Spatial and multi-omics transcriptomic dissects platinum resistance in lung adenocarcinoma: a five-gene predictive model with tumor microenvironment dynamics.

Cisplatin is a key drug for treating lung adenocarcinoma, and its sensitivity to cisplatin is directly related to prognosis. We aimed to reveal the roles of genes related to glutathione synthesis (glutamate-cysteine ligase catalytic subunit, GCLC) and cystine uptake (cystine/glutamate transporter, xCT and CD44v8-10) in cisplatin resistance and prognosis in lung adenocarcinoma. We established cell lines stably expressing GCLC, xCT, standard isoform of CD44, and CD44v8-10, and investigated their sensitivities to cisplatin. We also measured mRNA expression levels of these genes in the tumor tissues from 92 lung adenocarcinoma patients. Patients were divided into high-expression (upper quartile, N = 23) and low-expression groups (remaining patients, N = 69). Recurrence-free survival, overall survival (N = 92), and post-recurrence survival (N = 22) were selected as endpoints. Compared with the control green fluorescent protein-expressing cell line (inhibitory concentration 50:6.9 μM), all the stable cell lines were more resistant to cisplatin (12.9 μM, P = 0.025; 13.9 μM, P = 0.028; 26.7 μM, P = 0.001; 17.7 μM, P = 0.008, respectively). In contrast, there was no significant difference in recurrence-free or overall survival between the high- and low-expression groups for any of the genes. However, high expression of GCLC was a risk factor for poorer post-recurrence survival (hazard ratio, 6.26; 95% confidence interval, 1.37-28.7; P = 0.018). High expression levels of genes related to glutathione synthesis and cystine uptake promote cisplatin resistance in lung adenocarcinoma cell lines. High expression of GCLC in tumor tissue may be a potential predictor of treatment failure.

Background/Aims: Chemoresistance has been a major obstacle to the effective treatment of lung cancer. Previously, we found that contactin-1 (CNTN-1) is related to cisplatin resistance in lung adenocarcinoma. Here, we aimed to investigate the underlying mechanism behind the role of CNTN-1 in cisplatin resistance in lung adenocarcinoma. Methods: EMT-associated phenotypes, including alterations in cellular morphology and marker (E-cadherin, N-cadherin and Vimentin) expression, were compared between A549 cells and A549/DDP cells (a cisplatin-resistant cell line of lung adenocarcinoma with abnormal CNTN-1 expression) by using real-time time PCR and Western blotting. Other methods, including CNTN-1 overexpression in A549 cells and CNTN-1 knockdown in A549/DDP cells, were also used to investigate the role of CNTN-1 in mediating the EMT phenotype and thr resulting cisplatin resistance and malignant progression of cancer cells in vitro and in vivo. Results: A549/DDP cells exhibited an EMT phenotype and aggravated malignant behaviors. CNTN-1 knockdown in A549/DDP cells partly reversed the EMT phenotype, increased drug sensitivity, and attenuated the malignant progression whereas CNTN-1 overexpression in A549 cells resulted in the opposite trend. Furthermore, the PI3K/Akt pathway was involved in the effects of CNTN-1 on EMT progression in A549/DDP cells, verified by the xenograft mouse model. Conclusion: CNTN-1 promotes cisplatin resistance in human cisplatin-resistant lung adenocarcinoma through inducing the EMT process by activating the PI3K/Akt signaling pathway. CNTN-1 may be a potential therapeutic target to reverse chemoresistance in cisplatin-resistant lung adenocarcinoma.

Lung cancer is one of the most common malignancies and the leading cause of cancer-related death in the world. In patients with advanced lung adenocarcinoma who are negative for driver gene mutations, platinum-based chemotherapy represented by cisplatin remain the standard of care. Therefore, studying the mechanism behind inevitable cisplatin resistance in lung adenocarcinoma is still important. In this study, the potentially related differential expression gene for cisplatin resistance in lung adenocarcinoma was screened in the GEO database. The expression level of HEY1 in cell lines of lung adenocarcinoma was detected and HEY1 expression was up-regulated in cisplatin-resistant lung adenocarcinoma tissues and cell lines A549/DDP. Patients with high HEY1 expression have poor prognosis after cisplatin therapy. Gain and loss function assays uncovered that HEY1 could regulate the cisplatin sensitivity of NSCLC cells. In vivo experiments have confirmed that silence of HEY1 expression can induce cisplatin resistance, and epithelial-mesenchymal transition (EMT) changes occur during this process. Mechanically, HEY1 silencing significantly up-regulated E-cadherin expression and down-regulated Vimentin in A549/DDP cells. While up-regulation of HEY1 resulted in down-regulation of E-cadherin and up-regulation of Vimentin in A549 cells. Immunohistochemical experiments confirmed that E-cadherin was significantly decreased, and Vimentin expression was significantly up-regulated in cisplatin-resistant lung adenocarcinoma tissues. HEY1 can mediate the occurrence of cisplatin-acquired resistance in lung adenocarcinoma, and the possible mechanism is to regulate the EMT. The results of this study can provide a new direction and target for clinical research on the reversal of cisplatin resistance in lung adenocarcinoma.

