protein expression following NNMT knockdown in H1975 and H1975 osimertinib resistance (H1975OR) lung cancer cell lines. Gene expression patterns and their correlation with NNMT expression in lung cancer patients were analyzed using The Cancer Genome Atlas (TCGA) dataset. Additionally, a predictive model for lung cancer survival was developed via lasso regression analysis based on NNMT-associated gene expression. Drug sensitivity was assessed using the IC50 values and apoptosis ratio, and autophagy was evaluated through Western blot and flow cytometric analysis. Significant variations in the expression of 1,182 proteins were observed following NNMT knockdown, with a significant association with autophagy-related genes. Analysis of gene expression patterns unveiled a significant correlation between NNMT expression and specific changes in gene expression in lung cancer. The predictive model successfully forecasted lung cancer patient survival outcomes, highlighting the potential of NNMT-associated genes in predicting patient survival. Knockdown of NNMT reversed osimertinib resistance in H1975 cells, as evidenced by altered IC50 values and apoptosis ratio, and changes were observed in autophagy markers. Knockdown of NNMT in lung cancer cells enhances drug sensitivity by modulating autophagy, providing a promising therapeutic target to overcome chemoresistance in NSCLC. The study underscores the importance of NNMT in lung cancer pathology and underscores its potential as a predictive marker for clinical outcomes. Additionally, the developed predictive model further supports the clinical relevance of NNMT-associated gene expression in improving the prognosis of lung cancer patients.

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.

A redox-responsive nanosystem to suppress chemoresistant lung cancer through targeting STAT3

Cancer stem cells (CSCs) have been demonstrated to be involved in tumor initiation and relapse, and the presence of CSCs in the tumor tissue often leads to therapeutic failure. BBI608 has been identified to eliminate CSCs by inhibiting signal transducer and activator of transcription 3 (STAT3). In this study, we confirm that BBI608 can efficiently suppress the proliferation and migration of non-small cell lung cancer (NSCLC) cells, and specifically kill the stemness-high population in chemoresistant NSCLC cells. To improve its bioavailability and tumor accumulation, BBI608 is successfully encapsulated into redox-responsive PEGylated branched N-(2-hydroxypropyl) methacrylamide (HPMA)-deoxy cholic acid (DA) polymeric nanoparticles (BBI608-SS-NPs). The BBI608-SS-NPs can release the drug in response to high concentrations of intracellular glutathione, and exhibit cytotoxicity against lung cancer cells and CSCs comparable to the free drug BBI608. Furthermore, the BBI608-SS-NPs preferentially accumulate in tumor sites, resulting in a superior anti-tumor efficacy in both cisplatin-resistant cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models of NSCLC. Mechanistic studies demonstrate that BBI608-SS-NPs not only directly inhibit the downstream genes of the STAT3 pathway, but also indirectly inhibit the Wnt pathway. Overall, this stimuli-responsive polymeric nanoformulation of BBI608 shows great potential in the treatment of chemoresistant NSCLC by targeting CSCs.

Inhalable capsular polysaccharide-camouflaged gallium-polyphenol nanoparticles enhance lung cancer chemotherapy by depleting local lung microbiota.

Local lung microbiota is closely associated with lung tumorigenesis and therapeutic response. It is found that lung commensal microbes induce chemoresistance in lung cancer by directly inactivating therapeutic drugs via biotransformation. Accordingly, an inhalable microbial capsular polysaccharide (CP)-camouflaged gallium-polyphenol metal-organic network (MON) is designed to eliminate lung microbiota and thereby abrogate microbe-induced chemoresistance. As a substitute for iron uptake, Ga3+ released from MON acts as a "Trojan horse" to disrupt bacterial iron respiration, effectively inactivating multiple microbes. Moreover, CP cloaks endow MON with reduced immune clearance by masquerading as normal host-tissue molecules, significantly increasing residence time in lung tissue for enhanced antimicrobial efficacy. In multiple lung cancer mice models, microbe-induced drug degradation is remarkably inhibited when drugs are delivered by antimicrobial MON. Tumor growth is sufficiently suppressed and mouse survival is prolonged. Ourwork develops a novel microbiota-depleted nano-strategy to overcome chemoresistance in lung cancer by inhibiting local microbial inactivation of therapeutic drugs. This article is protected by copyright. All rights reserved.

