Lung Squamous Cell Carcinoma Cells Research Articles

Tenascin C as a novel zinc finger protein 750 target regulating the immunogenicity via DNA damage in lung squamous cell carcinoma

Modulation of DNA damage repair in lung squamous cell carcinoma (LUSC) can result in the generation of neoantigens and heightened immunogenicity. Therefore, understanding DNA damage repair mechanisms holds significant clinical relevance for identifying targets for immunotherapy and devising therapeutic strategies. Our research has unveiled that the tumor suppressor zinc finger protein 750 (ZNF750) in LUSC binds to the promoter region of tenascin C (TNC), leading to reduced TNC expression. This modulation may impact the malignant behavior of tumor cells and is associated with patient prognosis. Additionally, single-cell RNA sequencing (scRNA-seq) of LUSC tissues has demonstrated an inverse correlation between ZNF750/TNC expression levels and immunogenicity. Manipulation of the ZNF750-TNC axis in vitro within LUSC cells has shown differential sensitivity to CD8+ cells, underscoring its pivotal role in regulating cellular immunogenicity. Further transcriptome sequencing analysis, DNA damage repair assay, and single-strand break analyses have revealed the involvement of the ZNF750-TNC axis in determining the preference for homologous recombination (HR) repair or non-homologous end joining (NHEJ) repair of DNA damage. with involvement of the Hippo/ERK signaling pathway. In summary, this study sheds light on the ZNF750-TNC axis's role in DNA damage repair regulation in LUSC, laying a groundwork for future translational research in immune cell therapy for LUSC.

TNIK inhibition sensitizes TNIK-overexpressing lung squamous cell carcinoma to radiotherapy.

Most patients with lung squamous cell carcinoma (LSCC) undergo chemotherapy, radiotherapy, and adjuvant immunotherapy for locally advanced disease. The efficacy of these treatments is still limited due to dose-limiting toxicity or locoregional recurrence. New combination approaches and targets such as actionable oncogenic drivers are needed to advance treatment options for LSCC patients. Moreover, other options for chemotherapy-ineligible patients are also limited. As such there is a critical need for the development of selective and potent chemoradiosensitizers for locally advanced LSCC. Here, we investigated inhibiting TRAF2 and NCK-interacting protein kinase (TNIK), which is amplified in 40% of LSCC patients, as a strategy to sensitize LSCC tumors to chemo- and radiotherapy. Employing a range of human LSCC cell lines and the TNIK inhibitor NCB-0846, we investigated the potential of TNIK as a chemo- and radiosensitizing target with in vitro and in vivo preclinical models. The combination of NCB-0846 with cisplatin or etoposide was at best additive. Interestingly, pre-treating LSCC cells with NCB-0846 prior to ionizing radiation (IR) potentiated the cytotoxicity of IR in a TNIK-specific fashion. Characterization of the radiosensitization mechanism suggested that TNIK inhibition may impair the DNA damage response and promote mitotic catastrophe in irradiated cells. In a subcutaneous xenograft in vivo model, pretreatment with NCB-0846 significantly enhanced the efficacy of IR and caused elevated necrosis in TNIKhigh LK2 tumors but not TNIKlow KNS62 tumors. Overall, these results indicate that TNIK inhibition may be a promising strategy to increase the efficacy of radiotherapy in LSCC patients with high TNIK expression.

Expression of lncRNA LINC00943 in lung squamous cell carcinoma and its relationship with tumor progression

BackgroundMolecular biology has been applied to the diagnosis, prognosis and treatment of various diseases, and long noncoding RNA LINC00943 (lncRNA LINC00943; LINC00943) plays an important role in a variety of cancers. Therefore, this study explored the prognostic role of LINC00943 in lung squamous cell carcinoma (LUSC) and understood its impact on the development of LUSC.MethodsThere are 89 LUSC patients were involved in current assay. By detecting the expression of LINC00943 and miR-196b-5p in tissues and cells, LINC00943 and its correlation with the characteristics of clinical data were analyzed. The biological function of LINC00943 was studied by Transwell migration and invasion assays. In addition, Pearson correlation coefficient and luciferase activity experiments were chosen to characterize the relationship between LINC00943 and miR-196b-5p and explore the mechanism of LINC00943.ResultsCompared with normal controls, LINC00943 expression in LUSC tissues and cells was significantly reduced, miR-196b-5p was markedly increased, there was a negative correlation between LINC00943 and miR-196b-5p. According to the in vitro cell experiments, migration and invasion of LUSC cells were suppressed by overexpression of LINC00943. Besides, LINC00943 was demonstrated to have prognostic power and targeting miR-196b-5p was involved in the progression of LUSC.ConclusionsOverexpression of LINC00943 was molecular sponge for miR-196b-5p that controlled the deterioration of LUSC, which had great potential as a prognostic biomarker for LUSC.