Increasing evidence suggests that microRNAs' (miRNAs) abnormal expression is one of the main factors of chemotherapy resistance in various cancers. However, the role of miRNAs in lung adenocarcinoma (LUAD) resistance to cisplatin is still unclear. In this study, we analyzed a microarray dataset to investigate miRNAs related to cisplatin resistance in LUAD. The expression of miRNAs in LUAD tissues and cell lines was detected using real-time quantitative polymerase chain reaction (RT-qPCR). Special AT-Rich Sequence-Binding Protein 2 (SATB2) in LUAD cell lines was detected using RT-qPCR and Western blot. Cell proliferation was measured by CCK8 and colony formation assays, while cell cycle and apoptosis were measured by flow cytometry. A dual-luciferase reporter assay was performed to confirm that SATB2 is a target gene of microRNA-660 (miR-660). We showed that the expression of miR-660 was not only decreased in LUAD cells and tissues but also further decreased in the cisplatin-resistant A549 cell line. The overexpression of miR-660 increased cisplatin sensitivity in LUAD cells. In addition, we identified SATB2 as a direct target gene of miR-660. We also revealed that miR-660 increased cisplatin sensitivity in LUAD cells via targeting SATB2. In conclusion, miR-660/SATB2 axis is a key regulator of cisplatin resistance in LUAD.

Cisplatin is widely used as a conventional chemotherapy drug for lung adenocarcinoma (LUAD) patients. However, the chemical resistance greatly limits its therapeutic potential. The study aimed to uncover the specific role and new mechanisms of CPT1B in the cisplatin resistance of LUAD. Bioinformatics analysis was utilized to analyze the expression level and enriched pathway of CPT1B in LUAD. The expression of CPT1B in LUAD cells was determined by utilizing quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot (WB). The cisplatin resistance in LUAD was measured with IC50 values obtained from the CCK-8 assay. We used the corresponding reagent kit and WB analysis to determine the levels of triglycerides, cholesterol, phospholipids, fatty acid β-oxidation (FAO) rate, and expression of lipid metabolism-related proteins. Finally, the regulation relationship between CPT1B and ZNF263 was confirmed through bioinformatics analysis, dual-luciferase, and chromatin immunoprecipitation assays. The present investigation revealed that CPT1B was upregulated in LUAD, participating in fatty acid metabolism pathways. In vitro studies have shown that upregulation of CPT1B promoted cisplatin resistance in LUAD cells. This promotion effect induced by the high expression of CPT1B on cisplatin resistance in LUAD was weakened after the addition of the FAO inhibitor Etomoxir. Mechanistically, ZNF263 was capable of binding to the promoter of CPT1B to activate its transcription, thereby enhancing FAO and promoting cisplatin resistance in LUAD cells. In summary, ZNF263 enhances cisplatin resistance in LUAD cells by upregulating CPT1B expression. This study enriches the molecular mechanisms of LUAD chemotherapy resistance and provides new directions for exploring therapeutic targets for LUAD.

