Lung Adenocarcinoma Cell Line Research Articles

AHSG, a Gene Promoting Tumour Proliferation, Migration and Invasion, is an Independent Prognostic Factor for Poor Overall Survival in Lung Adenocarcinoma.

Recent studies have shown that the metabolic process-related gene AHSG is involved in multiple pathological processes of tumours. This study will explore the relationship between AHSG and lung adenocarcinoma. Expression analysis, survival analysis and co-expression analysis of AHSG were performed using a public database, and cytological and molecular biology assays were performed to explore the role of AHSG in lung adenocarcinoma. Compared with normal tissues, AHSG expression was significantly higher in cancer tissues in the TCGA-LUAD database, and pan-cancer analysis revealed abnormal AHSG expression in different kinds of tumours. Survival analysis revealed that compared with the low expression group, the patients in the high expression group had a significantly worse overall survival duration in the TCGA-LUAD database, and a subsequent study confirmed that AHSG expression could be an independent prognostic factor of overall survival in lung adenocarcinoma. AHSG-related genes are involved in multiple physiological and pathophysiological pathways. In subsequent cytological and molecular biology experiments, inhibition of AHSG expression suppressed proliferation, migration and invasion in lung adenocarcinoma cell lines, and the EMT process was blocked after knockdown of AHSG. AHSG could be used as a prognostic factor for OS in patients with lung adenocarcinoma. It can promote the biological behaviour of lung adenocarcinoma and may become a potential target for treatment, which is worthy of further study.

Exportin 1 inhibition prevents neuroendocrine transformation through SOX2 down-regulation in lung and prostate cancers.

In lung and prostate adenocarcinomas, neuroendocrine (NE) transformation to an aggressive derivative resembling small cell lung cancer (SCLC) is associated with poor prognosis. We previously described dependency of SCLC on the nuclear transporter exportin 1. Here, we explored the role of exportin 1 in NE transformation. We observed up-regulated exportin 1 in lung and prostate pretransformation adenocarcinomas. Exportin 1 was up-regulated after genetic inactivation of TP53 and RB1 in lung and prostate adenocarcinoma cell lines, accompanied by increased sensitivity to the exportin 1 inhibitor selinexor in vitro. Exportin 1 inhibition prevented NE transformation in different TP53/RB1-inactivated prostate adenocarcinoma xenograft models that acquire NE features upon treatment with the aromatase inhibitor enzalutamide and extended response to the EGFR inhibitor osimertinib in a lung cancer transformation patient-derived xenograft (PDX) model exhibiting combined adenocarcinoma/SCLC histology. Ectopic SOX2 expression restored the enzalutamide-promoted NE phenotype on adenocarcinoma-to-NE transformation xenograft models despite selinexor treatment. Selinexor sensitized NE-transformed lung and prostate small cell carcinoma PDXs to standard cytotoxics. Together, these data nominate exportin 1 inhibition as a potential therapeutic target to constrain lineage plasticity and prevent or treat NE transformation in lung and prostate adenocarcinoma.

Effects of Sufentanil Nanoparticles on Proliferation and Apoptosis of Human Lung Adenocarcinoma Cells

This research was aimed to investigate the effects of sufentanil nanoparticles (Suf-NPs) on the proliferation and apoptosis of human lung adenocarcinoma (LUAD) cells, and to provide theoretical support for future drug therapy. Based on the preparation method of solid lipid nanoparticles (SLN), stearic acid and lecithin were used as materials to prepare Suf-NPs. The physical and chemical properties of Suf-NPs were analyzed by scanning electron microscope and particle size analyzer. A549 human LUAD cell line was selected. According to different cell treatment methods, A549 cells were randomly divided into control (Con), sufentanil (Suf), blank SLN, and Suf-NPs groups. Con was cultured routinely, and Suf group was treated with 0.02 μg/mL sufentanil. Suf-NPs group was treated with Suf-NPs, and the concentration was the same as that in the Suf group. The SLN group was added with blank SLN suspension. CCK8 assay was adopted to test the proliferation inhibition rate (PIR) of A549 cells treated with SUF-NPs. Flow cytometry and dual-color fluorescence microscopy were adopted to detect the apoptosis rate. The expression levels of apoptosis-related proteins P53, Bax, and Bcl-2 were detected by Western blot. The results revealed that the average particle size of Suf-NPs was (113.86±2.03) nm, the zeta potential was (−28.44±1.52) mV, the encapsulation efficiency (PER) was (82.02±3.56) %, and the drug loading (DL) capacity was (50.31±6.49) %. As against Con, the PIR, apoptosis rate, P53, and Bax protein expression was significantly higher (P <0.001), and Bcl-2 protein was markedly inferior in Suf, SLN, and Suf-NPs groups (P <0.05). Compared with the Suf, the Suf-NPs group had markedly higher PIR, apoptosis rate, and expression of P53 and Bax proteins (P < 0.05), and markedly lower expression of Bcl-2 protein (P <0.05). Suf-NPs can markedly restrain A549 human LUAD cells, promote their apoptosis, and affect the expression of related genes and proteins. It provides a theoretical basis for the future development of Suf-NPs as a drug for the treatment of lung cancer (LC).