MiR-185-5p May Modulate the Chemosensitivity of LUSC to Cisplatin via Targeting PCDHA11: Multi-omics Analysis and Experimental Validation.

Drug resistance is the major difficulty in treatment of lung squamous cell carcinoma (LUSC). This study aims to explore drug response-related miRNAs (DRmiRNAs) based on multi-omics research. We identified DRmiRNAs of LUSC with a multi-omics integrated system that combines expression data of microRNA, lncRNA, mRNA, methylation levels, somatic mutations. After identifying DRmiRNAs, we screened and validated of the target mRNAs of DRmiRNAs through Targetscan and the miRDB database. Then, Real-time PCR and Western blot assays were used to estimate the expression of DRmiRNAs and target protein, and the dual-luciferase assays were used to confirm the interaction of DRmiRNAs and target mRNA. Furthermore, CCK-8 (Cell Counting Kit-8) assays were used to evaluate cell proliferation and drug sensitivity. After integrated analysis, hsa-miR-185-5p was identified as DRmiRNA based on multi-omics data. Through Targetscan and miRDB database, the possible target mRNAs were obtained and PCDHA11 was validated as a target mRNA of miR-185-5p by real-time PCR, Western blot assays and dual-luciferase assays. CCK-8 assays and clone formation assays showed that the proliferation of miR-185-5p mimics was significantly slower than that of miR-185-5p inhibitors, which means overexpression of miR-185-5p enhanced the anticancer effects of cisplatin, whereas the downregulation of miR-185-5p reduced the effects. Furthermore, the proliferation of silencing PCDHA11 was significantly slower than that of overexpression of PCDHA11, which means PCDHA11 overexpression weakened the anticancer effects of cisplatin, and silencing PCDHA11 enhanced the effects. This study demonstrated that miR-185-5p was involved in chemoresistance of LUSC cells to cisplatin partly via down-regulating PCDHA11, which may promote understanding the underlying molecular mechanisms of drug response.

Abstract 4040: Hypoxia-induced HIF-1α/USP3 pathway promoting immune escape in lung squamous cell carcinoma

Abstract Background: Immune escape poses a significant mechanism for tumor progress as well as enormous challenge for the treatment of lung squamous cell carcinoma (LUSC). The close association between hypoxic environments and tumor immune escape, with PD-L1 serving as a key marker, has garnered considerable attention. This study aims to delve into the molecular mechanisms underlying the regulation of PD-L1 deubiquitination by the hypoxia-induced HIF-1α/USP3 pathway, shedding light on the immune escape mechanisms in LUSC. Methods: NSCLC cells were cultured under hypoxia condition and the expression of USP3, HIF-1α and PD-L1 were measured by Western blot. Chromatin Immunoprecipitation (ChIP) and Co-immunoprecipitation (CO-IP) were employed to elucidate the direct regulatory mechanisms of the HIF-1α/USP3 pathway in PD-L1 deubiquitination. In vitro T cell-mediated killing assays was used to investigate the functional roles of USP3 in antitumor immunity in NSCLC. Results: We demonstrated that hypoxia score was increased in LUSC and negatively correlated with CD8+ T cell infiltration. Hypoxia as a major factor triggering LUSC cell immunosuppression and against T cell surveillance via stabilization of PD-L1. Mechanistically, USP3, induced by HIF-1α, is required for hypoxia-mediated PD-L1 stabilization in LUSC cells. USP3 inhibits the ubiquitination and degradation of PD-L1. The combination of USP3 inhibitors with PD-1/PD-L1 monoclonal antibodies significantly enhances the anti-LUSC effect, possibly through promoting the infiltration of CD8+ T and NK cells. Conclusion: This research unveils a novel mechanism by which hypoxia, through the HIF-1α/USP3 pathway, regulates PD-L1 deubiquitination, thereby promoting immune escape in LUSC. Furthermore, our study provides a new strategy involving USP3 inhibitors in combination with PD-1/PD-L1 monoclonal antibodies, offering novel insights for molecular targeted therapy and reversal of drug resistance in LUSC. Citation Format: Xiuwen Zhang, Hua Huang, Chen Ding, Chen Chen, Yongwen Li, Hongyu Liu, Chen Jun. Hypoxia-induced HIF-1α/USP3 pathway promoting immune escape in lung squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4040.