Circ_0010235 confers cisplatin resistance in lung cancer by upregulating E2F7 through absorbing miR-379-5p.

Cisplatin (DDP) treatment is one of the most predominant chemotherapeutic strategies for lung cancer patients. Circular RNAs (circRNAs) have been revealed to participate in the chemoresistance in lung cancer. Hence, the role and mechanism of circ_0010235 in cisplatin resistance in lung cancer was investigated. Expression levels of circ_0010235, microRNA (miR)-379-5p and E2F transcription factor 7 (E2F7) were analyzed using quantitative reverse transcription PCR (qRT-PCR) and western blot. Cell DDP sensitivity, proliferation, apoptosis, invasion, and migration were detected by cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine (EDU) assay, flow cytometry and western blot, respectively. The binding interaction was verified using dual-luciferase reporter assay. A murine xenograft model was established to investigate effects in vivo. Circ_0010235 was highly expressed in DDP-resistant lung cancer tissues and cells. Knockdown of circ_0010235 elevated DDP sensitivity, constrained proliferation, invasion and migration as well as fostered apoptosis in DDP-resistant lung cancer cells. Moreover, circ_0010235 silencing boosted DDP sensitivity and impeded tumor growth in lung cancer in vivo. Mechanistically, circ_0010235 acted as a sponge for miR-379-5p to elevate the expression of its target E2F7. Rescue experiments showed that miR-379-5p inhibition attenuated circ_0010235 knockdown-evoked reduction on DDP resistance of DDP-resistant cancer cells. In addition, miR-379-5p re-expression elevated DDP sensitivity and suppressed the malignant phenotype of DDP-resistant lung cancer cells through miR-379-5p. Circ_0010235 knockdown reduced DDP resistance and tumor growth via miR-379-5p/ E2F7 axis in lung cancer, suggesting an effective therapeutic target for lung cancer patients.

Chemotherapy-induced PDL-1 expression in cancer-associated fibroblasts promotes chemoresistance in NSCLC

ObjectivesA cure for cancer is out of reach for most patients due to chemoresistance. Cancer-associated fibroblasts (CAFs) play a vital role in cancer chemoresistance, but detailed understanding of the process particularly in chemoresistant lung cancer is lacking. In this study, we investigated programmed death-ligand 1 (PDL-1) as a potential biomarker for CAF-induced chemoresistance and evaluated its role and the underlying mechanisms of chemoresistance in non-small cell lung cancer (NSCLC). Materials and MethodsA systemic search of gene expression profiles of multiple tissues in NSCLC was carried out to determine the expression intensities of traditional fibroblast biomarkers and CAF-secreted protumorigenic cytokines. PDL-1 expression in CAFs was analyzed by ELISA, Western blotting, and flow cytometry. Human cytokine array was used to identify specific cytokines secreted from CAFs. Role of PDL-1 in NSCLC chemoresistance was assessed using CRISPR/Cas9 knockdown and various functional assays including MTT, cell invasion, sphere formation, and cell apoptosis. In vivo experiments were conducted using a co-implantation xenograft mouse model with live cell imaging and immunohistochemistry. ResultsWe demonstrated that chemotherapy-stimulated CAFs promoted tumorigenic and stem cell-like properties of NSCLC cells, which contribute to their chemoresistance. Subsequently, we revealed that PDL-1 expression is upregulated in chemotherapy-treated CAFs and is associated with poor prognosis. Silencing PDL-1 expression suppressed CAFs’ ability to promote stem cell-like properties and invasiveness of lung cancer cells, favoring chemoresistance. Mechanistically, an upregulation of PDL-1 in chemotherapy-treated CAFs led to an increase in hepatocyte growth factor (HGF) secretion, which stimulates cancer progression, cell invasion, and stemness of lung cancer cells, while inhibiting apoptosis. ConclusionOur results show that PDL-1-positive CAFs modulate stem cell-like properties of NSCLC cells by secreting elevated HGF, thereby promoting chemoresistance. Our finding supports PDL-1 in CAFs as a chemotherapy response biomarker and as a drug delivery and therapeutic target for chemoresistant NSCLC.