Genomic characterization and virulence of Streptococcus suis serotype 4 clonal complex 94 recovered from human and swine samples.

Streptococcus suis is a zoonotic pathogen that causes invasive infections in humans and pigs. Herein, we performed genomic analysis of seven S. suis serotype 4 strains belonging to clonal complex (CC) 94 that were recovered from a human patient or from diseased and clinically healthy pigs. Genomic exploration and comparisons, as well as in vitro cytotoxicity tests, indicated that S. suis CC94 serotype 4 strains are potentially virulent. Genomic analysis revealed that all seven strains clustered within minimum core genome group 3 (MCG-3) and had a high number of virulence-associated genes similar to those of virulent serotype 2 strains. Cytotoxicity assays showed that both the human lung adenocarcinoma cell line and HeLa cells rapidly lost viability following incubation for 4 h with the strains at a concentration of 106 bacterial cells. The human serotype 4 strain (ID36054) decreased cell viability profoundly and similarly to the control serotype 2 strain P1/7. In addition, strain ST1689 (ID34572), isolated from a clinically healthy pig, presented similar behaviour in an adenocarcinoma cell line and HeLa cells. The antimicrobial resistance genes tet(O) and ermB that confer resistance to tetracyclines, macrolides, and lincosamides were commonly found in the strains. However, aminoglycoside and streptothricin resistance genes were found only in certain strains in this study. Our results indicate that S. suis CC94 serotype 4 strains are potentially pathogenic and virulent and should be monitored.

The circadian clock circuitry deconvolutes colorectal cancer and lung adenocarcinoma heterogeneity in a dynamic time-related framework.

Increasing evidence imputes cancer progression and resistance to therapy to intra-tumor molecular heterogeneity set off by cancer cell plasticity. Re-activation of developmental programs strictly linked to epithelial-to-mesenchymal transition and gaining of stem cells properties are crucial in this setting. Many biological processes involved in cancer onset and progression show rhythmic fluctuations driven by the circadian clock circuitry. Novel cancer patient stratification tools taking into account the temporal dimension of these biological processes are definitely needed. Lung cancer and colorectal cancer (CRC) are the leading causes of cancer death worldwide. Here, by developing an innovative computational approach we named Phase-Finder, we show that the molecular heterogeneity characterizing the two deadliest cancers, CRC and lung adenocarcinoma (LUAD), rather than a merely stochastic event is the readout of specific cancer molecular states which correlate with time-qualified patterns of gene expression. We performed time-course transcriptome analysis of CRC and LUAD cell lines and upon computing circadian genes expression-based correlation matrices we derived pseudo-time points to infer time-qualified patterns in the transcriptomic analysis of real-world data (RWD) from large cohorts of CRC and LUAD patients. Our temporal classification of CRC and LUAD cohorts was able to effectively render time-specific patterns in cancer phenotype switching determining dynamical distribution of molecular subtypes impacting patient prognosis.

Tumor-suppressive action of miR-30a-5p in lung adenocarcinoma correlates with ABL2 inhibition and PI3K/AKT pathway inactivation.

