Lung Adenocarcinoma Cell Line Research Articles

A cuproptosis-related lncRNAs risk model to predict prognosis and guide immunotherapy for lung adenocarcinoma.

Cuproptosis, one of the newest forms of cell death induction, is attracting mounting attention. However, the role of cuproptosis in lung cancer is currently unclear. In this study, we constructed a prognostic signature utilizing cuproptosis-related long noncoding RNAs (CRL) in lung adenocarcinoma (LUAD) and researched its clinical and molecular function. RNA-related and clinical data were downloaded from The Cancer Genome Atlas (TCGA) database. Differentially expressed CRLs were screened using the 'limma' package of R software. We used coexpression analysis and univariate Cox analysis to further identify prognostic CRLs. Applying least absolute shrinkage and selection operator (LASSO) regression and Cox regression models, a prognostic risk model based on 16 prognostic CRLs was constructed. To validate prognostic CRL function in LUAD, vitro experiments were conducted to explore the expression of GLIS2-AS1, LINC01230, and LINC00592 in LUAD. Subsequently, according to a formula, patients in the training, test, and overall groups were split into high- and low-risk groups. Kaplan-Meier and receiver operating characteristic (ROC) analyses were applied to assess the predictability of the risk model. Finally, the associations between risk signature and immunity-related analysis, somatic mutation, principal component analysis (PCA), enriched molecular pathways, and drug sensitivity was investigated. A cuproptosis-related long noncoding RNAs (lncRNAs) signature was constructed. Using quantitative polymerase chain reaction (qPCR) trial, we verified that the expressions of GLIS2-AS1, LINC01230, and LINC00592 in LUAD cell lines and tissues were consistent with the above screening results. Based on this signature, a total of 471 LUAD samples from TCGA data set were split into two risk groups based on the computed risk score. The risk model showed a better capacity in predicting prognosis than traditional clinicopathological features. Moreover, significant differences were found in immune cell infiltration, drug sensitivity, and immune checkpoint expression between the two risk groups. The CRLs signature was shown to be a prospective biomarker to forecast prognosis in patients with LUAD and presents new insights for personalized treatment of LUAD.

Synthesis of Dendrimer Conjugates with Naproxen and their Anticancer Activity

Abstract: Here we report the easy synthesis of dendrimer conjugates with four and eight naproxen moieties at the periphery. The synthesis involved two steps protection-deprotection sequence joined with triethylene glycol. A comparison was made between the cytotoxicity of dendron-naproxen conjugates and that of G1.0 G2.0 dendrimer-naproxen conjugates. Cytotoxicity studies showed that dendron conjugates were toxic towards all cancer cell lines studied, and their toxicity was significantly lower than that of dendrimer conjugates. However, dendrimer conjugates showed higher cytotoxicity and selectivity against K-562 and SKLU- 1 than dendron conjugates.

Molecular Mechanism of Cyclin E1 in Lung Adenocarcinoma and Their Cellular Response

The purpose of this study was to explore the role and molecular mechanism of CCNE1 in lung adenocarcinoma (LUAD). CCNE1 expression was analyzed in LUAD tissues and cell lines by GEPIA and qRT-PCR, and its correlation with patient survival was studied using TCGA. CCNE1 overexpression was tested for its effects on cellular functions. METTL3 was predicted as a downstream gene of CCNE1 and their correlation was analyzed in lung adenocarcinoma. MeRIP was used to measure the m6A level of CCNE1 mRNA 3′-UTR after METTL3 overexpression. Dual luciferase reporter gene experiments were conducted to determine the effect of METTL3 knockdown on CCNE1 activity. Recovery experiments were performed to evaluate the CCNE1-METTL3 regulation. CCNE1 was up-regulated in LUAD cells and tissues and linked to patient prognosis. Highly expressed CCNE1 increased LUAD proliferation. METTL3 was also highly expressed in LUAD tissues and positively correlated with CCNE1 expression. METTL3 reduced CCNE1 methylation and increased expression. Additionally, METTL3 overexpression partially reversed the inhibitory effect of CCNE1 silencing on LUAD cell proliferation. This study found that CCNE1 was markedly overexpressed in lung adenocarcinoma. METTL3 can inhibit the methylation level of CCNE1. Highly expressed CCNE1 can promote the proliferation of lung adenocarcinoma.