Abstract 4888: Multi-modal knowledge graphs enhance patient stratification & biomarker discovery

Abstract Background: Multi-modal analysis is crucial for deeper understanding of disease subtypes and more meaningful patient selection. We developed a flexible Knowledge Graph (KG) framework that enables deep multi-omic analysis. It can be used to uncover the interrelationships between the layers of data in a population to inform patient selection or biomarker discovery. We present an application of our framework to non-small cell lung cancer (NSCLC) to identify and separate communities of patients based on their survival and identify the associated biomarkers. We identified potentially mislabeled patients that do not share all the characteristics of the cancer subtype to which they are assigned: either lung adenocarcinoma (LUAD) or lung squamous cell carcinoma (LUSC). Crucially, the community-based biomarkers for poor or long survivors were validated on the whole population. Methods: Our KG framework was leveraged by applying supervised community detection to NSCLC data from TCGA, specifically RNA expression and DNA methylation, with overall survival (OS) as the endpoint (n=999 subjects). Biomarkers associated to each community were ranked based on their prevalence inside against their prevalence outside the community. Results: We obtained 3 communities (391 + 229 + 379 patients) that are all significantly separated by their OS (p<0.05). While KG-derived communities largely overlapped with histology-labelled LUAD or LUSC (concordant LUAD (n=330)/concordant LUSC (n=342)), a small number of patients did not (discordant LUAD (n=37)/discordant LUSC (n=61)). Discordant LUAD patients had significantly lower OS than concordant LUAD (p=0.0198), despite being both labeled as LUAD. Many of the discordant LUSC lacked the 3q26 amplification commonly seen in LUSC and other squamous cell carcinomas. The results from our KG framework highlight its increased sensitivity in relation to existing tools (Cline, Sci Rep 3, 2652 (2013)) as we identified a discordant LUAD group in addition to a discordant LUSC group. Moreover, our tool can select the biomarkers most prevalent in each community, and these significantly separated long from poor survivors on the whole population (p=3.53e-5). Some of these KG-identified biomarkers are known regulators of progression and survival in NSCLC, whereas others are still not extensively studied. This highlights another advantage of our approach in prospective target discovery. Conclusions: Our KG framework allowed the observation of potential misclassifications of tumor subtypes in NSCLC (TCGA). This approach is a proof-of-concept of the value KGs have in identifying signals in muti-omic data that may improve patient stratification and uncover associated biomarker signatures. Our flexible end-to-end framework can take any type of ‘omic data and can be applied to any tumor type for such findings, which represents an alternative to pre-defined KG architectures with defined relationships. Citation Format: Miguel Goncalves, Jake Cohen-Setton, Ioannis Kagiampakis, Ben Sidders, Krishna Bulusu. Multi-modal knowledge graphs enhance patient stratification & biomarker discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4888.

HIF1A-dependent overexpression of MTFP1 promotes lung squamous cell carcinoma development by activating the glycolysis pathway