ABL2 contributes to the oncogenic potential of cancers, pointing to its inhibition as a possible strategy against malignant diseases. Bioinformatics prediction of upstream effector miR-30a-5p for ABL2 allowed us to hypothesize and then validate mechanistic actions of miR-30a-5p in lung adenocarcinoma (LUAD). The ABL2 expression in LUAD was analyzed in the TCGA data, clinical samples, and cell lines. The shRNA-mediated silencing of ABL2 was introduced to illustrate its effect on malignant phenotypes of LUAD cells. The binding affinity between ABL2 and miR-30a-5p was verified by luciferase activity and RNA pull-down assay. Ectopic expression, knockdown methods, and PI3K inhibitor LY294002 were used to investigate their effects on in vitro biological characteristics and in vivo tumor growth of LUAD cells. Using nude mouse lung adenocarcinoma in situ and brain metastasis models to validate the inhibitory effect of miR-30a-5p on LUAD by regulating the ABL2/PI3K/AKT signaling axis. High expression of ABL2 and poor expression of miR-30a-5p were noticed in LUAD tissues and cell lines. Importantly, miR-30a-5p was demonstrated to target and downregulate ABL2, subsequently inactivating the PI3K/AKT pathway. miR-30a-5p inhibited the malignant phenotypes of LUAD cells by inhibiting ABL2 expression and inactivating the PI3K/AKT pathway. For in vivo experiments, miR-30a-5p was substantiated to thwart tumor tumorigenesis by regulating the ABL2/PI3K/AKT axis. In addition, miR-30a-5p suppresses the occurrence and development of in situ lung cancer and brain metastasis via the ABL2/PI3K/AKT signaling pathway. This study underscores the inhibitory role of miR-30a-5p in LUAD through the ABL2/PI3K/AKT axis, which may be a viable target for LUAD treatment.

Screening and validation of plasma cell-derived, purinergic, and calcium signalling-related genetic signature to predict prognosis and PD-L1/PD-1 blockade responses in lung adenocarcinoma.

This study aims at screening and validation of prospective genetic signature for lung adenocarcinoma (LUAD) prognosis and treatment. The immune-related genes (IRGs) were obtained from The Cancer Genome Atlas (TCGA) dataset where a total of 535 LUAD and 59 control samples were included. A risk model was then developed for the risk stratification of LUAD patients. The immune cell infiltration, clinical outcomes, and the therapeuticefficacy of programmed cell death protein 1 (PD-1) and its ligand (PD-L1) blockade were compared between high and low-risk groups.Gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) were used to explore the biological processes and signalling pathways associated with the IRGs. Finally, IRGs mRNA levels were assayed by reverse transcription quantitative real-time PCR (RT-qPCR)in LUAD and relevant cell lines. Two IRGs, P2RX1 (purinergic receptor P2X 1) and PCP4 (Purkinje cell protein 4), were screened from a module that possesses the highest correlation with plasma cells. RT-qPCR verified the expression of the two IRGs in plasmacytoma cell RPMI 8226 but not in LUAD cells. A higher risk score is associated with a lower infiltration of immune cells. Kaplan-Meier and nomogram analysis showed that the high-risk group has a lower survival rate than the low-risk cohort. Furthermore, the high-risk group had a worse response rate to PD-L1/PD-1 blockade. GSVA and GSEA-GO results indicated that a lower risk score is linked to signalling pathways and biological functions promoting immune response and inflammation. In contrast, a higher risk score is associated with signalling cascades promoting tumour growth. The immune-related prognostic model based on P2RX1 and PCP4 is conducive to predicting the therapeutic response of PD-L1/PD-1 blockade and clinical outcomes of LUAD.

Long non-coding RNA GNAS-AS1 knockdown inhibits proliferation and epithelial-mesenchymal transition of lung adenocarcinoma cells via the microRNA-433-3p/Rab3A axis.

The goal of this study was to demonstrate the functions and specific mechanism of long non-coding RNA (lncRNA) GNAS-AS1 in lung adenocarcinoma. Levels of lncRNA GNAS-AS1, microRNA (miR)-433-3p, and Rab3A were assessed by quantitative real-time PCR (qRT-PCR). The target-binding sites of lncRNA GNAS-AS1, miR-433-3p, and Rab3A were predicted and confirmed by bioinformatics tool (StarBase) and a dual-luciferase reporter system. Cell proliferation and apoptosis were checked using MTT and flow cytometry, respectively. Additionally, the levels of apoptosis-related and epithelial-mesenchymal transition (EMT)-associated genes in A549 cells were analyzed by qRT-PCR and western blot. We found that lncRNA GNAS-AS1 was upregulated, miR-433-3p was low-expressed, and Rab3A was overexpressed in lung adenocarcinoma tissues and cell lines. LncRNA GNAS-AS1 interacted with miR-433-3p and negatively regulated miR-433-3p levels. Rab3A was a direct target of miR-433-3p. Downregulation of lncRNA GNAS-AS1 remarkably suppressed cell proliferation, promoted cell apoptosis, decreased B-cell lymphoma-2 (Bcl-2) expression, enhanced the Bcl-2-Associated X (Bax) level, promoted E-cadherin expression, and reduced N-cadherin and Rab3A levels. However, the miR-433-3p inhibitor reversed all these findings. Similarly, the inhibitory effects of miR-433-3p mimic on A549 cells were reversed by the Rab3A-plasmid. In conclusion, lncRNA GNAS-AS1 downregulation suppressed lung adenocarcinoma cell proliferation and EMT through the miR-433-3p/Rab3A axis.