ADCY9 functions as a novel cancer suppressor gene in lung adenocarcinoma.

The process of lucubrating into lung adenocarcinoma (LUAD) is of profound clinical and practical significance in improving the prognosis of LUAD patients. Multiple biomarkers are reportedly involved in the proliferation or metastasis of adenocarcinoma. However, whether the ADCY9 gene influences the development of LUAD remains unknown. Therefore, we aimed to elucidate the relationship between the expression of ADCY9 and the proliferation and migration of LUAD. The ADCY9 gene was filtered via a survival analysis of LUAD acquired from the Gene Expression Omnibus (GEO). Then, we conducted a validation analysis and ADCY9-microRNA, microRNA-lncRNA, and ADCY9-lncRNA targeting relationship analysis through the data obtained from The Cancer Genome Atlas (TCGA) dataset. The survival curve, correlation, and prognostic analysis were implemented through bioinformatics methods. Both protein and mRNA expression levels of LUAD cell lines and LUAD patient samples (80 pairs) were detected using western blot assays and quantitative real-time polymerase chain reaction (qRT-PCR). An immunohistochemistry assay was performed to display the correlation between the expression level of the ADCY9 gene and prognosis in LUAD patients (2012-2013; n=115). Overexpression of cell lines SPCA1 and A549 were used for a series of cell function assays. Compared with the expression level in adjacent normal tissues, ADCY9 expression was downregulated in LUAD tissues. Based on the result of the survival curve analysis, high expression of ADCY9 may lead to a better prognosis and may be seen as an independent predictor for LUAD patients. High expression of ADCY9-related microRNA hsa-miR-7-5p may lead to a worse prognosis, and high expression of hsa-miR-7-5p-related lncRNAs may lead to the opposite effects. Overexpression of ADCY9 restrained the proliferation, invasion, and migration abilities of SPCA1, A549 cells. Results indicate that the ADCY9 gene acts as a tumor suppressor to restrain the proliferation, migration, and invasion in LUAD and can lead to a better survival or prognosis in LUAD patients.

The Atypical MAP Kinase MAPK15 Is Required for Lung Adenocarcinoma Metastasis via Its Interaction with NF-κB p50 Subunit and Transcriptional Regulation of Prostaglandin E2 Receptor EP3 Subtype

Studying the relatively underexplored atypical MAP Kinase MAPK15 on cancer progression/patient outcomes and its potential transcriptional regulation of downstream genes would be highly valuable for the diagnosis, prognosis, and potential oncotherapy of malignant tumors such as lung adenocarcinoma (LUAD). Here, the expression of MAPK15 in LUAD was detected by immunohistochemistry and its correlation with clinical parameters such as lymph node metastasis and clinical stage was analyzed. The correlation between the prostaglandin E2 receptor EP3 subtype (EP3) and MAPK15 expression in LUAD tissues was examined, and the transcriptional regulation of EP3 and cell migration by MAPK15 in LUAD cell lines were studied using the luciferase reporter assay, immunoblot analysis, qRT-PCR, and transwell assay. We found that MAPK15 is highly expressed in LUAD with lymph node metastasis. In addition, EP3 is positively correlated with the expression of MAPK15 in LUAD tissues, and we confirmed that MAPK15 transcriptionally regulates the expression of EP3. Upon the knockdown of MAPK15, the expression of EP3 was down-regulated and the cell migration ability was decreased in vitro; similarly, the mesenteric metastasis ability of the MAPK15 knockdown cells was inhibited in in vivo animal experiments. Mechanistically, we demonstrate for the first time that MAPK15 interacts with NF-κB p50 and enters the nucleus, and NF-κB p50 binds to the EP3 promoter and transcriptionally regulates the expression of EP3. Taken together, we show that a novel atypical MAPK and NF-κB subunit interaction promotes LUAD cell migration through transcriptional regulation of EP3, and higher MAPK15 level is associated with lymph node metastasis in patients with LUAD.