IntroductionMitochondrial fission process 1 (MTFP1) is an inner mitochondrial membrane (IMM) protein implicated in the development and progression of various tumors, particularly lung squamous cell carcinoma (LUSC). This study aims to provide a more theoretical basis for the treatment of LUSC. MethodsThrough bioinformatics analysis, MTFP1 was identified as a novel target gene of HIF1A. MTFP1 expression in LUSC was examined using The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and Proteomics Data Commons (PDC) databases. The Kaplan-Meier plotter (KM plotter) database was utilized to evaluate its correlation with patient survival. Western blot and chromatin immunoprecipitation (ChIP) assays were employed to confirm the regulatory relationship between MTFP1 and HIF1A. Additionally, cell proliferation, colony formation, and migration assays were conducted to investigate the mechanism by which MTFP1 enhances LUSC cell proliferation and metastasis. ResultsOur findings revealed that MTFP1 overexpression correlated with poor prognosis in LUSC patients(P < 0.05). Moreover, MTFP1 was closely associated with hypoxia and glycolysis in LUSC (R = 0.203; P < 0.001, R = 0.391; P < 0.001). HIF1A was identified as a positive regulator of MTFP1. Functional enrichment analysis demonstrated that MTFP1 played a role in controlling LUSC cell proliferation. Cell proliferation, colony formation, and migration assays indicated that MTFP1 promoted LUSC cell proliferation and metastasis by activating the glycolytic pathway (P < 0.05). ConclusionsThis study establishes MTFP1 as a novel HIF1A target gene that promotes LUSC growth by activating the glycolytic pathway. Investigating MTFP1 may contribute to the development of effective therapies for LUSC patients, particularly those lacking targeted oncogene therapies.

LncRNA DGUOK-AS1 Promotes Cell Progression in Lung Squamous Cell Carcinoma by Regulation of miR-653-5p/SLC6A15 Axis.

Long noncoding RNA (lncRNA) plays a key role in regulating cancer development. LncRNA deoxyguanosine kinase antisense RNA 1 (DGUOK-AS1) has been reported as a promoter in tumor. The work was designed to further investigate the mechanism of action of DGUOK-AS1 in lung squamous cell carcinoma (LUSC). DGUOK-AS1 level in LUSC cells was measured using RT-qPCR. Counting Kit-8 assays and colony forming assays were performed to evaluate LUSC cell viability and proliferation. Transwell assays were performed to detect cell migration and invasion. Luciferase reporter and RNA pulldown assays were used to verify the binding capacity of DGUOK-AS1 and miR-653-5p. RNA immunoprecipitation assays were performed to verify the relationship of DGUOK-AS1, miR-653-5p, and SLC6A15. DGUOK-AS1 was highly expressed in LUSC cells. DGUOK-AS1 knockdown suppressed LUSC cell proliferation, migration, and invasion. SLC6A15 was demonstrated to be targeted by miR-653-5p, and DGUOK-AS1 interacted with miR-653-5p to modulate SLC6A15 level in LUSC cells. Overexpression of SLC6A15 reversed the suppressive effects of DGUOK-AS1 knockdown on LUSC cell processes. In conclusion,DGUOK-AS1 promotes malignant behaviors of LUSC cells by upregulating SLC6A15 level through interaction with miR-653-5p.

Novel nanoparticle AuNCs conjugated with Desmoglein-3 antibody for FL/CT dual-mode targeted imaging and precise treatment of lung squamous cell carcinoma

Due to lack of effective, early and non-invasive diagnostic as well as treatment tools, the surgical treatment opportunities for lung squamous cell carcinoma (SCC) are limited, resulting in high mortality rates. Therefore, the combination of targeted recognition and precise treatment of lung SCC is of great significance. In this study, a multifunctional nanoparticle is designed and synthesized, which specifically identifies lung SCC cells for target imaging and therapy. Desmoglein-3 (Dsg-3), a transmembrane glycoprotein found in desmosomes, is highly expressed in lung SCC cells. Gold nanoclusters (AuNCs) conjugated with Dsg-3 antibodies to form Au-Dsg-3 through coupling reaction. The results showed that the fluorescence imaging (FI) intensity and computed tomography (CT) signal of Au-Dsg-3 significantly increased within 6 h in vitro and in vivo, achieving dual-modal imaging to detect lung SCC effectively. Besides, Au-Dsg-3 even integrates targeted photothermal therapy (PTT) characteristics in a single nanoparticle. When exposed to near-infrared radiation (NIR), the temperature of the tumor site increased rapidly and reached a high temperature of 53.3 °C after 600 s, causing tumor ablation and growth inhibition. In summary, Au-Dsg-3 provides a key platform for targeted biological imaging and collaborative PTT, which demonstrates good performance on lung SCC.

DNA-PK-Mediated Cytoplasmic DNA Sensing Stimulates Glycolysis to Promote Lung Squamous Cell Carcinoma Malignancy and Chemoresistance.