Lung adenocarcinoma (LUAD) is the predominant subtype within the spectrum of lung malignancies. CTHRC1 has a pro-oncogenic role in various cancers. Here, we observed the upregulation of CTHRC1 in LUAD, but its role in cisplatin resistance in LUAD remains unclear. Bioinformatics analysis was employed to detect CTHRC1 and SRY-related HMG-box 4 (SOX4) expression in LUAD. Gene Set Enrichment Analysis predicted the enriched pathways related to CTHRC1. JASPAR and MotifMap databases predicted upstream transcription factors of CTHRC1. Pearson analysis was conducted to analyze the correlation between genes of interest. The interaction and binding relationship between CTHRC1 and SOX4 were validated through dual-luciferase and chromatin immunoprecipitation assays. Quantitative real-time polymerase chain reaction determined the expression of CTHRC1 and SOX4 genes. CCK-8 was performed to assess cell viability and calculate IC50 value. Flow cytometry examined the cell cycle. Comet assay and western blot assessed DNA damage. CTHRC1 and SOX4 were upregulated in LUAD. CTHRC1 exhibited higher expression in cisplatin-resistant A549 cells compared to cisplatin-sensitive A549 cells. Knockdown of CTHRC1 enhanced DNA damage during cisplatin treatment and increased the sensitivity of LUAD cells to cisplatin. Additionally, SOX4 modulated DNA damage repair (DDR) by activating CTHRC1 transcriptional activity, promoting cisplatin resistance in LUAD cells. SOX4 regulated DDR by activating CTHRC1, thereby enhancing cisplatin resistance in LUAD cells. The finding provides a novel approach to address clinical cisplatin resistance in LUAD, with CTHRC1 possibly serving as a candidate for targeted therapies in addressing cisplatin resistance within LUAD.

BackgroundLung cancer is the most common malignant tumor in the world. The Whole-proteome microarray showed that ubiquitin ligase chromatin assembly factor 1 subunit B (CHAF1B) expression in A549/DDP cells is higher than in A549 cells. Our study explored the molecular mechanism of CHAF1B affecting cisplatin resistance in lung adenocarcinoma (LUAD).MethodsProteome microarray quantify the differentially expressed proteins between LUAD cell line A549 and its cisplatin-resistant strain A549/DDP. Quantitative real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot (WB) confirmed the CHAF1B expression. Public databases analyzed the prognosis of LUAD patients with varied LUAD expression followed by the substrates prediction of CHAF1B. Public databases showed that nuclear receptor corepressor 2 (NCOR2) may be substrates of CHAF1B. WB detected that CHAF1B expression affected the expression of NCOR2. Cell and animal experiments and clinical data detected function and integrating mechanism of CHAF1B compounds.ResultsProteome chips results indicated that CHAF1B, PPP1R13L, and CDC20 was higher than A549 in A549/DDP. Public databases showed that high expression of CHAF1B, PPP1R13L, and CDC20 was negatively correlated with prognosis in LUAD patients. PCR and WB results indicated higher CHAF1B expression in A549/DDP cells than that in A549 cells. NCOR2 and PPP5C were confirmed to be substrates of CHAF1B. CHAF1B knockdown significantly increased the sensitivity of cisplatin in A549/DDP cells and the upregulated NCOR2 expression. CHAF1B and NCOR2 are interacting proteins and the position of interaction between CHAF1B and NCOR2 was mainly in the nucleus. CHAF1B promotes ubiquitination degradation of NCOR2. Cells and animal experiments showed that under the action of cisplatin, after knockdown of CHAF1B and NCOR2 in A549/DDP group compared with CHAF1B knockdown alone, the cell proliferation and migratory ability increased and apoptotic rate decreased, and the growth rate and size of transplanted tumor increased significantly. Immunohistochemistry suggested that Ki-67 increased, while apoptosis-related indicators caspase-3 decreased significantly. Clinical data showed that patients with high expression of CHAF1B are more susceptible to cisplatin resistance.ConclusionUbiquitin ligase CAHF1B can induce cisplatin resistance in LUAD by promoting the ubiquitination degradation of NCOR2.

AimThis study aimed to explore the mechanism of LncRNA urothelial carcinoma-associated 1 (UCA1) promoting cisplatin resistance in lung adenocarcinoma (LUAD).MethodThe UCA1 expression level in LUAD cell lines was detected by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). We overexpressed UCA1 in A549 cells and downregulated UCA1 in A549/DDP cells by the lentivirus‑mediated technique. Subsequently, in vitro, and in vivo functional experiments were performed to investigate the functional roles of UCA1 in the growth and metastasis of LUAD cell lines. Furthermore, RNA pulldown, mass spectrometry, and RNA immunoprecipitation technique were performed to analyze various downstream target factors regulated by UCA1.ResultsThe results revealed a higher UCA1 expression level in A549/DDP cells and LUAD tissues than in A549 cells and adjacent cancer tissues. UCA1 expression was significantly associated with distant metastasis, clinical stage, and survival time of patients with LUAD. UCA1 overexpression significantly increased the proliferation, invasion, clone formation, and cisplatin resistance ability and enhanced the expression levels of proliferating cell nuclear antigen and excision repair cross-complementing gene 1 in A549 cells. However, these trends were mostly reversed after the knockdown of UCA1 in A549/DDP cells. Tumorigenic assays in nude mice showed that UCA1 knockdown significantly inhibited tumor growth and reduced cisplatin resistance. Enolase 1 was the RNA-binding protein (RBP) of UCA1.ConclusionBased on the results, we concluded that UCA1 promoted LUAD progression and cisplatin resistance and hence could be a potential diagnostic marker and therapeutic target in patients with LUAD.