Evaluation of CAR-T Cells’ Cytotoxicity against Modified Solid Tumor Cell Lines

In recent years, adoptive cell therapy has gained a new perspective of application due to the development of technologies and the successful clinical use of CAR-T cells for the treatment of patients with malignant B-cell neoplasms. However, the efficacy of CAR-T therapy against solid tumor remains a major scientific and clinical challenge. In this work, we evaluated the cytotoxicity of 2nd generation CAR-T cells against modified solid tumors cell lines-lung adenocarcinoma cell line H522, prostate carcinoma PC-3M, breast carcinoma MDA-MB-231, and epidermoid carcinoma A431 cell lines transduced with lentiviruses encoding red fluorescent protein Katushka2S and the CD19 antigen. A correlation was demonstrated between an increase in the secretion of proinflammatory cytokines and a decrease in the confluence of tumor cells' monolayer. The proposed approach can potentially be applied to preliminarily assess CAR-T cell efficacy for the treatment of solid tumors and estimate the risks of developing cytokine release syndrome.

LncRNA PCAT6 promotes proliferation, migration, invasion, and epithelial-mesenchymal transition of lung adenocarcinoma cell by targeting miR-545-3p

BackgroundLung cancer is a high incidence cancer on a worldwide basis and has become a major public health problem. Lung adenocarcinoma (LUAD) makes up approximately half of all lung cancers and is a threat to human health. Long non-coding RNAs (lncRNAs) is an important regulator of the development and progression of lung adenocarcinoma. In this manuscript we examined the role and potential mechanism of lncRNA PCAT6 in the development of LUAD.Methods and resultsDifferences in lncRNA PCAT6 levels between LUAD samples and normal samples were first explored in the GEPIA database. We found that lncRNA PCAT6 expression was elevated, which was also validated in lung adenocarcinoma tissues and cell lines. Using western blotting, CCK-8, EdU, wound healing and transwell assays, we found that knockdown of lncRNA PCAT6 inhibited EMT, proliferation, migration, and invasion of LUAD cells. We noted a predicted a binding site for lncRNA PCAT6 and miR-545-3p through conducting bioinformatic analyses, and their binding was subsequently verified by a dual-luciferase reporter assay. Rescue experiments confirmed that miR-545-3p inhibitor partially abolished the inhibition function of lncRNA PCAT6 knockdown on LUAD cells. In addition, we predicted the downstream target genes of miR-545-3p and verified them by RT-qPCR. We found that EGFR was reduced in the silence of lncRNA PCAT6 and upregulated after miR-545-3p inhibition.ConclusionThis study demonstrates that lncRNA PCAT6 promotes a more aggressive LUAD phenotype by sponging miR-545-3p. This finding may provide new ideas for the treatment of lung cancer.

Antioxidant, Antibacterial, and Anticancer Activity of Ultrasonic Nanoemulsion of Cinnamomum Cassia L. Essential Oil.

Cinnamomum cassia (C. assia) has long been used in traditional holistic medicine for its medicinal properties. It is used as an antioxidant, antibacterial, anti-inflammatory and anticancer agent. Cinnamon, in particular, the essential oil of C. cassia, has significant biological properties. Despite this, the volatility, stability, and insolubility of C. cassia essential oil (CEO) remain the main disadvantages that limit its application, ultimately affecting its pharmacological efficacy. To find a solution to this problem, we developed the CEO nanoemulsion (CEO-NE). For lipophilic compounds, insoluble nanoemulsion-based formulations are a popular delivery strategy. In this research work, a highly stable dosage form named CEO-NE was successfully developed using polysorbate 80 and water. The findings show that the synthesized CEO has a uniform shape with a PDI of 0.380 and an adequate particle size of 221.8 nm. The antioxidant outcomes show excellent results for CEO-NE compared to CEO against DPPH and hydrogen peroxide. The obtained antibacterial activity of CEO-NE was more efficient than that of CEO against Klebsiella pneumonia (MTCC 8911) with 0.025% and 0.05%, respectively. The CEO-NE preparation was tested against an alveolar lung adenocarcinoma cell line (A549) with an IC50 of 50.21 µg/mL for CEO and 18.05 µg/mL for CEO-NE, respectively. These results are encouraging for future translational studies on CEO-NE use in lung cancer therapy due to its excellent antioxidant, antibacterial, and killing kinetic properties.