N6-methyladenine-mediated aberrant activation of the lncRNA SOX2OT-GLI1 loop promotes non-small-cell lung cancer stemness

Despite the advent of precision medicine and immunotherapy, mortality due to lung cancer remains high. The sonic hedgehog (SHH) cascade and its key terminal factor, glioma-associated oncogene homolog 1 (GLI1), play a pivotal role in the stemness and drug resistance of lung cancer. Here, we investigated the molecular mechanism of non-canonical aberrant GLI1 upregulation. The SHH cascade was upregulated in stem spheres and chemo-resistant lung cancer cells and was accountable for drug resistance against multiple chemotherapy regimens. GLI1 and the long non-coding RNA SOX2OT were positively regulated, and the GLI1-SOX2OT loop mediated the proliferation of parental and stem-like lung cancer cells. Further mechanistic investigation revealed that SOX2OT facilitated METTL3/14/IGF2BP2-mediated m6A modification and stabilization of the GLI1 mRNA. Additionally, SOX2OT upregulated METTL3/14/IGF2BP2 by sponging miR-186-5p. Functional analysis corroborated that GLI1 acted as a downstream target of METTL3/14/IGF2BP2, and GLI1 silencing could block the oncogenicity of lung cancer stem-like cells. Pharmacological inhibition of the loop remarkably inhibited the oncogenesis of lung cancer cells in vivo. Compared with paired adjacent normal tissues, lung cancer specimens exhibited consistently upregulated GLI1/SOX2OT/METTL3/14/IGF2BP2. The m6A-modified GLI1-SOX2OT loop may serve as a potential therapeutic target and prognostic predictor for lung cancer therapy and diagnosis in the clinic.

M6A-mediated upregulation of lncRNA-AC026356.1 promotes cancer stem cell maintenance in lung adenocarcinoma via activating Wnt signaling pathway.

LncRNA plays a pivotal role in the stemness and drug resistance of lung cancer. Here, we found that lncRNA-AC026356.1 was upregulated in stem spheres and chemo-resistant lung cancer cells. Our fish assay also shows that AC026356.1 was predominantly located in the cytoplasm of lung cancer cells and does not have protein-coding potential. Silencing AC026356.1 significantly inhibited proliferation and migration but increased apoptosis in A549-cisplatin (DDP) cells. Additionally, IGF2BP2 and the lncRNA-AC026356.1 positively regulated the proliferation and stemness of stem-like lung cancer cells. Further mechanistic investigation revealed that METTL14/IGF2BP2-mediated m6A modification and stabilization of the AC026356.1 RNA. Functional analysis corroborated that AC026356.1 acted as a downstream target of METTL14/IGF2BP2 and AC026356.1 silencing could block the oncogenicity of lung cancer stem-like cells. AC026356.1 expression was correlated with immune cell infiltration and T cell exhaustion. Compared with paired adjacent normal tissues, lung cancer specimens exhibited consistently upregulated METTL14/IGF2BP2/AC026356.1. M6A-modified METTL14/IGF2BP2/AC026356.1 loop may serve as a potential therapeutic target and prognostic predictor for lung cancer therapy and diagnosis in the clinic.

Targeting transient receptor potential canonical 1 reduces non‑small cell lung cancer chemoresistance and stemness via inhibition of PI3K/AKT signaling.