Dipeptidase‑2 is a prognostic marker in lung adenocarcinoma that is correlated with its sensitivity to cisplatin.

Lung cancer accounts for the highest percentage of cancer morbidity and mortality worldwide, and lung adenocarcinoma (LUAD) is the most prevalent subtype. Although numerous therapies have been developed for lung cancer, patient prognosis is limited by tumor metastasis and more effective treatment targets are urgently required. In the present study, gene expression profiles were extracted from the Gene Expression Omnibus database and mRNA expression data were downloaded from The Cancer Genome Atlas database. In addition, TIMER 2.0 database was used to analyze the expression of genes in normal and multiple tumor tissues. Protein expression was confirmed using the Human Protein Atlas database and LUAD cell lines, sphere formation assay, western blotting, and a xenograft mouse model were used to confirm the bioinformatics analysis. Dipeptidase‑2 (DPEP2) expression was significantly decreased in LUAD and was negatively associated with prognosis. DPEP2 overexpression substantially inhibited epithelial‑mesenchymal transition (EMT) as well as LUAD cell metastasis, and limited the expression of the cancer stem cell transformation markers, CD44 and CD133. In addition, DPEP2 improved LUAD sensitivity to cisplatin by inhibiting EMT; this was verified invitro and invivo. These data indicated that DPEP2 upregulates E‑cadherin, thereby regulating cell migration, cancer stem cell transformation, and cisplatin resistance, ultimately affecting the survival of patients with LUAD. Overall, the findings of the present suggest that DPEP2 is important in the development of LUAD and can be used both as a prognostic marker and a target for future therapeutic research.

Identification of lysosomal genes associated with prognosis in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer, representing 40% of all cases of this tumor. Despite immense improvements in understanding the molecular basis, diagnosis, and treatment of LUAD, its recurrence rate is still high. RNA-seq data from The Cancer Genome Atlas (TCGA) LUAD cohort were download from Genomic Data Commons Portal. The GSE13213 dataset from Gene Expression Omnibus (GEO) was used for external validation. Differential prognostic lysosome-related genes (LRGs) were identified by overlapping survival-related genes obtained via univariate Cox regression analysis with differentially expressed genes (DEGs). The prognostic model was built using Kaplan-Meier curves and least absolute shrinkage and selection operator (LASSO) analyses. In addition, univariate and multivariate Cox analyses were employed to identify independent prognostic factors. The responses of patients to immune checkpoint inhibitors (ICIs) were further predicted. The pRRophetic package and rank-sum test were used to compute the half maximal inhibitory concentrations (IC50) of 56 chemotherapeutic drugs and their differential effects in the low- and high-risk groups. Moreover, quantitative real-time polymerase chain reaction, Western blot, and human protein atlas (HPA) database were used to verify the expression of the four prognostic biomarkers in LUAD. Of the nine candidate differential prognostic LRGs, GATA2, TFAP2A, LMBRD1, and KRT8 were selected as prognostic biomarkers. The prediction of the risk model was validated to be reliable. Cox independent prognostic analysis revealed that risk score and stage were independent prognostic factors in LUAD. Furthermore, the nomogram and calibration curves of the independent prognostic factors performed well. Differential analysis of ICIs revealed CD276, ICOS, PDCD1LG2, CD27, TNFRSF18, TNFSF9, ENTPD1, and NT5E to be expressed differently in the low- and high-risk groups. The IC50 values of 12 chemotherapeutic drugs, including epothilone.B, JNK.inhibitor.VIII, and AKT.inhibitor.VIII, significantly differed between the two risk groups. KRT8 and TFAP2A were highly expressed, while GATA2 and LMBRD1 were poorly expressed in LUAD cell lines. In addition, KRT8 and TFAP2A were highly expressed, while GATA2 and LMBRD1 were poorly expressed in tumor tissues. Four key prognostic biomarkers-GATA2, TFAP2A, LMBRD1, and KRT8-were used to construct a significant prognostic model for LUAD patients.