Comprehensive analysis of BUBs gene family in lung adenocarcinoma with immunological analysis.

Lung adenocarcinoma (LUAD) is one of the most commonly malignant tumors, and major challenges remain in the treatment of LUAD. Budding uninhibited by benzimidazole 1/3 (BUB1/3) play significant roles in the process of spindle-assembly checkpoint (SAC) during mitosis. However, their roles in LUAD have not been established. Here, we performed an immunological analysis of BUB1/3 in LUAD using a comprehensive bioinformatics approach, quantitative real-time-PCR and Western blotting technique. Our results indicated that the expression levels of BUB1 and BUB3 in LUAD samples were higher than the expression levels in the control groups and were associated with some clinicopathologic parameters in patients with LUAD. BUB1/3 and their related genes were enriched in cell immune, and the immune infiltration analysis revealed that the BUB1/3 expression profile was significantly correlated with characteristics of immune cell infiltration. Survival analysis showed that the disease-free survival and overall survival of patients with LUAD decreased with an increase in the BUB1/3 expression levels. The mRNA and protein expression levels of BUB1 and BUB3 in each of the LUAD cell lines were upregulated to varying degrees. BUB1 and BUB3 are the potential immunological therapeutic intervention targets for patients with LUAD.

Synthesis, Antiproliferative Evaluation, and Molecular Docking Study of Novel Longifolene-Derived Tetraline Fused Thiazole-Amide Derivatives.

Twenty novel longifolene-derived tetraline fused thiazole-amide compounds were synthesized from longifolene, a renewable natural resource. Their structures were characterized by FT-IR, NMR, ESI-MS, and elemental analysis. The in vitro antiproliferative activity of these compounds against SK-OV-3 ovarian cancer cell lines, MCF-7 human breast cancer cell lines, HepG2 human liver cancer cell lines, A549 human lung adenocarcinoma cell lines, and T-24 human bladder cancer cell lines was tested by MTT assay. Compounds 6a-6c displayed significant and broad-spectrum antiproliferative activity against almost the tested cancer cell lines with IC50 in the range of 7.84 to 55.88 μM, of which compound 6c exhibited excellent antiproliferative activities with 7.84 μM IC50 against SKOV-3, 13.68 μM IC50 against HepG2, 15.69 μM IC50 against A549, 19.13 μM IC50 against MCF-7, and 22.05 μM IC50 against T-24, showing better and broad-spectrum antiproliferative effect than that of the positive control 5-FU. Furthermore, the action model was analyzed by the molecular docking study. Some intriguing structure-activity relationships were found and discussed herein by DFT theoretical calculation.

CDCA4 interacts with IGF2BP1 to regulate lung adenocarcinoma proliferation via the PI3K/AKT pathway.

Lung adenocarcinomas (LUAD) remain the leading cause of death in many countries. In this study, we investigated the role of division cycle-associated 4 (CDCA4) in the carcinogenesis of LUADs. Real-time fluorescent quantitative polymerase chain reaction and western blot were performed to detect the messenger RNA and protein levels of CDCA4 in cells. Cell counting kit 8, real-time cell analysis, clone formation, EdU assays, and cell-cycle assays were used to preliminarily investigate the proliferation and cell-cycle-related functions of CDCA4 in lung adenocarcinoma. Immunoprecipitation assays were used to identify possible targets of CDCA4. A xenograft model was used to examine how CDCA4 knockdown affects LUAD cells growth in vivo. We found that the expression of CDCA4 was upregulated in LUAD cell lines. When CDCA4 was knocked out, the ability of LUAD cells to proliferate was dramatically reduced, and the cell cycle was stalled in the S phase. Meanwhile, boosting the CDCA4 expression had the opposite effect. The critical protein levels of phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) signaling pathway were subsequently examined. The findings demonstrated that elevated CDCA4 lowered the phosphate and tensin homolog expression and increased the p-PI3K and p-AKT levels. Moreover, we demonstrated that CDCA4 favorably regulated IGF2BP1, a downstream target. The downregulation of the IGF2BP1 expression could reverse the proliferation promotion effect induced by the CDCA4 overexpression. CDCA4 can operate as an oncogenic factor to control the growth of lung adenocarcinoma via the PI3K/AKT pathway.