DNA-PK is a cytoplasmic DNA sensor that activates ZAK/AKT/mTOR signaling and boosts glycolysis to enhance malignancy and chemoresistance of lung squamous cell carcinoma.

Deubiquitylase USP31 Induces Autophagy and Promotes the Progression in Lung Squamous Cell Carcinoma Cells by Stabilizing E2F1 Expression.

Autophagy exerts a vital role in the progression of lung squamous cell carcinoma (LUSC). Ubiquitin-specific peptidase 31 (USP31) has recently been found to be involved in the development of a variety of cancers. However, whether USP31 modulates autophagy in LUSC remains unclear. This study revealed that high levels of USP31 were discovered in LUSC tissue samples employing the Gene Expression Profiling Interactive Analysis (GEPIA) database, quantitative real- time PCR (qRT-PCR), and Western blot analysis. Cell proliferation was tested via cell counting kit 8 (CCK-8) as well as colony formation, demonstrating that USP31-stable knockdown reduced cell viability. Immunofluorescence analysis illustrated that USP31 knockdown blocked the occurrence of LUSC autophagy. Meanwhile, USP31 has been shown to stabilize the expression of E2F transcription factor 1 (E2F1) through the proteasome pathway. Furthermore, overexpressed E2F1 effectively eliminated the effect of USP31 knockdown on LUSC cell proliferation and autophagy. In summary, this investigation proved that USP31 promoted LUSC cell growth and autophagy, at least in part by stabilizing E2F1 expression, which provided a potential therapeutic gene for the treatment of LUSC.

Effects of methionine deficiency on B7H3-DAP12-CAR-T cells in the treatment of lung squamous cell carcinoma

Lung squamous cell carcinoma (LUSC) is a subtype of lung cancer for which precision therapy is lacking. Chimeric antigen receptor T-cells (CAR-T) have the potential to eliminate cancer cells by targeting specific antigens. However, the tumor microenvironment (TME), characterized by abnormal metabolism could inhibit CAR-T function. Therefore, the aim of this study was to improve CAR-T efficacy in solid TME by investigating the effects of amino acid metabolism. We found that B7H3 was highly expressed in LUSC and developed DAP12-CAR-T targeting B7H3 based on our previous findings. When co-cultured with B7H3-overexpressing LUSC cells, B7H3-DAP12-CAR-T showed significant cell killing effects and released cytokines including IFN-γ and IL-2. However, LUSC cells consumed methionine (Met) in a competitive manner to induce a Met deficiency. CAR-T showed suppressed cell killing capacity, reduced cytokine release and less central memory T phenotype in medium with lower Met, while the exhaustion markers were up-regulated. Furthermore, the gene NKG7, responsible for T cell cytotoxicity, was downregulated in CAR-T cells at low Met concentration due to a decrease in m5C modification. NKG7 overexpression could partially restore the cytotoxicity of CAR-T in low Met. In addition, the anti-tumor efficacy of CAR-T was significantly enhanced when co-cultured with SLC7A5 knockdown LUSC cells at low Met concentration. In conclusion, B7H3 is a prospective target for LUSC, and B7H3-DAP12-CAR-T cells are promising for LUSC treatment. Maintaining Met levels in CAR-T may help overcome TME suppression and improve its clinical application potential.

Identification of tumor-suppressor genes in lung squamous cell carcinoma through integrated bioinformatics analyses.

Lung cancer is a prevalent malignancy, and fatalities of the disease exceed 400,000 cases worldwide. Lung squamous cell carcinoma (LUSC) has been recognized as the most common pathological form of lung cancer. The comprehensive understanding of molecular features related to LUSC progression has great significance in LUSC prognosis assessment and clinical management. In this study, we aim to identify a panel of signature genes closely associated with LUSC, which can provide novel insights into the progression of LUSC. Gene expression profiles were retrieved from public resources including gene expression omnibus (GEO) and the cancer genome atlas (TCGA) database. Differentially expressed genes (DEGs) between LUSC specimens and normal lung tissues were identified by bioinformatics analyses. A total of 66 DEGs were identified based on two cohorts of data. CytoHubba plugin of Cytoscape software was utilized for the further analyses of the top 10 candidate hub genes including OGN, ABI3BP, MAMDC2, FGF7, FAM107A, SPARCL1, DCN, COL14A1, and MFAP4 and CHRDL1, which showed significant downregulation in LUSC. Two LUSC cell lines were used to validate the functions of CHRDL1 and FAM107A through overexpression experiment. Together, our data revealed novel candidate tumor-suppressor genes in LUSC, suggesting previously unappreciated mechanisms in the progression of LUSC.

