Cisplatin In Non-small Cell Lung Cancer Research Articles

Abstract 7599: Evaluate the treatment efficacy of magnolol combined with cisplatin in non-small cell lung cancer

Abstract Lung cancer is one of the five most common cancers in the world. Statistics indicate that approximately 80% to 90% of lung cancer cases fall under the classification of non-small cell lung cancer (NSCLC). For several decades, non-small cell lung cancer (NSCLC) has often been treated with chemotherapy drugs. However, lung cancer has high incidence and recurrence rates, mainly due to drug resistance and side effects during chemotherapy and radiotherapy. This is the hinder problem in the treatment of NSCLC, which highlight the necessity of developing new drugs or new treatments for NSCLC. Cisplatin is a chemotherapy drug that can treat about 50% of solid tumors, but the cisplatin-induced cytoprotective autophagy is the resistance to clinical treatment. It is urgent to elucidate the underlying molecular mechanisms regulating cisplatin resistance to improve its therapeutic value in lung cancer. Magnolol is a phenolic component isolated from the roots and bark of Magnolia officinalis and is used in traditional Chinese medicine. With a wide range of pharmacological activities, various studies have focused on the anti-cancer properties of magnolol in recent years. The effect of the drug is to keep cells in the G2/M phase, induce mitochondrial dysfunction and ROS production, affect the supply of cellular energy, and then lead to cell apoptosis. In this study, we investigated the ability of magnolol enhance cisplatin-induced cytotoxicity, showing dose dependence in NSCLC cell by MTT assay. We utilized the immunofluorescence staining and western blotting assay to confirm the downgrade expression of LC3B and the upregulate expression of p62 that define magnolol is able to suppress cisplatin-induced cytoprotective autophagy in NSCLC. We also detected the extrinsic/intrinsic apoptosis induction ability of magnolol combined with cisplatin in NSCLC. In the result, the cisplatin combined with magnolol have more significantly triggered the loss of mitochondrial membrane potential, and activate more protein cleaved caspase-3, -8 and -9 than monotherapy. We also used western blotting assay to confirm the expression of apoptosis inhibitory protein Bcl-2 and pro-apoptotic protein Bax to measure the degree of apoptosis. The migration and invasion effects of magnolol and cisplatin were identified by transwell invasion and wound healing on NSCLC. The magnolol combined with cisplatin extended the mouse survival rate and can inhibit tumor progression. These results suggest that combinational therapy of cisplatin and magnolol could be a promising treatment strategy for NSCLC. The potential regulation of magnolol on NSCLC is associated with triggering cisplatin-induced apoptosis and diminishing cisplatin-induce cytoprotective autophagy. Citation Format: Rong-Er Lu, Bo-Xun Chen, Fei-Ting Hsu, I-Tsang Chiang. Evaluate the treatment efficacy of magnolol combined with cisplatin in non-small cell lung cancer [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 7599.

Unveiling Novel ERCC1-XPF Complex Inhibitors: Bridging the Gap from In Silico Exploration to Experimental Design.

Modifications in DNA repair pathways are recognized as prognostic markers and potential therapeutic targets in various cancers, including non-small cell lung cancer (NSCLC). Overexpression of ERCC1 correlates with poorer prognosis and response to platinum-based chemotherapy. As a result, there is a pressing need to discover new inhibitors of the ERCC1-XPF complex that can potentiate the efficacy of cisplatin in NSCLC. In this study, we developed a structure-based virtual screening strategy targeting the inhibition of ERCC1 and XPF interaction. Analysis of crystal structures and a library of small molecules known to act against the complex highlighted the pivotal role of Phe293 (ERCC1) in maintaining complex stability. This residue was chosen as the primary binding site for virtual screening. Using an optimized docking protocol, we screened compounds from various databases, ultimately identifying more than one hundred potential inhibitors. Their capability to amplify cisplatin-induced cytotoxicity was assessed in NSCLC H1299 cells, which exhibited the highest ERCC1 expression of all the cell lines tested. Of these, 22 compounds emerged as promising enhancers of cisplatin efficacy. Our results underscore the value of pinpointing crucial molecular characteristics in the pursuit of novel modulators of the ERCC1-XPF interaction, which could be combined with cisplatin to treat NSCLC more effectively.