Stenotrophomonas maltophilia promotes lung adenocarcinoma progression by upregulating histone deacetylase 5.

Lung cancer is the leading cause of cancer death worldwide, and lung adenocarcinoma (LADC) is the most common lung cancer. Lung cancer has a distinct microbiome composition correlated with patients' smoking status. However, the causal evidence of microbial impacts on LADC is largely unknown. We investigated microbial communities' differences in Formalin-Fixed Paraffin-Embedded tissues of ever-smoke (n = 22) and never-smoke (n = 31) patients with LADC through bacterial 16S rRNA gene high-throughput sequencing. Then nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung cancer mouse model and A549 cells were used to study the effect of Stenotrophomonas maltophilia (S. maltophilia) in LADC. We found a significant increase of genus Stenotrophomonas in LADC tissues of patients with primary tumor size greater than 3 cm and never-smoker patients. We further found that intratracheal infection with S. maltophilia promoted tumor progression in the NNK-induced lung cancer mouse model. We performed RNA-seq analysis on lung tissues and found that S. maltophilia treatment drove inflammation and upregulated tumor associated cell signaling, including Apelin signaling pathway. Mechanistically, histone deacetylase 5 (HDAC5) gene expression was significantly upregulated in S. maltophilia treated groups, and was required for S. maltophilia induced cell proliferation and migration in LADC cell line A549. Therefore, we provide in vivo and in vitro evidence to demonstrate that S. maltophilia promotes LADC progression, in part, through HDAC5.

LncRNA FOXD2-AS1 Increased Proliferation and Invasion of Lung Adenocarcinoma via Cell-Cycle Regulation.

Long non-coding RNA FOXD2 antisense RNA 1 (FOXD2-AS1) has been reported in many malignancies. However, the molecular mechanism of many actions is not clarified. This study was conducted to investigate the function of FOXD2-AS1 in lung adenocarcinoma and its molecular mechanism. Bioinformatics and in vitro analysis including RT-qPCR, CFU, CCK8, Transwell, Cell Apoptosis and Cell Cycle Assay were used for the analysis of gene expression and related effects. It revealed increased expression of lncRNA FOXD2-AS1 in lung adenocarcinoma cell lines (A549 cells), and abundant expression of lncRNA FOXD2-AS1 was also observed in the acquired lung adenocarcinoma tissues. In vitro results showed that knockdown of lncRNA FOXD2-AS1 in A549 cells weakened cell proliferation, invasion and increased apoptosis. At the same time, we found that reducing the expression of lncRNA FOXD2-AS1 caused cell cycle arrest in the G1/S phase. Differential gene analysis of lung adenocarcinoma and adjacent normal tissues showed that the cell cycle and its related process regulation were significantly enriched. Gene Set Enrichment Analysis (GSEA) analysis showed that miR-206, miR-143, lL6-JAK-STAT3 signalling pathway, STAT3, E2F targets, EZH2, P53 signalling pathway and E2F3 targets interacting with lncRNA FOXD2-AS1 were also enriched. This study demonstrates the role and mechanism of the lncRNA FOXD2-AS1 in lung adenocarcinoma and provides a better understanding for the treatment of lung adenocarcinoma, which indicates that interfering with lncRNA FOXD2-AS1 expression may be a novel strategy.

A novel prognostic model related to oxidative stress for treatment prediction in lung adenocarcinoma.