C-Rel is a Novel Oncogene in Lung Squamous Cell Carcinoma Regulating Cell Proliferation and Migration.

Lung squamous cell carcinoma (LUSC) accounts for approximately 25% to 30% of lung cancers, but largely no targeted therapy is available against it, calling for identification of new oncogenes in LUSC growth for new therapeutic targets. In this study, REL was identified through a screening for oncogenes that are highly amplified in human LUSC. Its expression was associated with poor prognosis in LUSC patients. Furthermore, knockdown of c-Rel in LUSC cell lines lead to significant decrease in cell proliferation and migration. Mechanistically, c-Rel knockdown suppressed NFκB pathway by blocking phosphorylation of IκB. Consistently, pharmaceutic inhibition of c-Rel also. In orthotopic xenograft lung cancer mouse model, c-Rel knockdown inhibited the tumor growth. Cancer cell proliferation and epithelial-mesenchymal-transition (EMT) of the tumors were impaired by c-Rel knockdown. Finally, it's confirmed in precision-cut tumor slices of LUSC that deletion of c-Rel inhibits the NFκB pathway and cancer cell growth. Accordingly, we hypothesize that c-Rel promotes the activation of the NFκB pathway by promoting the phosphorylation of IκB in LUSC. Our study reveals REL as a novel LUSC oncogene and provides new insights into the molecular regulation of LUSC, which will provide new therapeutic targets for the treatment of squamous lung cancer.

Acrolein suppresses anticancer drug-induced toxicity mediated by activating claudin-1 and Nrf2 axis in a spheroid model of human lung squamous cell carcinoma cells

Tobacco smoke contains various carcinogenic ingredients such as nicotine, acrolein, and benzopyrene; however, their effects on cancer treatment are not fully understood. Claudin-1 (CLDN1), a component of tight junctions, is involved in the increased resistance to anticancer drugs. In this study, we found that acrolein increases the mRNA and protein levels of CLDN1 in RERF-LC-AI cells derived from human lung squamous cell carcinoma (SCC). Acrolein increased the p-extracellular signal-regulated kinase (ERK) 1/2 levels without affecting the p-Akt level. The acrolein-induced elevation of CLDN1 expression was attenuated by U0126, a mitogen-activated protein kinase kinas (MEK) inhibitor. These results indicate that the activation of MEK/ERK pathway is involved in the acrolein-induced elevation of CLDN1 expression. In a spheroid model, acrolein suppressed the accumulation and toxicity of doxorubicin (DXR), which were rescued by CLDN1 silencing. The acrolein-induced effects were also observed in lung SCC-derived EBC-1 and LK-2 cells. Acrolein also increased the expression level of nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor that regulates antioxidant and detoxifying genes, which were inhibited by CLDN1 silencing. In spheroid cells, the levels of reactive oxygen species were enhanced by acrolein, which was inhibited by CLDN1 silencing. Taken together, acrolein may reduce the anticancer drug-induced toxicity in human lung SCC cells mediated by high CLDN1 expression followed by the upregulation of Nrf2 signaling pathway.

Development of an Anoikis-Related Gene Signature for Lung Squamous Cell Carcinomato Predict Prognosis, Immune Landscape, and Immunotherapy Response