Our previous experiments have demonstrated that lncRNA UCA1 (UCA1) promoted cisplatin resistance in lung adenocarcinoma (LUAD). This study aimed to explore the potential downstream target genes regulated by UCA1 and how this downstream gene promotes cisplatin resistance in LUAD. Here, we measured the expression level of Heme oxygenase1 (HO1) in LUAD cell lines by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) based on UCA1 overexpression cell lines and UCA1 knockdown cell lines. HO1 was knocked down in the UCA1 overexpression cell line, and HO1 was overexpressed in the UCA1 knockdown cell line, and the half maximal inhibitory concentration (IC50) trends were observed by adding cisplatin containing a certain concentration gradient. Cell functional assays were performed to observe the changes in the biological behavior of HO1 after overexpression and knockdown, and the tumorigenic assay in nude mice was performed to verify the effect of UCA1 in regulating the growth and cisplatin resistance of HO1 on LUAD cells in vivo. The results showed that HO1 and UCA1 expression were both upregulated in LUAD tissues and LUAD cisplatin-resistant cell lines, and there was a significant positive correlation between the expression of HO1 and UCA1. In vitro experiments showed that HO1 overexpression could reverse the reduced sensitivity to cisplatin caused by UCA1 knockdown in A549/DDP cells, and HO1 knockdown could reduce cisplatin resistance in A549 UCA1 overexpressing cells. Tumorigenic assays in nude mice further confirmed the role of HO1 in the regulation of UCA1 by activating the NRF2/HO1 pathway against LUAD cisplatin resistance. Our findings suggested that UCA1 regulates HO1 targets the UCA1/NRF2-HO1 pathway to exert cisplatin resistance in LUAD.

We previously used a pulse-based in vitro assay to unveil targetable signalling pathways associated with innate cisplatin resistance in lung adenocarcinoma (Hastings et al., 2020). Here, we advanced this model system and identified a non-genetic mechanism of resistance that drives recovery and regrowth in a subset of cells. Using RNAseq and a suite of biosensors to track single-cell fates both in vitro and in vivo, we identified that early S phase cells have a greater ability to maintain proliferative capacity, which correlated with reduced DNA damage over multiple generations. In contrast, cells in G1, late S or those treated with PARP/RAD51 inhibitors, maintained higher levels of DNA damage and underwent prolonged S/G2 phase arrest and senescence. Combined with our previous work, these data indicate that there is a non-genetic mechanism of resistance in human lung adenocarcinoma that is dependent on the cell cycle stage at the time of cisplatin exposure.

We previously used a pulse-based in vitro assay to unveil targetable signalling pathways associated with innate cisplatin resistance in lung adenocarcinoma (Hastings et al., 2020). Here, we advanced this model system and identified a non-genetic mechanism of resistance that drives recovery and regrowth in a subset of cells. Using RNAseq and a suite of biosensors to track single-cell fates both in vitro and in vivo, we identified that early S phase cells have a greater ability to maintain proliferative capacity, which correlated with reduced DNA damage over multiple generations. In contrast, cells in G1, late S or those treated with PARP/RAD51 inhibitors, maintained higher levels of DNA damage and underwent prolonged S/G2 phase arrest and senescence. Combined with our previous work, these data indicate that there is a non-genetic mechanism of resistance in human lung adenocarcinoma that is dependent on the cell cycle stage at the time of cisplatin exposure.

MiR-320a is associated with cisplatin resistance in lung adenocarcinoma and its clinical value in non-small cell lung cancer: A comprehensive analysis based on microarray data