Lactate promoted cisplatin resistance in NSCLC by modulating the m6A modification-mediated FOXO3/MAGI1-IT1/miR-664b-3p/IL-6R axis

BackgroundCisplatin resistance is one of the major obstacles in non-small cell lung cancer (NSCLC) treatment. Intriguingly, elevated lactate levels were observed in cisplatin-resistant cells, which spurred further investigation into their underlying biological mechanisms. MethodsLactate levels were measured by lactate detection kit. Cisplatin-resistance NSCLC cells were established using progressive concentration of cisplatin. Cell viability, proliferation, and apoptosis were detected by CCK-8, EdU, and flow cytometry, respectively. Cell proliferation in vivo was determined by immunohistochemistry of Ki67 and apoptotic cells were calculated by the TUNEL. MeRIP-PCR was used to measure FOXO3 m6A levels. The interactions of genes were analyzed via RIP, ChIP, Dual-luciferase reporter, and RNA pull-down, respectively. ResultsElevated lactate levels were observed in both NSCLC patients and cisplatin-resistance cells. Lactate treatment increased cisplatin-resistance cell viability in vitro and promoted tumor growth in vivo. Mechanistically, lactate downregulated FOXO3 by YTHDF2-mediated m6A modification. FOXO3 transcriptionally reduced MAGI1-IT1 expression. FOXO3 overexpression inhibited the lactate-induced promotion of cisplatin resistance in NSCLC, which were reversed by MAGI1-IT1 overexpression. MAGI1-IT1 and IL6R competitively bound miR-664b-3p. FOXO3 overexpression or MAGI1-IT1 knockdown repressed lactate-mediated cisplatin resistance in vivo. ConclusionLactate promoted NSCLC cisplatin resistance through regulating FOXO3/MAGI1-IT1/miR-664b-3p/IL6R axis in YTHDF2-mediated m6A modification.

Long non-coding RNA PANDAR promoted radiation and cisplatin-induced DNA damage repair through ATR/CHK1 in NSCLC.

DNA-damaging agents, including radiation and platinum-based chemotherapy, are indispensable treatments for non-small cell lung cancer (NSCLC) patients. However, cancer cells tend to be resistant to both radiation and chemotherapy, thus resulting in treatment failure or recurrence. The purpose of this study was to explore the effect and mechanism of long non-coding RNA (lncRNA) PANDAR (promoter of CDKN1A antisense DNA damage-activated RNA) on NSCLC sensitivity to radiation and chemotherapy. Cell counting kit (CCK-8), colony formation and flow cytometry were respectively performed to determine the cell cycle and apoptosis of NSCLC cells treated with γ-ray radiation and cisplatin. The extent of DNA damage was evaluated using a comet assay and immunofluorescence staining against γH2AX. In addition, we explored the role of PANDAR in DNA damage response pathways through western blot analysis. Finally, a nude mouse subcutaneous xenograft model was established to assess the sensitivity to radiation and chemotherapy in vivo. In cell experiments, PANDAR knockdown can increase the sensitivity of NSCLC cells to radiation and cisplatin. The CCK-8 results showed that cell viability was significantly increased in the overexpression group after radiation and cisplatin treatments. The overexpression group also showed more colonies, less apoptosis and DNA damage, and G2/M phase arrest was aggravated to provide the time necessary for DNA repair. Contrary to PANDAR overexpression, the trends were reversed in the PANDAR knockdown group. Furthermore, PANDAR knockdown inhibited radiation and cisplatin-activated phosphorylation levels of ATR and CHK1 in NSCLC cells. Finally, our in vivo model showed that targeting PANDAR significantly sensitized NSCLC to radiation and cisplatin. Our study showed that PANDAR knockdown promoted sensitivity to radiation and cisplatin in NSCLC by regulating the ATR/CHK1 pathway, thus providing a novel understanding as well as a therapeutic target for NSCLC treatment. In NSCLC cells, lncRNA PANDAR negatively regulates sensitivity to radiation and cisplatin. PANDAR can promote the repair of radiation and cisplatin-induced DNA damage and activation of the G2/M checkpoint through the ATR/CHK1 pathway. PANDAR knockdown results in defects in DNA damage repair accompanied by more cell apoptosis.

LncRNA ITGB2-AS1 promotes cisplatin resistance of non-small cell lung cancer by inhibiting ferroptosis via activating the FOSL2/NAMPT axis

ABSTRACT Cisplatin resistance is a major therapeutic challenge in non-small cell lung cancer (NSCLC). Herein, the regulatory role of long non-coding RNA (lncRNA) ITGB2-AS1 in regulating NSCLC cisplatin resistance was investigated. NSCLC cisplatin resistance cells were constructed using A549 and H1975 cells. Cell viability and proliferation were detected by MTT assay and colony formation assay, respectively. Cell apoptosis and cell cycle were examined by flow cytometry. GSH, MDA, ROS, and Fe2+ levels were measured by the corresponding kits. The expressions of ferroptosis-negative regulation genes (GPX4 and SLC7A11) were determined by qRT-PCR and western blot. Molecular interactions were analyzed by RNA pull-down, RIP, ChIP, and dual-luciferase reporter assays. The effects of ITGB2-AS1 silencing on NSCLC cisplatin resistance in vivo were elevated by the tumor xenograft experiment. ITGB2-AS1 expression was increased in NSCLC patients and cisplatin-resistant NSCLC cells, which was positively correlated with ferroptosis-negative regulation genes. ITGB2-AS1 knockdown suppressed resistant cell proliferation and promoted cell apoptosis and ferroptosis. ITGB2-AS1 increased NAMPT expression by binding to FOSL2, thereby repressing p53 expression. The ITGB2-AS1 knockdown also inhibited NSCLC cisplatin resistance in vivo. ITGB2-AS1 promoted NSCLC cisplatin resistance by inhibiting p53-mediated ferroptosis via activating the FOSL2/NAMPT axis.