Background. Anoikis, a form of programed cell death, plays a pivotal role in the invasion and metastasis of various tumors, including lung squamous cell carcinoma (LUSC). This study aims to construct a prognostic model for LUSC, leveraging anoikis-related genes (ARGs). Methods. A total of 357 ARGs were extracted from the GeneCards database and Harmonizome portals. Subsequently, ARGs influencing LUSC patients’ prognosis were identified using univariate Cox regression analysis. Unsupervised clustering analysis was carried out utilizing the “consensusplus” R package, and LASSO regression was deployed to craft a risk regression model. The ‘IBOR’ R package quantified the immune cell infiltration abundance. Moreover, the “maftools” R package, paired with the GISTIC online tool, facilitated the assessment of gene copy number variations. Experimental validation was conducted through RT-PCR, evaluating the differential expression of eight pivotal genes, and cellular functional assays discerned the influences of the CHEK2 and SDCBP genes on LUSC cells’ migratory and invasive capabilities. Results. Fifteen survival-associated ARGs delineated three molecular subtypes within the TCGA-LUSC cohort. An eight ARG-based risk prognostic model was constructed, delineating significant survival disparities between high and low-risk groups. Notably, the low-risk group manifested a diminished propensity for immune therapy evasion and gene mutations. A comprehensive nomogram, incorporating risk scores and clinical attributes, was fashioned, exemplifying remarkable predictive acumen. Cellular functional assays substantiated that the modulation of CHEK2 and SDCBP expressions conspicuously influenced the migratory and invasive propensities of LUSC cells. Conclusions. This rigorous study unveils novel anoikis-related biomarkers integral to LUSC prognostication. The meticulously constructed risk prognostic model, underscored by these biomarkers, augurs a potent predictive tool for enhancing the LUSC patient prognosis and therapeutic strategies.

LINC01936 inhibits the proliferation and metastasis of lung squamous cell carcinoma probably by EMT signaling and immune infiltration

Purpose To discover the biological function and potential mechanism of LINC01936 in the development of lung squamous cell carcinoma (LUSC). Methods Transcriptome data of LUSC from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were used to analyze the differentially expressed lncRNAs in LUSC and normal tissues by R “DEseq2”, “edgeR” and “limma” packages. The subcellular localization of LINC01936 was predicted by lncLocator. Cell proliferation and apoptosis were measured by CCK-8, MTT assay and Hoechst fluorescence staining. The migration and invasion were detected by Transwell assay. The function and pathway enrichment analysis were performed by Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and gene set variation analysis (GSVA). The downstream targets of LINC01936 were predicted using RNA-Protein Interaction Prediction (RPISeq) program. The effect of LINC01936 on tumor immune infiltration was analyzed using Pearson Correlation Analysis using R “ggpubr” package. Results Based on the gene expression data of LUSC from TCGA database, 1,603, 1,702 and 529 upregulated and 536, 436 and 630 downregulated lncRNAs were obtained by DEseq2, edgeR and limma programs, respectively. For GSE88862 dataset, we acquired 341 differentially expressed lncRNAs (206 upregulated and 135 downregulated). Venn plot for the intersection of above differential expressed lncRNAs showed that there were 29 upregulated and 23 downregulated genes. LINC01936 was one of downregulated lncRNAs in LUSC tissues. The biological analysis showed that the overexpression of LINC01936 significantly reduced proliferation, migration and invasion of LUSC cells, and promoted cell apoptosis. The knockdown of LINC01936 promoted cell proliferation and metastasis. Pathway and GSVA analysis indicated that LINC01936 might participated in DNA repair, complement, cell adhesion and EMT, etc. LINC01936 was predicted to interact with TCF21, AOC3, RASL12, MEOX2 or HSPB7, which are involved in EMT and PI3K-AKT-MTOR pathway, etc. The expression of LINC01936 was also positively correlated with the infiltrating immune cells in LUSC. Conclusions LINC01936 is downregulated in LUSC. LINC01936 affected proliferation, migration and invasion of LUSC cells probably by EMT and immune infiltration, which might serve as a new target for the treatment of LUSC.

Characterization and verification of CD81 as a potential target in lung squamous cell carcinoma

CD81 is a cell surface transmembrane protein of the tetraspanin family, which critically regulates signal transduction and immune response. Growing evidence has shown that CD81 plays important roles in tumorigenesis and influences immunotherapy response. Here, combining bio-informatics and functional analysis, we find that CD81 is a risk factor in lung squamous cell carcinoma (LUSC), whereas a protective factor in lung adenocarcinoma. In LUSC with high expression of CD81, the autophagy and JAK-STAT signaling pathway are activated. Meanwhile, the expression level of CD81 is negatively correlated with tumor mutational load (TMB), microsatellite instability (MSI), and neoantigen (NEO). Furthermore, patients with LUSC and high expression of CD81 do not respond to immunotherapy drugs, but can respond to chemotherapy drugs. Importantly, depletion of CD81 suppresses the proliferation of LUSC cell, and enhances the sensitivity to cisplatin. Our findings suggest that CD81 represents a potential target for cisplatin-based chemotherapy in patients with LUSC.