The prognostic model based on oxidative stress for lung adenocarcinoma (LUAD) remains unclear. The information of LUAD patients were acquired from TCGA dataset. We also collected two external datasets from GEO for verification. Oxidative stress-related genes (ORGs) were extracted from Genecards. We performed machine learning algorithms, including Univariate Cox regression, Random Survival Forest, and Least Absolute Shrinkage and Selection Operator (Lasso) analyses on the ORGs to build the OS-score and OS-signature. We drew the Kaplan-Meier and time-dependent receiver operating characteristic curve (ROC) to evaluate the efficacy of the OS-signature in predicting the prognosis of LUAD. We used GISTIC 2.0 and maftool algorithms to explore Genomic mutation of OS-signature. To analyze characteristic of tumor infiltrating immune cells, ESTIMATE, TIMER2.0, MCPcounter and ssGSEA algorithms were applied, thus evaluating the immunotherapeutic strategies. Chemotherapeutics sensitivity analysis was based on pRRophetic package. Finally, PCR assays was also used to detect the expression values of related genes in the OS-signature in cell lines. Ten ORGs with prognostic value and the OS-signature containing three prognostic ORGs were identified. The significantly better prognosis of LUAD patients was observed in LUAD patients. The efficiency and accuracy of OS-signature in predicting prognosis for LUAD patients was confirmed by survival ROC curves and two external validation data sets. It was clearly observed that patients with high OS-scores had lower immunomodulators levels (with a few exceptions), stromal score, immune score, ESTIMATE score and infiltrating immune cell populations. On the contrary, patients with higher OS-scores were more likely to have higher tumor purity. PCR assays showed that, MRPL44 and CYCS were significantly higher expressed in LUAD cell lines, while CAT was significantly lower expressed. The novel oxidative stress-related model we identified could be used for prognosis and treatment prediction in lung adenocarcinoma.

FAM83B promotes the invasion of primary lung adenocarcinoma via PI3K/AKT/NF-κB pathway

ObjectsThe family with sequence similarity 83B (FAM83B) is one of the markers for poor prognosis in several carcinomas, but the expression and the mechanism resulted in malignant phenotype in lung adenocarcinoma (LUAD) remain to be elucidated.Methods Data of RNA-seq in LUAD were downloaded from the cancer genome atlas (TCGA) database for differential expression and survival analysis, and immunohistochemistry was employed to analyze the protein expression of FAM83B in 126 cases of primary LUAD. The LUAD cell lines were collected for the detection of the effects on migration and invasion. Then, western blot was performed to measure the expression of tissue inhibitor of metalloproteinase (TIMP)-1 and activation of PI3K/AKT/NF-κB pathway.ResultsFAM83B was overexpressed in multiple types of carcinomas; The differential expression analysis revealed that the level of FAM83B was higher in LUAD than that in para-carcinoma; The patients with overexpression of FAM83B were with shorter overall survival (OS), disease specific survival (DSS) and progress free interval (PFI); Enrichment analysis suggested it was related to the focal adhesion of LUAD. Immunohistochemistry analysis demonstrated that higher FAM83B expression was positively related to lymph node metastasis in primary. Scratch assay and Borden chamber assay showed that the overexpression of FAM83B promoted migration and invasion activity in vitro. Furthermore, high level of FAM83B accelerated the tumorigenesis in vivo. Western blot showed that TIMP-1 was upregulated in H1299/FAM83B OE cells accompanying by the activation of PI3K/AKT/NF-κB pathway.ConclusionsFAM83B was a marker for poor prognosis of LUAD and it might promote the expression of TIMP-1 by activating PI3K/AKT/NF-κB pathway and then affect the ECM balance, which resulted in the migration and invasion of LUAD.

In Vitro Anticancer Activity of Novel Ciprofloxacin Mannich Base in Lung Adenocarcinoma and High-Grade Serous Ovarian Cancer Cell Lines via Attenuating MAPK Signaling Pathway.

Novel drugs are desperately needed in order to combat a significant challenge due to chemo-therapeutic resistance and bad prognosis. This research aimed to assess the anticancer activity of a newly synthesized ciprofloxacin Mannich base (CMB) on ovarian cancer (OVCAR-3) and lung cancer (A-549) cell lines and to investigate probable involved molecular mechanisms. The cytotoxic and pro-apoptotic impact of CMB on both cell lines was investigated using MTT assay, Annexin V assay, and cell cycle analysis, as well as caspase-3 activation. Western blotting was carried out to evaluate downstream targets of the MAPK pathway, while qRT PCR was used to evaluate the gene expression pattern of the p53/Bax/Bcl2 pathway. CMB treatment showed significantly reduced cell proliferation in both OVCAR-3 and A-549 cells with half maximum inhibitory concentration (IC50) = 11.60 and 16.22 µg/mL, respectively. CMB also induced apoptosis, S phase cell cycle arrest, and up-regulated expression of p53, p21, and Bax while down-regulated Bcl2 expression. CMB also halted cell proliferation by deactivating the MAPK pathway. In conclusion, CMB may be regarded as a potential antiproliferative agent for lung and ovarian cancers due to anti-proliferative and pro-apoptotic actions via inhibition of the MAPK pathway and p53/Bax/Bcl2.