Anticancer activity of manganese dioxide/reduced graphene oxide nanocomposites against A549 human lung adenocarcinoma cell line

A modified version of Hummers’ process was used to synthesize graphene oxide (GO), whereas solar radiation was used to create reduced graphene oxide (rGO) and MnO2/rGO (1%, 3%, 5%) nanostructures were synthesized using a facile hydrothermal method. The X-ray diffraction (XRD) pattern revealed the tetragonal structure of α-MnO2 and determined the crystallite size of MnO2/rGO as 14.36 nm. A scanning electron microscope (SEM) analysis confirms the growth of MnO2 nanorods on the rGO surface. MnO2/rGO (1%, 3%, 5%) contains elements such as carbon, oxygen and manganese, as verified by energy-dispersive X-ray analysis (EDAX). When the rGO percentage was increased, the surface area of MnO2/rGO was decreased to 16.340 m2/g, which enhances the interaction between A549 cells, according to Brunauer–Emmett–Teller (BET) analysis. The allotropes of carbon and the functional groups were examined using Raman spectroscopy and Fourier transform infrared spectroscopy (FTIR). A (dimethyl thiazolyl tetrazolium bromide) MTT assay was used to assess the cytotoxicity of GO, rGO and MnO2/rGO (1%, 3%, 5%) nanostructures against the human lung adenocarcinoma cell line (A549) and human lung epithelial cell line (L132). The half maximum inhibitory concentration (IC50) value for MnO2/5% rGO towards A549 cells was determined to be 120 μg/mL, which shows appreciable results when compared to GO (180 μg/mL), rGO (160 μg/mL), MnO2/1% rGO (180 μg/mL) and MnO2/3% rGO (160 μg/mL). The morphological changes in A549 cells were investigated using an AO/EtBr dual stain, the intracellular reactive oxygen species produced were evaluated using a DCFH-DA stain and the fragmented nuclei were examined using a Hoechst stain. According to the findings, the synthesized MnO2/rGO nanocomposites can be efficiently used for cytotoxicity against A549 cells and possess significant potential for the human lung cancer cell line.

LINC01117 inhibits invasion and migration of lung adenocarcinoma through influencing EMT process.

Studying the mechanism of action of LncRNAs in lung adenocarcinoma (LUAD) is of great importance for an in-depth understanding of the molecular mechanism of lung adeno carcinogenesis and development. The aim is to identify a long non-coding RNA LINC01117 that is specifically and highly expressed in LUAD cells and to investigate its biological functions and molecular mechanisms in LUAD cells, providing a new potential target for targeting LUAD therapy. This study used publicly available data downloaded from The Cancer Genome Atlas (TCGA) database. Construction of siRNA and overexpression plasmid-packed lentiviral constructs were used to knock down and increase the expression of LINC01117 in LUAD cells. The effect of LINC01117 on LUAD cell migration and invasion was verified by scratch assays and Transwell assays. Western blot assays were performed to verify the effect of knocking down LINC01117 expression on key proteins of the EMT process. The effect of overexpression and knockdown LINC01117 expression on key proteins of the EMT process and the nuclear and cytoplasmic distribution of YAP1, a key effector molecule of the Hippo pathway, was verified by Western blot assays. LINC01117 expression was upregulated in LUAD tissues and cell lines. Clinical correlation and prognostic analyses showed that LINC01117 was associated with poorer clinical features (staging and N classification) and poorer prognosis and could be analyzed as an independent prognostic factor. Cell migration and invasion were significantly inhibited in the knockdown group compared to the control group; in contrast, cell migration and invasion were promoted in the overexpression group. Overexpression of LINC01117 resulted in down-regulation of E-cadherin expression and increased expression levels of N-cadherin, vimentin, ZEB1, snail and slug; in contrast, knockdown of LINC01117 appeared to have the opposite effect. Furthermore, knockdown of LINC01117 increased the enrichment of YAP1 protein in the cytoplasm and reduced its level in the nucleus; overexpression of LINC01117 produced the opposite intracellular distribution results. LINC01117 was highly expressed in LUAD, and knockdown of LINC01117 significantly inhibited the migration and invasion of LUAD cells, while overexpression of LINC01117 significantly promoted the migration and invasion of LUAD cells, and affected the EMT process, and was able to alter the distribution of YAP1 in the nucleus and cytoplasm. This suggests that LINC01117 may regulate the activity of the Hippo pathway by altering the nuclear and cytoplasmic distribution of YAP1, which in turn induces the EMT process in lung adenocarcinoma cells and thus exerts a pro-cancer effect. It suggests that LINC01117 may play a key role in the occurrence and development of LUAD.