TRPC1 enhances cell proliferation and migration in non-small cell lung cancer (NSCLC); however, its effect on NSCLC chemoresistance and stemness remains to be determined. The aim of the current study was to investigate the effect of TRPC1 on NSCLC chemoresistance and stemness and to determine the underlying mechanism of action. Cisplatin-resistant A549 (A549/CDDP) and H460 (H460/CDDP) cells were first established and were then transfected with negative control small interfering (si)RNA (si-NC) or TRPC1 siRNA (si-TRPC1). Cells were then treated with 740 Y-P, a PI3K/Akt agonist. Subsequently, the sensitivity of A549/CDDP and H460/CDDP cells to CDDP was evaluated. Furthermore, the expression levels of CD133 and CD44, and sphere formation ability were also determined. The results showed that the half-maximal inhibitory concentration (IC50) of CDDP was significantly higher in A549/CDDP cells compared with A549 cells and in H460/CDDP cells compared with H460 cells. TRPC1 silencing decreased the IC50 value of CDDP compared with the si-NC group in A549/CDDP (11.78 vs. 21.58 µM; P<0.01) and H460/CDDP (23.76 vs. 43.11 µM; P<0.05) cells. Additionally, TRPC1 knockdown in both cell lines decreased the number of spheres formed compared with the si-NC group. Furthermore, compared with the si-NC group, A549/CDDP cells transfected with si-TRPC1 exhibited decreased levels of both CD133 (P<0.01) and CD44 (P<0.05). However, only CD133 (P<0.05) was downregulated in TRPC1-depleted H460/CDDP cells compared with the si-NC group. In addition, TRPC1 knockdown repressed PI3K/AKT signaling compared with the si-NC group in both A549/CDDP and H460/CDDP cells (all P<0.05). Finally, cell treatment with 740 Y-P reversed the effect of TRPC1 knockdown on PI3K/AKT signaling, chemoresistance, and cancer stemness in A549/CDDP and H460/CDDP cells (all P<0.05). In conclusion, the results of the current study suggested that targeting TRPC1 could attenuate cancer stemness and chemoresistance via suppression of PI3K/AKT signaling in NSCLC.

Abstract 529: Novel inhibitors of the NRF2 pathway also arrest lung cancer cells in G2/M

Abstract Background: Non-small cell lung (NSCLC) constitutes approximately 80% of all diagnosed cases of lung cancer. Up to 30% of NSCLC acquire mutations that result in constitutive NRF2 pathway activity, which correlates with worse patient prognosis. NRF2 regulates antioxidant genes that confer cytoprotection and chemoresistance. Inhibiting NRF2 activity increases tumor susceptibility to chemotherapy in preclinical lung cancer models; however, there are no FDA-approved NRF2 inhibitors available. Because current inhibitors have low potency and specificity, we developed novel compounds that target the NRF2 pathway. Previously, a high throughput screen identified MSU-38225, which was efficacious in sensitizing chemoresistant lung cancer to chemotherapy in a mouse xenograft model. We sought to improve the poor pharmacokinetics of this compound and developed and characterized a series of novel derivatives with increased solubility. Hypothesis: Derivatives of MSU-38225 inhibit the NRF2 pathway and decrease cancer cell viability. Methods: The efficacy of our NRF2 pathway inhibitors was assessed in A549 and H460 human lung cancer cells with constitutive NRF2 pathway activity using western blotting, RT-PCR, and MTT or Cell Titer Glow viability assays. Cell death and cell cycle analysis were evaluated using flow cytometry. Data were evaluated with one or two-way ANOVA and the Dunnett post-hoc test. Results: The NRF2 pathway inhibitors decreased NRF2 protein level in A549 and H460 cells by approximately 60%, as measured by western blotting. As a result, NRF2-mediated transcription of cytoprotective genes NQO1, GCLC, GCLM, and AKR1C2 were decreased by 30-60% (p&amp;lt;0.05) by qPCR. Further, these compounds decreased lung cancer cell viability, as measured in an MTT assay, by 50% in A549 cells and by 90% in H460 (p&amp;lt;0.05). We then evaluated a specific inhibitor, MSU-71, to determine the cause of decreased viability and found it did not induce annexin-V positive apoptosis. However, subsequent analysis of the cell cycle with propidium iodide univariate analysis showed MSU-71 arrests lung cancer cells. 50% of H460 and 75% of A549 cells were arrested in G2/M (p&amp;lt;0.05). Finally, we developed several “click” chemistry tool compounds that have equitable NRF2 pathway inhibition, which will aid in target identification of the new inhibitors. Conclusion: Taken together, the new NRF2 pathway inhibitors are efficacious in blocking NRF2 activation. Optimization of these inhibitors could lead to a new therapy to treat lung cancer. Citation Format: Christopher John Occhiuto, Lizbeth Lockwood, Zhilin Hou, Aaron Odom, Karen Liby. Novel inhibitors of the NRF2 pathway also arrest lung cancer cells in G2/M [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 529.