CBX5 loss drives EGFR inhibitor resistance and results in therapeutically actionable vulnerabilities in lung cancer

Although epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFRi) are approved for treating EGFR-mutant lung adenocarcinoma (LUAD), emergence of acquired resistance limits their clinical benefits. Several mechanisms for acquired resistance to EGFRi in LUAD have been identified; however, the molecular basis for this resistance remains unknown in ~30% of LUAD. Chromatin and DNA modifiers and their regulators play important roles in determining response to anticancer therapies. Therefore, to identify nongenetic mechanisms of EGFRi resistance in LUAD, we performed an epigenome-wide shRNA screen targeting 363 human epigenetic regulator genes. This screen identified loss of the transcriptional repressor chromobox homolog 5 (CBX5) as a driver of EGFRi resistance in EGFR-mutant LUAD. Loss of CBX5 confers resistance to multiple EGFRi in both cell culture and mice. We found that CBX5 loss in EGFR-mutant LUAD cells leads to increased expression of the transcription factor E2F1, which in turn stimulates expression of the antiapoptotic gene BIRC5 (survivin). This E2F1-mediated upregulation of BIRC5 in CBX5-knockdown LUAD cells attenuates apoptosis induction following EGFRi treatment. Consistent with these results, knockdown of E2F1 or BIRC5 partly rescues CBX5-knockdown-induced EGFRi resistance in cell culture and mice. EGFRi-resistant LUAD cell lines show reduced CBX5 expression compared to parental lines; however, bromo- and extra-terminal (BET)-domain inhibitors (BETi) restore CBX5 expression in these cells and sensitize them to EGFRi/BETi combination therapy. Similarly, treatment with a BIRC5 inhibitor suppresses growth of EGFRi-resistant LUAD cells. Collectively, these studies identify CBX5 loss as a driver of EGFRi resistance and reveal therapeutic opportunities for treating EGFRi-resistant LUAD.

PU.1 negatively regulates tumorigenesis in non-small-cell lung cancer.

PU.1 is a key transcription factor that modulates hematopoietic cell differentiation and is involved in various physiological and pathological processes. PU.1 has been described to have multiple roles in a diverse range of cancers, but its contribution in non-small-cell lung cancer (NSCLC) has not been clearly elucidated. Fifty pairs of lung adenocarcinoma (LUAD) tissues and paraneoplastic tissues were collected. RT-qPCR assay was used to test PU.1 expression. Expression of PU.1 in LUAD cell lines and control cell lines was detected by RT-qPCR, and the role of PU.1 in LUAD was investigated by in vitro experiment. Levels of the major proteins in the apoptotic pathway were also detected by Western blot. The expression of PU.1 was remarkably downregulated in LUAD. Overexpression of PU.1 impaired the viability of LUAD cells as well as their metastatic function. In addition, PU.1 promoted apoptosis of LUAD cells by decreasing Bcl2 and increasing Bax/Bak1 expression. PU.1 plays an inhibitory role in LUAD, mainly promoting the apoptosis of LUAD cells.

Genetic and phenotypic determinants of morphologies in 3D cultures and xenografts of lung tumor cell lines.