Synthesis and in-vitro study of a novel ligustrazine diselenide as a potential chemotherapy drug for lung adenocarcinoma

A novel ligustrazine diselenide, 1,2-bis ((3,5,6-trimethylpyrazin-2-yl) methyl) diselenide (Se2), for potential treatment on adenocarcinoma of lung cancer was successfully synthesized and fully characterized by various analytical approaches. Cytotoxic, antiproliferative and apoptosis-triggering mechanism of Se2 compound have been investigated through human lung adenocarcinoma (LUAD) cell line A549. The study found that Se2 significantly inhibit the proliferation of A549 cells in a dose-dependent manner. Flow cytometry showed that Se2 induced cell arrest and apoptosis in S and G2/M phase, and the apoptotic effect of Se2 were associated with the increase of caspase 3 and PARP-1 level approved by western blot assay. Further mechanism study results suggested that Se2 suppressed the migration,invasion and colony formation of A549 cells, significantly inhibited the PI3K/Akt/m-TOR signaling pathway. The study indicated that Se2 is a bioactive substance that can induce apoptosis of A549 cells in-vitro, and it is a potent candidate drug for LUAD.

Defining the first bona fide cell model for SMARCA4-deficient, undifferentiated tumor.

The World Health Organization's tumor classification guidelines are frequently updated and renewed as knowledge of cancer biology advances. For instance, in 2021, a novel lung tumor subtype named SMARCA4-deficient, undifferentiated tumor (SMARCA4-dUT, code 8044/3) was included. To date, there is no defined cell model for SMARCA4-dUT that could be used to help thoracic clinicians and researchers in the study of this newly defined tumor type. As this tumor type was recently described, it is feasible that some cell models formerly classified as lung adenocarcinoma (LUAD) could now be better classified as SMARCA4-dUT. Thus, in this work, we aimed to identify a bona fide cell model for the experimental study of SMARCA4-dUT. We compared the differential expression profiles of 36 LUAD-annotated cell lines and 38 cell lines defined as rhabdoid in repositories. These comparative results were integrated with the mutation and expression profiles of the SWI/SNF complex members, and they were surveyed for the presence of the SMARCA4-dUT markers SOX2, SALL4, and CD34, measured by RT-qPCR and western blotting. Finally, the cell line with the paradigmatic SMARCA4-dUT markers was engrafted into immunocompromised mice to assess the histological morphology of the formed tumors and compare them with those formed by a bona fide LUAD cancer cell line. NCI-H522, formerly classified as LUAD, displayed expression profiles nearer to rhabdoid tumors than LUAD tumors. Furthermore, NCI-H522 has most of the paradigmatic features of SMARCA4-dUT: hemizygous inactivating mutation of SMARCA4, severe SMARCA2 downregulation, and high-level expression of stem cell markers SOX2 and SALL4. In addition, the engrafted tumors of NCI-H522 did not display a typical differentiated glandular structure as other bona fide LUAD cell lines (A549) do but had rather a largely undifferentiated morphology, characteristic of SMARCA4-dUT. Thus, we propose the NCI-H522 as the first bona fide cell line model of SMARCA4-dUT. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Downregulation of AHNAK2 inhibits cell cycle of lung adenocarcinoma cells by interacting with RUVBL1.

Lung adenocarcinoma (LUAD) is the leading cause of death among cancer diseases. The tumorigenic functions of AHNAK2 in LUAD have attracted more attention in recent years, while there are few studies which have reported its high molecular weight. The mRNA-seq data of AHNAK2 and corresponding clinical data from UCSC Xena and GEO was analyzed. LUAD cell lines were transfected with sh-NC and sh-AHNAK2, and cell proliferation, migration and invasion were then detected by in vitro experiments. We performed RNA sequencing and mass spectrometry analysis to explore the downstream mechanism and interacting proteins of AHNAK2. Finally, western blot, cell cycle analysis and CO-IP were used to confirm our assumptions regarding previous experiments. Our study revealed that AHNAK2 expression was significantly higher in tumors than in normal lung tissues and higher AHNAK2 expression led to a poor prognosis, especially in patients with advanced tumors. AHNAK2 suppression via shRNA reduced the LUAD cell lines proliferation, migration and invasion and induced significant changes in DNA replication, NF-kappa B signaling pathway and cell cycle. AHNAK2 knockdown also caused G1/S phase cell cycle arrest, which could be attributed to the interaction of AHNAK2 and RUVBL1. In addition, the results from gene set enrichment analysis (GSEA) and RNA sequencing suggested that AHNAK2 probably plays a part in the mitotic cell cycle. AHNAK2 promotes proliferation, migration and invasion in LUAD and regulates the cell cycle via the interaction with RUVBL1. More studies of AHNAK2 are still needed to reveal its upstream mechanism.