CircANKRD28 inhibits cisplatin resistance in non-small-cell lung cancer through the miR-221-3p/SOCS3 axis.

Non-small-cell lung cancer (NSCLC) is a common cancer. Chemotherapeutic drug resistance limits the therapeutic effect of NSCLC and leads to a poor prognosis. As a result, new specific targets may be better identified by studying the mechanism of drug resistance to cisplatin in NSCLC. In the present study, we performed a quantitative real-time polymerase chain reaction and western blotting to detect mRNA and protein levels. The proliferation of cells was analyzed by a Cell Counting Kit-8 and colony formation assays. Cell invasion was measured via the Transwell assay. A scratch assay was performed to measure cell migration in cisplatin (DDP)-resistant NSCLC cells. Apoptosis of cells was examined using flow cytometry. We found that circANKRD28 was notably decreased in NSCLC. The results showed that circANKRD28 expression was not affected, and its half-life was more than 12 h. Functional experiments revealed that circANKRD28 overexpression inhibited DDP resistance in NSCLC cells in vitro. Mechanistic findings demonstrated that circANKRD28 regulated tumor cell progression and DDP sensitivity through the miR-221-3p/SOCS3 axis. The present study revealed the regulatory effects and molecular mechanism of circANKRD28 on the development and cisplatin resistance in NSCLC, which may provide experimental basis and theoretical support to identify new targets for therapy of DDP resistance in NSCLC.

Efficacy and safety of endostar combined with cisplatin in treatment of non-small cell lung cancer with malignant pleural effusion: A meta-analysis

Although there are new treatments for non-small cell lung cancer with malignant pleural effusion, these therapies are prone to recurrent pleural effusion and poor in efficacy. And recombinant human endostatin is a new type of anti-tumor angiogenesis drug independently developed in my country. It has the effect of inhibiting tumor angiogenesis, inhibiting tumor proliferation and differentiation, and effectively inhibiting the formation and recurrence of malignant pleural effusion. Therefore, this study is aim to systematically review the efficacy and safety of intrapleural injection of endostar combined with cisplatin in the treatment of non-small cell lung cancer (NSCLC) with malignant pleural effusion. Databases including Cochrane Library, PubMed, CBM, Embase, CNKI, and WanFang Data were searched to collect randomized controlled trials about endostar combined with cisplatin for NSCLC with malignant pleural effusion from inception to April 2022. Two reviewers independently screened the literature, extracted data, and assessed the risk of bias in included studies. Finally, the meta-analysis was made by using RevMan 5.4.1 software. A total of 11 randomized controlled trials involving 814 patients were finally included. The results of the meta-analysis showed that: The overall response rate and the improvement rate of quality of life in the endostar combined with cisplatin group were higher than that of the cisplatin alone group (relative risk = 1.58, 95% confidence interval = 1.42-1.76, P < .00001; relative risk = 1.63, 95% confidence interval = 1.38-1.93, P < .00001, respectively). Meanwhile, there were no significant differences between the 2 groups in the incidence of gastrointestinal reaction, the incidence of leucopenia, the incidence of thrombocytopenia, and the incidence of hypodynamia (all P values > .05). Compared with cisplatin, intrapleural injection of endostar combined with cisplatin could improve the overall response rate and the quality of life of NSCLC patients with malignant pleural effusion. Due to the limited quality and quantity of included studies, more high-quality studies are needed to verify the above conclusion.