We previously proposed the classification of lung adenocarcinoma into two groups: the bronchial epithelial phenotype (BE phenotype) with high-level expressions of bronchial epithelial markers and actionable genetic abnormalities of tyrosine kinase receptors and the non-BE phenotype with low-level expressions of bronchial Bronchial epithelial (BE) epithelial markers and no actionable genetic abnormalities of tyrosine kinase receptors. Here, we performed a comprehensive analysis of tumor morphologies in 3D cultures and xenografts across a panel of lung cancer cell lines. First, we demonstrated that 40 lung cancer cell lines (23 BE and 17 non-BE) can be classified into three groups based on morphologies in 3D cultures on Matrigel: round (n=31), stellate (n=5), and grape-like (n=4). The latter two morphologies were significantly frequent in the non-BE phenotype (1/23 BE, 8/17 non-BE, p=0.0014), and the stellate morphology was only found in the non-BE phenotype. SMARCA4 mutations were significantly frequent in stellate-shaped cells (4/4 stellate, 4/34 non-stellate, p=0.0001). Next, from the 40 cell lines, we successfully established 28 xenograft tumors (18 BE and 10 non-BE) in NOD/SCID mice and classified histological patterns of the xenograft tumors into three groups: solid (n=20), small nests in desmoplasia (n=4), and acinar/papillary (n=4). The latter two patterns were characteristically found in the BE phenotype. The non-BE phenotype exhibited a solid pattern with significantly less content of alpha-SMA-positive fibroblasts (p=0.0004) and collagen (p=0.0006) than the BE phenotype. Thus, the morphology of the tumors in 3D cultures and xenografts, including stroma genesis, reflects the intrinsic properties of the cancer cell lines. Furthermore, this study serves as an excellent resource for lung adenocarcinoma cell lines, with clinically relevant information on molecular and morphological characteristics and drug sensitivity.

Downregulation of Linc00173 increases BCL2 mRNA stability via the miR-1275/PROCA1/ZFP36L2 axis and induces acquired cisplatin resistance of lung adenocarcinoma

BackgroundLINC00173 had been reported as a cisplatin (cis-diamminedichloroplatinum, DDP) chemotherapy-resistant inducer in small-cell lung cancer (SCLC) and lung squamous cell carcinoma (LUSC). This study aimed to display reverse data for LINC00173 as a DDP chemosensitivity-inducing factor in lung adenocarcinoma (LUAD).MethodsLINC00173 was screened from the Gene Expression Omnibus database (GSE43493). The expression level of LINC00173 in LUAD tissues and cell lines was detected using in situ hybridization and quantitative reverse transcription–polymerase chain reaction. Colony formation, cell viability, half-maximal inhibitory concentration, flow cytometry, and xenograft mouse model were used to evaluate the role of LINC00173 in the chemosensitivity of LUAD to DDP. The mechanism of LINC00173 in DDP resistance by mediating miR-1275/PROCA1/ZFP36L2 axis to impair BCL2 mRNA stability was applied, and co-immunoprecipitation, chromatin immunoprecipitation, RNA antisense purification, RNA immunoprecipitation, and luciferase reporter assays were performed.ResultsLINC00173 downregulation in patients with DDP-resistant LUAD was correlated with poor prognosis. Further, LINC00173 expression was significantly reduced in DDP-resistant LUAD cells and DDP-treated human LUAD tissues. Suppressed LINC00173 expression in LUAD cells enhanced DDP chemoresistance in vivo and in vitro, while restored LINC00173 expression in DDP-resistant LUAD cells markedly regained chemosensitivity to DDP. Mechanistically, DDP-resistant LUAD cells activated PI3K/AKT signal and further elevated the c-Myc expression. The c-Myc, as an oncogenic transcriptional factor, bound to the promoter of LINC00173 and suppressed its expression. The reduced LINC00173 expression attenuated the adsorption of oncogenic miR-1275, downregulating the expression of miR-1275 target gene PROCA1. PROCA1 played a potential tumor-suppressive role inducing cell apoptosis and DDP chemosensitivity via recruiting ZFP36L2 to bind to the 3′ untranslated region of BCL2, reducing the stability of BCL2 mRNA and thus activating the apoptotic signal.ConclusionsThis study demonstrated a novel and critical role of LINC00173. It was transcriptionally repressed by DDP-activated PI3K/AKT/c-Myc signal in LUAD, promoting DDP-acquired chemotherapeutic resistance by regulating miR-1275 to suppress PROCA1/ZFP36L2-induced BCL2 degradation, which led to apoptotic signal reduction. These data were not consistent with the previously described role of LINC00173 in SCLC or LUSC, which suggested that LINC00173 could play fine-tuned DDP resistance roles in different pathological subtypes of lung cancer. This study demonstrated that the diminished expression of LINC00173 might serve as an indicator of DDP-acquired resistance in LUAD.