Mechanism of Ziyuglycoside II-mediated Ferroptosis-related Proteins on the Proliferation and Metastasis of Human Lung Adenocarcinoma Cell Lines

Background Ferroptosis is a novel type of regulated cell death and targeting ferroptosis may be a potential treatment strategy for lung cancer. Ziyuglycoside II (ZYG II) has a significant inhibitory effect on the growth of lung cancer cells. However, the selective anti-tumor effect of the ZYG II against lung cancer has not been systemically studied. Objectives We combined ferrostatin-1 and erastin to explore the potential therapeutic mechanism of the ZYG II for lung adenocarcinoma. Materials and Methods A549 and H1299 cells were randomly divided into the control, ZYG II, ferroptosis inhibitor group (ZYG II+ ferrostatin-1), and erastin group (ZYG II+ erastin). Cell proliferation was detected using the CCK-8 method. Cell migration and invasion were evaluated using the Transwell assay. The protein expression levels of Glutathione Peroxidase 4 (GPX4), Solute Carrier Family 7 Member 11 (SLC7A11), and Transferrin receptor 1 (TFR1) were measured using western blotting. Results Compared with the control group, the cell proliferation, migration, and invasion abilities of the ZYG II group significantly decreased, the protein expression levels of GPX4 and SLC7A11 in the ZYG II group declined significantly, and the expression of TFR1 increased significantly ( p < 0.05). After adding ferrostatin 1 (ZYG II+ Ferrostatin 1), the cell proliferation, migration, and invasion abilities of the inhibited cells were significantly increased, the expression of GPX4 and SLC7A11 increased significantly and the expression of TFR1 decreased significantly ( p < 0.05). However, after adding the erastin (ZYG II+ erastin), the cell viability was further inhibited in A549, the expression levels of GPX4 and SLC7A11 were further inhibited and the expression of TFR1 was further increased ( p < 0.05). Conclusion ZYG II significantly inhibited the survival rate, proliferation, migration, and invasion ability of A549 and H1299 cells, possibly by inducing ferroptosis.

MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34.

Lung adenocarcinoma (LUAD) represents the most prevalent subtype of lung cancer and typically has high incidence and fatality rates. Motor neuron and pancreas homeobox 1 (MNX1) and coiled-coil domain-containing 34 (CCDC34) serve as oncogenes in multiple types of cancer. However, their role in LUAD remains to be elucidated. In the present study, bioinformatics analysis and LUAD cell lines were adopted to examine the expression of MNX1 and CCDC34. The proliferation, migration and invasion abilities of A549 cells were determined using Cell Counting Kit-8, colony formation, wound-healing and Transwell assay, and flow cytometry was conducted to assess cell cycle distribution and apoptosis. The interaction between MNX1 and CCDC34 was verified by luciferase reporter and chromatin immunoprecipitation assays. In addition, an in vivo animal model of LUAD was established for validation. The results demonstrated that both MNX1 and CCDC34 were upregulated in LUAD cell lines. MNX1 knockdown significantly suppressed cell proliferation, migration and invasion, hindered cell cycle progression and promoted cell apoptosis in vitro and inhibited tumor growth in vivo. However, the antitumor effect of MNX1 knockdown was weakened by simultaneous CCDC34 overexpression in vitro. In terms of mechanism, MNX1 was demonstrated to directly bind to the CCDC34 promoter and transcriptionally activate CCDC34 expression. In conclusion, the present study highlighted a critical role of the MNX1/CCDC34 axis in regulating LUAD progression, providing novel therapeutic targets for LUAD treatment.

Clinicopathological significance of unraveling mitochondrial pathway alterations in non-small-cell lung cancer.

Early detection, accurate monitoring, and therapeutics are major problems in non-small-cell lung cancer (NSCLC) patients. We identified genomic copy number variation of a unique panel of 40 mitochondria-targeted genes in NSCLCs (GEOGSE #29365). Validation of mRNA expression of these molecules revealed an altered panel of 34 genes in lung adenocarcinomas (LUAD) and 36 genes in lung squamous cell carcinomas (LUSC). In the LUAD subtype (n = 533), we identified 29 upregulated and 5 downregulated genes, while in the LUSC subtype (n = 502), a panel of 30 upregulated and 6 downregulated genes were discovered. The majority of these genes are associated with mitochondrial protein transport, ferroptosis, calcium signaling, metabolism, OXPHOS function, TCA cycle, apoptosis, and MARylation. Altered mRNA expression of SLC25A4, ACSF2, MACROD1, and GCAT was associated with poor survival of the NSCLC patients. Progressive loss of SLC25A4 protein expression was confirmed in NSCLC tissues (n = 59), predicting poor survival of the patients. Forced overexpression of SLC25A4 in two LUAD cell lines inhibited their growth, viability, and migration. A significant association of the altered mitochondrial pathway genes with LC subtype-specific classical molecular signatures was observed, implicating the existence of nuclear-mitochondrial cross-talks. Key alteration signatures shared between LUAD and LUSC subtypes including SLC25A4, ACSF2, MACROD1, MDH2, LONP1, MTHFD2, and CA5A could be helpful in developing new biomarkers and therapeutics.