Identifying Biomarkers of Cisplatin Sensitivity in Non-Small Cell Lung Cancer via Comprehensive Integrative Analysis

Background: Only 30-40% of non-small cell lung cancer (NSCLC) patients are clinically sensitive to cisplatin-based chemotherapy. Thus, it is necessary to identify biomarkers for personalized cisplatin chemotherapy in NSCLC. However, data heterogeneity and low-value density make it challenging to detect reliable cisplatin efficacy biomarkers using traditional analysis methods. Objective: This paper aims to find reliable cisplatin efficacy biomarkers for NSCLC patients using comprehensive integrative analysis. Method: We searched online resources and collected six NSCLC transcriptomics data sets with responses to cisplatin. The six data sets are divided into two groups: the learning group for biomarker identification and the test group for independent validation. We performed comprehensive integrative analysis under two kinds of frameworks, i.e., one-level and two-level, with three integrative models. Pathway analysis was performed to estimate the biological significance of the resulting biomarkers. For independent validation, logrank statistic was employed to test how significant the difference of Kaplan- Meier (KM) curves between two patient groups is, and the Cox proportional-hazards model was used to test how the expression of a gene is associated with patients’ survival time. Especially, a permutation test was performed to verify the predictive power of a biomarker panel on cisplatin efficacy. For comparison, we also analyzed each learning data set individually, in which three popular differential expression models, Limma, SAM, and RankSum, were used. Results: A total of 318 genes were identified as a core panel of cisplatin efficacy markers for NSCLC patients, exhibiting consistent differential expression between cisplatin-sensitive and –resistant groups across studies. A total of 129 of 344 KEGG pathways were found to be enriched in the core panel, reflecting a picture of the molecular mechanism of cisplatin resistance in NSCLC. By mapping onto the KEGG pathway tree, we found that a KEGG pathway-level I module, genetic information processing, is most active in the core panel with the highest activity ratio in response to cisplatin in NSCLC as expected. Related pathways include mismatch repair, nucleotide excision repair, aminoacyl-tRNA biosynthesis, and basal transcription factors, most of which respond to DNA double-strand damage in patients. Evaluation on two independent data sets demonstrated the predictive power of the core marker panel for cisplatin sensitivity in NSCLC. Also, some single markers, e.g., MST1R, were observed to be remarkably predictive of cisplatin resistance in NSCLC. Conclusion: Integrative analysis is more powerful in detecting biomarkers for cisplatin efficacy by overcoming data heterogeneity and low-value density in data sets, and the identified core panel (318 genes) can help develop personalized medicine of cisplatin chemotherapy for NSCLC patients.

Reversal of Chemotherapy Resistance to Cisplatin in NSCLC by miRNA-195-5p via Targeting the FGF2 Gene.

ObjectiveTo explore the mechanism of miR-195-5p in the pathogenesis non-small cell lung cancer (NSCLC) and cisplatin resistance.MethodsThe function of miR-195-5p in NSCLC and cisplatin resistance were determined by MTT, scratch assay, transwell assay, and nude mice xenograft experiments. miR-195-5p target gene was identified by dual-luciferase reporter assays and real-time PCR analysis.ResultsmiR-195-5p content was lower in A549/DDP than that in A549 cells, with reduced chemotherapy sensitivity and increased cell invasion and migration ability. The loss-of-function and gain-of-function assays illustrated that miR-195-5p might have increased the chemosensitivity to cisplatin in the A549/DDP cells and decreased cell migration and invasion. FGF2 is a negatively correlated action target of miR-195-5p. miR-195-5p might affect EMT by inhibiting FGF2. Overexpression of FGF2 resulted in enhanced cisplatin resistance in the cells, while miR-195-5p might have reversed this resistance.ConclusionOverall, miR-195-5p might target FGF2 to reduce cisplatin resistance in A549/DDP cells and enhance chemosensitivity.

Synergistic effects of olaparib combined with ERCC1 on the sensitivity of cisplatin in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a common malignant tumor. ERCC excision repair 1 endonuclease non-catalytic subunit (ERCC1) is a key mediator of nucleotide excision repair. The present study aimed to explore the synergistic effects of the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib combined with ERCC1 on the sensitivity of NSCLC cells to cisplatin. Preliminary experiments were performed to identify the optimal concentrations of cisplatin and olaparib for cellular treatment and subsequently NCI-H1299 and SK-MES-1 cells were treated with 20 µg/ml cisplatin combined with 50 µg/ml olaparib and 50 µg/ml cisplatin combined with 70 µg/ml olaparib, respectively. Subsequently, transfections were carried out to overexpress or knockdown the expression of ERCC1 in NSCLC cell lines, including NCI-H1299 and SK-MES-1. The transfection efficiency was evaluated using reverse transcription-quantitative PCR and western blotting. The results demonstrated that cells with ERCC1 overexpression and ERCC1 knockdown were successfully constructed. Finally, the cell viability and apoptosis were determined using the Cell Counting Kit-8 and Annexin V-FITC cell apoptosis assays, respectively. In NCI-H1299 or SK-MES-1 cells treated with cisplatin combined with olaparib for 24 h, the cell viability significantly increased following ERCC1 overexpression compared with the GV230 group (P<0.05), but significantly inhibited following ERCC1 knockdown compared with the siRNA-NC group (P<0.05). However, ERCC1 overexpression or knockdown had the opposite effect on apoptosis. In conclusion, olaparib combined with ERCC1 expression may enhance the sensitivity of cisplatin in NSCLC. These findings may provide novel insight for the improvement of platinum drug sensitivity and treatment of NSCLC.