Molecular characterization of STEAP3 in lung squamous cell carcinoma: Regulating EGFR to affect cell proliferation and ferroptosis

Six-transmembrane epithelial antigen of the prostate 3 (STEAP3) has been reported to play a regulatory role in various types of cancers. However, its involvement in lung squamous cell carcinoma (LUSC) remains understudied. Here, we aimed to explore the biological functions and underlying mechanisms of STEAP3 in LUSC. Intersection genes associated with LUSC and ferroptosis were analyzed using the Venn method, STRING, GEPIA and UALCAN databases. The expression of STEAP3 was detected by qPCR and western blotting assay. Cell proliferation and viability were determined using the cell counting kit-8 assay and EDU staining. Oxidative stress and lipid peroxidation were measured by corresponding kits and DCFH-DA staining. Ferroptosis was evaluated by Phen Green SK and Western blot assay. The correlation between STEAP3 and EGFR was predicted by the TIMER and starBase database. Co-immunoprecipitation was conducted to verify the binding of STEAP3 and EGFR. The data demonstrated a significant upregulation of STEAP3 expression in LUSC cell lines. Silencing of STEAP3 suppressed H2170 cell viability and proliferation while promoting oxidative stress and lipid peroxidation through increased levels of MDA and ROS, as well as inhibited SOD activity. In addition, knockdown of STEAP3 induced ferroptosis through the regulation of ferroptosis-related proteins. Moreover, the binding between STEAP3 and EGFR was predicted and confirmed in LUSC. EGFR overexpression reversed the effects of STEAP3 silencing on H2170 cell viability, proliferation, oxidative stress, and ferroptosis. To summarize, the inhibition of STEAP3/EGFR may serve as a promising therapeutic target for LUSC treatment, as it can suppress LUSC proliferation and promote lipid peroxidation and ferroptosis.

TSTA3 overexpression promotes malignant characteristics in LUSC by regulating LAMP2-mediated autophagy and tumor microenvironment

BackgroundTSTA3 gene encoding GDP-l-fucose synthase has recently been proved to be closely related to the prognosis of patients with various tumors. However, its role in lung cancer is still unclear. The purpose of this study is to explore the expression level, prognostic effect, potential function and mechanism of TSTA3 in lung cancer.MethodsBased on TCGA database, Kaplan–Meier and COX regression was used to analyze the relationship between TSTA3 expression and prognosis of lung cancer patients. Immunohistochemistry was used to determine the TSTA3 protein expression in lung cancer and normal tissues. The function of TSTA3 in lung squamous cell carcinoma (LUSC) cell was determined by CCK8, colony formation, transwell assay in vitro and subcutaneous xenografts in vivo. Transcriptome analysis, Lyso-Tracker Red staining and rescue experiment were used to explore the possible underlying mechanism.ResultsThe expression of TSTA3 was significantly increased in lung cancer, especially in LUSC, and was significantly correlated with the malignant characteristics of LUSC. COX regression analysis showed that the high expression of TSTA3 was an independent prognostic factor in LUSC patients. This was also confirmed by immunohistochemical staining. Compared with the control group, the proliferation, colony formation, invasion and migration ability of LUSC cells with TSTA3 overexpression was enhanced. Similarly, the ability of cell proliferation, colony formation, invasion and migration were weakened after transient knockdown of TSTA3. In vivo experiment showed that compared with control group, TSTA3 overexpression significantly promoted the growth of tumor and shortened survival time. In addition, transcriptome sequencing analysis showed that the differentially expressed genes between TSTA3 overexpression and control group was mainly concentrated in the lysosome pathway. Further study found that TSTA3 might affect the proliferation, invasion and migration of LUSC by regulating the expression of lysosome-associated membrane protein 2 (LAMP2) in LUSC.ConclusionThe expression level of TSTA3 in LUSC is significantly higher than that in normal tissues. High expression of TSTA3 is associated with poor prognosis of LUSC patients. TSTA3 may affect the proliferation, invasion and migration of LUSC by regulating LAMP2.