Effect of circ_0000009 on lung adenocarcinoma progression by regulating PDZD2 in a ceRNA- and RBP- dependent manner

Accumulating evidence now demonstrated that circular RNAs (circRNAs) are closely related to the pathogenesis of lung adenocarcinoma (LUAD). Through GEO2R online analysis, we screened hsa_circ_0000009 (circ_0000009) from the GEO database (GSE158695), and its expression in LUAD cancer tissues and cell lines was detected by RT-qPCR. The looping structure of circ_0000009 was tested by RNase R and actinomycin D experiments. The changes of proliferation were tested by CCK-8 or EdU assay. And the changes of apoptosis in A549 and H1299 cells were measured via flow cytometry. The A549 BALB/c tumor model was established to evaluate the influence of circ_0000009 on LUAD cell growth in vivo. In addition, experiments connected with ceRNA direction (mainly including bioinformatics prediction and luciferase reporter assay) and RNA Binding Protein (RBP) direction (mainly including RNA pull-down assay, RIP assay and mRNA stability assay) were further developed to reveal the regulatory mechanism of circ_0000009. The gene and protein levels in this project were assessed by RT-qPCR and western blotting analysis, respectively. The data manifested that circ_0000009 was in low expression in LUAD. The in vitro and in vivo experiments threw light on that overexpression of circ_0000009 dramatically suppressing LUAD tumorigenesis. Mechanistically, circ_0000009 promoted the expression of PDZD2 by sponging miR-154-3p. Furthermore, circ_0000009 stabilized PDZD2 by recruiting IGF2BP2. This study illustrated the mechanism that overexpressing of circ_0000009 suppressed LUAD progression by upregulating PDZD2 expression, providing an original treatment direction for LUAD.

Ginsenoside Rg5 enhances the radiosensitivity of lung adenocarcinoma via reducing HSP90-CDC37 interaction and promoting client protein degradation

Ginsenoside Rg5 is a rare ginsenoside showing promising tumor-suppressive effects. This study aimed to explore its radio-sensitizing effects and the underlying mechanisms. Human lung adenocarcinoma cell lines A549 and Calu-3 were used for in vitro and in vivo analysis. Bioinformatic molecular docking prediction and following validation by surface plasmon resonance (SPR) technology, cellular thermal shift assay (CETSA), and isothermal titration calorimetry (ITC) were conducted to explore the binding between ginsenoside Rg5 and 90 kD heat shock protein alpha (HSP90α). The effects of ginsenoside Rg5 on HSP90-cell division cycle 37 (CDC37) interaction, the client protein stability, and the downstream regulations were further explored. Results showed that ginsenoside Rg5 could induce cell-cycle arrest at the G1 phase and enhance irradiation-induced cell apoptosis. It could bind to HSP90α with a high affinity, but the affinity was drastically decreased by HSP90α Y61A mutation. Co-immunoprecipitation (Co-IP) and ITC assays confirmed that ginsenoside Rg5 disrupts the HSP90-CDC37 interaction in a dose-dependent manner. It reduced irradiation-induced upregulation of the HSP90-CDC37 client proteins, including SRC, CDK4, RAF1, and ULK1 in A549 cell-derived xenograft (CDX) tumors. Ginsenoside Rg5 or MRT67307 (an IKKε/TBK1 inhibitor) pretreatment suppressed irradiation-induced elevation of the LC3-II/β ratio and restored irradiation-induced downregulation of p62 expression. In A549 CDX tumors, ginsenoside Rg5 treatment suppressed LC3 expression and enhanced irradiation-induced DNA damage. In conclusion, ginsenoside Rg5 may be a potential radiosensitizer for lung adenocarcinoma. It interacts with HSP90α and reduces the binding between HSP90 and CDC37, thereby increasing the ubiquitin-mediated proteasomal degradation of the HSP90-CDC37 client proteins.