CircRNA_100565 contributes to cisplatin resistance of NSCLC cells by regulating proliferation, apoptosis and autophagy via miR-337-3p/ADAM28 axis.

Circular RNAs (circRNAs) have been revealed to involve in the chemoresistance of various cancers, including non-small cell lung cancer (NSCLC). Here, we further investigate the role of circRNA_100565 in NSCLC cisplatin (DDP) resistance. The expression of circRNA_100565 and microRNA (miR)-337-3p, and ADAM metallopeptidase domain 28 (ADAM28) mRNA was detected using quantitative real-time polymerase chain reaction. Cell viability and apoptosis were measured by cell counting kit-8 assay and flow cytometry, respectively. Western blot was used to detect the level of ADAM28 and autophagy-related protein. The interaction between miR-337-3p and circRNA_100565 or ADAM28 was confirmed by dual-luciferase reporter assay or pull-down assay. In vivo experiments were conducted via the murine xenograft model. We found CircRNA_100565 was up-regulated in NSCLC DDP-resistant tissues and cell lines, and its high expression was associated with shorter overall survival of NSCLC patients. CircRNA_100565 deletion mitigated DDP resistance, reflected by the suppression of proliferation and autophagy, the reduction of IC50 value, as well as enhancement of apoptosis in DDP-resistant NSCLC cells. MiR-377-3p was confirmed to directly bind to circRNA_100565 or ADAM28 3'-UTR. Moreover, circRNA_100565 indirectly regulated ADAM28 expression by sponging miR-377-3p in NSCLC cells. Additionally, circRNA_100565 deletion-induced sensitivity of NSCLC resistant cells to DDP could be remarkably attenuated by miR-377-3p inhibition or ADAM28 re-expression. Meanwhile, circRNA_100565 knockdown contributed to the anti-tumor effects of DDP on NSCLC in vivo.CONCLUSION: CircRNA_100565 was an independent prognostic factor for NSCLC patient survival, and enhanced the resistance of NSCLC cells to cisplatin by regulating cell proliferation, apoptosis and autophagy via miR-337-3p/ADAM28 axis, shedding light on the development of a novel therapeutic strategy to boost the effectiveness of NSCLC chemotherapy.

GOLM1 predicts poor prognosis of patients with NSCLC and is associated with the proliferation and chemo-sensitivity of cisplatin in NSCLC cells: bioinformatics analysis and laboratory validation.

Lung cancer is one of the deadliest malignant tumors with non-small cell lung cancer (NSCLC) being the most prevalent type. Patients with NSCLC usually were diagnosed at the advance clinical stages, and these patients often had high rate of tumor-recurrence, thus leading to poor prognosis. Yet, the molecular mechanisms underlying NSCLC progression and recurrence are largely unknown. This study aimed to identify potential hub genes associated with the pathophysiology of NSCLC by bioinformatics analysis and laboratory validation. The GSE51852, GSE52248 and GSE75037 datasets were downloaded from the Gene Expression Omnibus database. The overlapping differentially expressed genes (DEGs) were analyzed by GEO2R tool. Gene Ontology (GO) and KEGG pathway enrichment analysis were performed on these overlapping DEGs. The protein-protein interaction network was constructed to identify hub genes from DEGs. The expression and survival analysis of these hub genes were performed by using the integrated bioinformatics tools. Finally, the effects of GOLM1 on the proliferation and chemo-sensitivity of NSCLC cells were determined by in vitro functional assays. A total of 197 overlapping DEGs (37 up-regulated and 160 down-regulated) were identified from the microarray datasets. Furthermore, the PPI network with 89 nodes and 768 edges was constructed and 17 hub genes were identified from PPI network by using MCODE analysis. The survival analysis revealed that the expression of 5 hub genes (FGF2, GOLM1, GPC3, IL6 and SPP1) were significantly correlated with overall survival of patients with lung cancer. Furthermore, the in vitro functional studies showed that GOLM1 overexpression promoted the NSCLC cell proliferation and colony formation; while GOLM1 knockdown exerted the opposite effects. Importantly, GOLM1 overexpression reduced the chemo-sensitivity of cisplatin in NSCLC cells by attenuating the inhibitory effects of cisplatin on the cell proliferation and colony formation. In conclusion, the present study showed that 5 hub genes including FGF2, GOLM1, GPC3, IL6 and SPP1 were deregulated in NSCLC tissues and may predict the prognosis of patients with NSCLC. GOLM1 may play an important role in regulating the cell proliferation and chemo-sensitivity of cisplatin in NSCLC.