Background: Previously, microRNA (miR)-7 has been reported to function as a tumor suppressor in human cancers, but the correlations of miR-7 expression with prognosis and cisplatin (CDDP) resistance in lung adenocarcinoma (LA) are unclear. Here, our aim is to determine the prognostic significance of miR-7 and its roles in the regulation of CDDP resistance in LA. Methods: Quantitative real-time PCR (qRT-PCR) assay was performed to determine miR-7 expression in 108 paired of LA tissues and analyze its correlations with clinicopathological factors of patients. The patient survival data were collected retrospectively by Kaplan-Meier analyses, and multivariate analysis was performed using the Cox proportional hazards model to determine the prognostic significance of miR-7 expression. The effects of miR-7 expression on the chemosensitivity of LA cells to CDDP and its possible mechanisms were evaluated by MTT, flow cytometry, Western blot and luciferase assays. Results: It was observed that the relative expression level of miR-7 in LA tissues was significantly lower than that in the adjacent normal tissues and low miR-7 expression level was closely associated with poorer tumor differentiation, advanced pathological T-factor, higher incidence of lymph node metastasis and advanced p-TNM stage. Also, patients with low miR-7 expression showed a shorter overall survival than those with high miR-7 expression, and multivariate analysis indicated that status of miR-7 expression was an independent molecular biomarker for predicting the overall survival (OS) of LA patients. In addition, upregulation of miR-7 increases the sensitivity of LA cells to CDDP via induction of apoptosis by targeting Bcl-2. Conclusions: Our finding for the first time demonstrates that low miR-7 expression may be an independent poor prognostic factor and targeting miR-7 may be a potential strategy for the reversal of CDDP resistance in LA.

The aim of this study was to explore the roles of GPX2, a member of the glutathione peroxidase family (GPXs, GSH-Px), in cisplatin (DDP) resistance in lung adenocarcinoma (LUAD). GPX2 was found to be the most significantly upregulated gene in a DDP-resistant A549/DDP cell line compared with the parental A549 cell line by RNA sequencing. The knockdown of GPX2 expression in A549/DDP cells inhibited cell proliferation in vitro and in vivo, decreased the IC50 values of DDP, induced apoptosis, inhibited the activities of GSH-Px and superoxide dismutase (SOD), inhibited ATP production and glucose uptake, and increased malondialdehyde (MDA) and reactive oxygen species (ROS) production; while GPX2 overexpression in A549 cells resulted in the opposite effects. Using gene set enrichment analysis (GSEA), we found that GPX2 may be involved in DDP resistance through mediating drug metabolism, the cell cycle, DNA repair and energy metabolism, and the regulation of an ATP-binding cassette (ABC) transporters member ABCB6, which is one of the hallmark genes in glycolysis. Moreover, immunohistochemistry revealed that GPX2 was upregulated in 58.6% (89/152) of LUAD cases, and elevated GPX2 expression was correlated with high expression of ABCB6, high 18-fluorodeoxyglucose (18F-FDG) uptake, and adverse disease-free survival (DFS) in our cohort. The Cancer Genome Atlas (TCGA) data also indicated that GPX2 expression was higher in LUAD than it was in normal lung tissues, and the mRNA expression levels of GPX2 and ABCB6 were positively correlated. In conclusion, our study demonstrates that GPX2 acts as oncogene in LUAD and promotes DDP resistance by regulating oxidative stress and energy metabolism.

ABSTRACTHOXA11, which is a member of the homeobox (HOX) gene family, and its natural antisense transcript (NAT) HOXA11-AS have been reported to be closely related to the development of lung cancer. We aimed to investigate their specific roles in cisplatin (DDP) resistance in lung adenocarcinoma (LUAD). First, we found that HOXA11 is hypermethylated and significantly downregulated in a DDP-resistant A549 cell line (A549/DDP) and LUAD tissues, while the HOXA11-AS expression level is elevated. Although HOXA11 and HOXA11-AS mRNA overlap in the 5ʹ-untranslated region (5ʹ UTR) and share two CpG islands, DNA methylation only regulates the expression of HOXA11. Then, we found that HOXA11 and HOXA11-AS have an inverse interaction by transfecting their siRNAs and overexpression vectors into A549 and A549/DDP cells. A dual-luciferase reporter assay further confirmed that the overlapping 5ʹUTR is essential for the bidirectional regulation between HOXA11 and HOXA11-AS. Functional analysis showed that knockdown of HOXA11 expression in A549 cells induced DDP resistance and activated Akt/β-catenin signaling, while overexpression of HOXA11 in A549/DDP cells increased DDP sensitivity and inhibited Akt/β-catenin signaling. Moreover, HOXA11-AS knockdown in A549 cells increased DDP sensitivity and inhibited Akt/β-catenin signaling, while the overexpression of HOXA11-AS in A549/DDP cells induced DDP resistance and activated Akt/β-catenin signaling. In conclusion, our study demonstrates that the inverse interaction between HOXA11 and HOXA11-AS promotes DDP resistance in LUAD.