Carbon Dots-Based Nanozyme for Drug-Resistant Lung Cancer Therapy by Encapsulated Doxorubicin/siRNA Cocktail.

Nanomaterials exhibited intrinsic enzyme-like properties due to the unique properties compared with natural enzyme. Carbon dots (CDs) are an important kind of quantum-sized nanomaterials, which have enormous application potential in bio-imaging, drug carrier, and nanosystems. Carbon dots possess intrinsic enzyme-like properties, such as glutathione (GSH) oxidase or peroxidase activities. A co-delivery nanosystem that could carry siRNA and doxorubucin (DOX) simultaneously has been studied in this work. The co-delivery based on carbon dots was surface-modified with poly-ethylenimine (PEI) and loaded the siMRP1 with chemotherapeutics on the surface with pH-triggered drug release. The CD-PEI was synthesized by one-step microwave assisted method; the PEI was raw materials and passivator during the reaction process that makes CDs exhibit excellent optical property. The CD-PEI was capable of loading and delivering siMRP1 and DOX to tumors and releasing them synchronously in cells in an acid-triggered manner. The particles exhibited GSH oxidase-like catalytic property, oxidizing GSH to oxidized glutathione with concomitant increase of reactive oxygen species (ROS). We found that silencing of MRP1 by co-delivery system antagonized chemoresistance by increasing DOX accumulation and significantly enhancing the inhibitory effect of cell viability induced by CD-PEI-DOX. The co-delivery system dramatically inhibited tumor growth in xenograft model, and CDs counteracted MRP1 function by siRNA-mediated knockdown of MRP1. Taken together, we uncover the potential role of CDs with a combination of siRNA and chemotherapeutics in overcoming chemoresistance of lung cancer by suppressing MRP1 and oxidation of GSH. Our findings imply its potential of antagonizing chemoresistance to enhance therapeutic efficiency of doxorubicin in clinical practices of lung cancer treatment.

Emerging role of non-coding RNAs in resistance to platinum-based anti-cancer agents in lung cancer.

Platinum-based drugs are the first line of therapeutics against many cancers, including lung cancer. Lung cancer is one of the leading causes of cancer-related death worldwide. Platinum-based agents target DNA and prevent replication, and transcription, leading to the inhibition of cell proliferation followed by cellular apoptosis. About twenty-three platinum-based drugs are under different stages of clinical trials, among cisplatin, carboplatin, and oxaliplatin are widely used for the treatment of various cancers. Among them, cisplatin is the most commonly used drug for cancer therapy, which binds with RNA, and hinders the cellular RNA process. However, long-term use of platinum-based drugs can cause different side effects and has been shown to develop chemoresistance, leading to poor clinical outcomes. Chemoresistance became an important challenge for cancer treatment. Platinum-based chemoresistance occurs due to the influence of intrinsic factors such as overexpression of multidrug resistance proteins, advancement of DNA repair mechanism, degradation, and deactivation of intracellular thiols. Recently, epigenetic modifications, especially non-coding RNAs (ncRNAs) mediated gene regulation, grasp the attention for reversing the sensitivity of platinum-based drugs due to their reversible nature without altering genome sequence. ncRNAs can also modulate the intrinsic and non-intrinsic mechanisms of resistance in lung cancer cells. Therefore, targeting ncRNAs could be an effective approach for developing novel therapeutics to overcome lung cancer chemoresistance. The current review article has discussed the role of ncRNA in chemoresistance and its underlying molecular mechanisms in human lung cancer.