The long intergenic noncoding RNA GAS5 reduces cisplatin-resistance in non-small cell lung cancer through the miR-217/LHPP axis.

Long noncoding RNAs (lncRNAs) are known to exert their effects to tumor progression. In this study, the role of the lncRNA GAS5 (growth arrest specific 5) was confirmed in reducing non-small cell lung cancer (NSCLC) cisplatin (DDP) resistance. In NSCLC tissue samples, GAS5 expression decreased significantly. Low GAS5 levels were positively correlated with NSCLC characteristics including TNM, tumor size and lymphatic metastasis. Functionally, GAS5 significantly reduced NSCLC/DDP cell migration, invasion and epithelial-mesenchymal transition (EMT) progression in vitro. In vivo, GAS5 upregulation inhibited remarkably NSCLC/DDP cell tumor growth. Mechanism analysis suggested that GAS5 was a molecular sponge of miR-217, inhibiting the expression of phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP). In conclusion, this study reveals that the GAS5/miR-217/LHPP pathway reduces NSCLC cisplatin resistance and that LHPP may serve as a potential therapeutic target for NSCLC cisplatin resistance.

Characterization of a novel HDAC/RXR/HtrA1 signaling axis as a novel target to overcome cisplatin resistance in human non-small cell lung cancer

BackgroundCisplatin is a first-line drug for the treatment of human non-small cell lung cancer (NSCLC); however, the majority of patients will develop drug resistance after treatment. In order to overcome cisplatin resistance, it is important to understand the mechanisms underlying the resistance.MethodsA gene microarray was used to screen for genes related to cisplatin resistance in NSCLC cell lines. Subsequently, the correlation between the HDAC, RXR and HtrA1 genes, in NSCLC, were verified using gene manipulation. Immunohistochemical staining was used to detect HDAC, RXR and HtrA1 expression in NSCLC specimens. Proliferation, migration and invasion assays were performed in vitro and in vivo to determine the role of the HDAC/RXR/HtrA1 signaling axis in cisplatin resistance, and luciferase reporter analysis and ChIP assays were performed to ascertain the mechanisms by which HDAC and RXR regulate the expression of HtrA1. Furthermore, in vitro and in vivo experiments were conducted in NSCLC cisplatin-resistant NSCLC to elucidate the effect of the low molecular weight compound, DW22, which targets the NSCLC cisplatin resistance HDAC/RXR/HtrA1 signaling pathway.ResultsHtrA1 was identified as a cisplatin resistance-related gene in NSCLC cells. The regulation of HtrA1 by HDAC and RXR significantly decreased the efficacy of cisplatin in NSCLC cells resistant to cisplatin. Immunohistochemistry results showed a negative relationship between HDAC1 and HtrA1, and a positive relationship between RXRα and HtrA1 in NSCLC patients’ tissues. Notably, the expression of HDAC1 and HtrA1 can be considered as biomarkers for the efficacy of platinum-based drugs and prognosis in NSCLC patients. Mechanistically, the heterodimers of the nuclear receptor RXR, in combination with the enzyme, HDAC, regulate the transcription of HtrA1 in NSCLC cells. The rescue of HtrA1 expression by dual targeting of HDAC and RXR with the compound, DW22, significantly inhibited the proliferation, migration and invasion of NSCLC cells resistant to cisplatin, and induced NSCLC cell apoptosis.ConclusionOur results indicate that HtrA1, a cisplatin resistance-related gene, is synergistically regulated by HDAC and RXR in NSCLC. Targeting the HDAC/RXR/HtrA1 signaling axis can rescue HtrA1 expression and reverse cisplatin resistance in NSCLC.

Inhibition of DNA‑PK by gefitinib causes synergism between gefitinib and cisplatin in NSCLC.

Lung cancer has the highest incidence and mortality rates among the malignant tumor types world-wide. Platinum-based chemotherapy is the main treatment for advanced non-small-cell lung cancer (NSCLC), and epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have greatly improved the survival of patients with EGFR-sensitive mutations. However, there is no standard therapy for treating patients who are EGFR-TKI resistant. Combining EGFR-TKIs and platinum-based chemotherapy is the most popular strategy in the clinical practice. However, the synergistic mechanism between EGFR-TKIs and platinum remains unknown. Therefore, the aim of the present study was to determine the synergistic mechanism of gefitinib (an EGFR-TKI) and cisplatin (a main platinum-based drug). MTT assay, apoptosis analysis, tumorsphere formation and an orthotropic xenograft mouse model were used to examine the combination effects of gefitinib and cisplatin on NSCLC. Co-immunoprecipitation and immunofluorescence were used to identify the underlying mechanism. It was found that gefi-tinib could selectively inhibit EGFR from entering the nucleus, decrease DNA-PK activity and enhance the cytotoxicity of cisplatin on NSCLC. Collectively, the results suggested that inhibition of DNA-dependent protein kinase by gefitinib may be due to the synergistic mechanism between gefitinib and cisplatin. Thus, the present study provides a novel insight into potential biomarkers for the selection of combination therapy of gefitinib and cisplatin.

YBX1 mediates autophagy by targeting p110\u03b2 and decreasing the sensitivity to cisplatin in NSCLC

Y-box binding protein 1 (YBX1) is involved in the development of multiple types of tumors. However, the relationship between YBX1 and autophagy in non-small cell lung cancer (NSCLC) remains unclear. In this study, we analyzed the expression and clinical significance of YBX1 and markers of autophagy (LC3I/II) in NSCLC and examined their roles in regulating sensitivity to cisplatin in NSCLC. The retrospective analysis of patients with NSCLC indicated that YBX1 was positively correlated with autophagy. Increased levels of YBX1 or autophagy also observed in NSCLC cells compared with those in 16HBE cells. Compared to the controls, the knockdown of YBX1 expression suppressed autophagy, increased drug sensitivity and promoted apoptosis in response to cisplatin in NSCLC cells by targeting the p110β promoter and inhibiting p110β/Vps34/beclin1 signaling pathways. We also demonstrated in an in vivo study that the overexpressed YBX1 effectively increased NSCLC growth and progression and decreased the sensitivity to cisplatin by inducing autophagy in a xenograft tumor model, and these effects were concomitant with the increasing of p110β and beclin1 expression. Collectively, these results show that YBX1 plays an essential role in autophagy in NSCLC.

MicroRNA-497-5p negatively regulates the proliferation and cisplatin resistance of non-small cell lung cancer cells by targeting YAP1 and TEAD1

Background: MicroRNAs (miRNAs) are crucial regulators in the pathological processes and drug resistance of lung cancer. In this study, we investigated the role of miR-497-5p in modulating the function of non-small cell lung cancer (NSCLC). Methods: MiR-497-5p expression in lung cancer tissues and cells was evaluated by qRT-PCR. Cell proliferation was evaluated by CCK-8 assay and colony-formation assay. Cell cycle and cell apoptosis were detected by flow cytometry. The effect of miR-497-5p on the expression of Yes-associated protein 1 (YAP1) and TEA domain family member 1 (TEAD1) was analyzed by qRT-PCR, Western blot and luciferase activity assay. Results: The expression of miR-497-5p was significantly downregulated in lung cancer tissues and cells compared with paired normal tissues and cells. Overexpression of miR-497-5p induced growth retardation and apoptosis of A549 lung cancer cells. Mechanistically, YAP1 and TEAD1 were targeted and downregulated by miR-497-5p. Finally, we found that miR-497-5p increased cisplatin chemosensitivity in A549 cells. Conclusions: MiR-497-5p suppresses cell proliferation and resistance to cisplatin in NSCLC by downregulating the expression of YAP1 and TEAD1.

MicroRNA-497-5p negatively regulates the proliferation and cisplatin resistance of non-small cell lung cancer cells by targeting YAP1 and TEAD1.

BackgroundMicroRNAs (miRNAs) are crucial regulators in the pathological processes and drug resistance of lung cancer. In this study, we investigated the role of miR-497-5p in modulating the function of non-small cell lung cancer (NSCLC).MethodsMiR-497-5p expression in lung cancer tissues and cells was evaluated by qRT-PCR. Cell proliferation was evaluated by CCK-8 assay and colony-formation assay. Cell cycle and cell apoptosis were detected by flow cytometry. The effect of miR-497-5p on the expression of Yes-associated protein 1 (YAP1) and TEA domain family member 1 (TEAD1) was analyzed by qRT-PCR, Western blot and luciferase activity assay.ResultsThe expression of miR-497-5p was significantly downregulated in lung cancer tissues and cells compared with paired normal tissues and cells. Overexpression of miR-497-5p induced growth retardation and apoptosis of A549 lung cancer cells. Mechanistically, YAP1 and TEAD1 were targeted and downregulated by miR-497-5p. Finally, we found that miR-497-5p increased cisplatin chemosensitivity in A549 cells.ConclusionsMiR-497-5p suppresses cell proliferation and resistance to cisplatin in NSCLC by downregulating the expression of YAP1 and TEAD1.

Restoration of miR-26b expression partially reverses the cisplatin resistance of NSCLC by targeting tafazzin.

BackgroundDysregulation of microRNAs has been reported to be responsible for drug resistance of cancers. However, the association between aberrant expression of miR-26b and cisplatin resistance in non-small cell lung cancer (NSCLC) remains unclear.MethodsPC9 and A549 were used to establish the cisplatin resistance models on NSCLC. Expression of miR-26b in cisplatin-resistant PC9 and A549 cells (PC9/R and A549/R) was detected by quantitative real-time PCR assays. Drug sensitivity and mitochondrial apoptosis were detected by Cell Counting Kit-8 assay and flow cytometry assay, respectively. The target relationship between miR-26b and tafazzin (TAZ) was validated by dual-luciferase reporter assay.ResultsObvious downregulation of miR-26b was observed in PC9/R and A549/R cells. Restoration of miR-26b partially reversed the cisplatin resistance of PC9/R and A549/R cells. Expression of TAZ was increased in PC9/R and A549/R cells compared to the parental PC9 and A549 cells. Results of dual-luciferase reporter assays verified that TAZ was targeted by miR-26b. We showed that restoration of miR-26b expression inhibited the TAZ expression and thus expanded the mitochondrial pathway of apoptosis induced by cisplatin in PC9/R and A549/R cells.ConclusionRestoration of miR-26b expression partially reverses the cisplatin resistance of NSCLC by targeting TAZ. miR-26b/TAZ axis may represent a potential strategy to reverse the cisplatin in NSCLC.

Abstract 2119: DCLK1 regulates ATR-DNA damage response for KRAS mutant lung cancer drug resistance and stemness

Abstract Background: Lung cancer is the deadliest cancer in both men and women. About 85% of lung cancers are non-small cell lung cancer (NSCLC) and almost 25% of all types of NSCLC contain KRAS mutations, which remain as an undruggable challenge. Doublecortin-like kinase 1 (DCLK1) regulate many key oncogenes including KRAS in various solid cancers is overexpressed in lung cancer, raising the possibility to selectively target DCLK1 expression to combat the undruggable KRAS mutant NSCLC and its drug resistance. Experimental Procedure: H460 and A549 (KRAS mutant NSCLC cell lines) and H1299 (WT KRAS NSCLC cell line) were used. Cisplatin resistance KRAS mutant NSCLC cells (CR-A549) were generated. Transfection of siRNA against DCLK1 in NSCLC cells and CR-A549 cells were carried out. Tumor cell apoptosis, DNA damage, DNA damage response (DDR) and tumor cell self-renewal were assessed. Protein and mRNA expressions by western-blot and RT-PCR, apoptosis by FACS, cell death by COMET assay and self-renewal by clonogenic assay. Results: Here, we report that increased expression of DCLK1 is the key to develop chemoresistance and self-renewal in KRAS mutant NSCLC. We observed increased immunostaining for DCLK1 in patient lung adenocarcinoma compared to normal lung tissue. We observed an increased expression of DCLK1 protein and mRNA in KRAS mutant NSCLC cells compared to WT KRAS NSCLC cells. To investigate the role of DCLK1 in KRAS mutant NSCLC tumor progression, we knockdown DCLK1 in KRAS mutant NSCLC cells, which resulted in reduced cell proliferation/survival and self-renewal. We generated KRAS mutant NSCLC cisplatin resistance cells (CR-A549) and found that CR-A549 cells acquired higher DCLK1 expression with enhanced self-renewal capacity and display ~9fold higher IC50 for cisplatin treatment compared to parental cells. However, silencing DCLK1 in CR-A549 cells reversed the tumor cell resistance to cisplatin and increased cell death and reduced self-renewal. To further investigate mechanistically, we examine the expression of DDR signaling in CR-A549 cells. We observed an increased expression of ATR-DDR in CR-A549 cells. Furthermore, DCLK1 knockdown reduced the expression of ATR and treatment of siATR plus cisplatin (5µM) to CR-A549 cells demonstrated similar effects observed in the treatment of siDCLK1 plus cisplatin (5uM), which resulted in increased sensitivity of CR-A549 cells to cisplatin, and thus increased cell death and complete abrogation of self-renewal capacity, suggesting that DCLK1 mediated cisplatin resistance occurs via ATR-DDR dependent mechanism. Conclusion: Our data demonstrate that the increased expression of DCLK1 is associated with enhanced cancer stem cell-like features, ATR-DDR signaling and chemoresistance in KRAS mutant NSCLC. Targeting DCLK1 alone or in combination with cisplatin may represent a novel therapeutic strategy for the effective treatment of undruggable KRAS mutant NSCLC. Citation Format: Courtney Houchen, Janani Panneerselvam, Priyanga Mohandoss, Nathaniel Weygant, Randal May, Dongfeng Qu, Naushad Ali, Timothy Wang, Chinthalapally Rao, Michael Bronze, Parthasarathy Chandrakesan. DCLK1 regulates ATR-DNA damage response for KRAS mutant lung cancer drug resistance and stemness [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 2119.