Progression Of Lung Squamous Cell Carcinoma Research Articles

PAK2 promotes proliferation, migration, and invasion of lung squamous cell carcinoma through LIMK1/cofilin signaling pathway.

Although the p21-activated kinase 2 (PAK2) is an essential serine/threonine protein kinase, its role in lung squamous cell carcinoma (LUSC) progression has yet to be fully understood. We analyzed PAK2 mRNA levels and DNA copy numbers as well as protein levels by quantitative real-time PCR and immunohistochemical staining, respectively, in human LUSC tissues and adjacent normal tissues. Then, we used colony formation assays, cell counting kit-8 assays, matrigel invasion assays, wound healing assays and xenograft models in nude mice to investigate the functions of PAK2 in LUSC progression. We demonstrated that the mRNA levels, DNA copy numbers, and protein levels of PAK2 were up-regulated in human LUSC tissues than in adjacent normal tissues. In addition, a higher PAK2 expression was correlated with a poorer prognosis in LUSC patients. In the in vitro study, we found that PAK2 promoted cell growth, migration, invasion, EMT process, and cell morphology regulation in LUSC cells. Furthermore, PAK2 enhanced tumor cell proliferation, migration, and invasion by regulating actin dynamics through the LIMK1/cofilin signaling. Our findings implicated that the PAK2/LIMK1/cofilin signaling pathway is likely a potential clinical marker and therapeutic target for LUSC.

Diagnostic Value of GDF10 for the Tumorigenesis and Immune Infiltration in Lung Squamous Cell Carcinoma.

Lung squamous cell carcinoma (LUSC) is associated with a low survival rate. Evidence suggests that bone morphogenetic proteins (BMPs) and their receptors (BMPRs) play crucial roles in tumorigenesis and progression. However, a comprehensive analysis of their role in LUSC is lacking. Our study aimed to explore the relationship between BMPs/BMPRs expression levels and the tumorigenesis and prognosis of LUSC. The "R/Limma" package was utilized to analyze the differential expression characteristics of BMPs/BMPRs in LUSC, using data from TCGA, GTEx, and GEO databases. Concurrently, the "survminer" packages were employed to investigate their prognostic value and correlation with clinical features in LUSC. The core gene associated with LUSC progression was further explored through weighted gene correlation network analysis (WGCNA). LASSO analysis was conducted to construct a prognostic risk model for LUSC. Clinical specimens were examined by immunohistochemical analysis to confirm the diagnostic value in LUSC. Furthermore, based on the tumor immune estimation resource database and tumor-immune system interaction database, the role of the core gene in the tumor microenvironment of LUSC was explored. GDF10 had a significant correlation only with the pathological T stage of LUSC, and the protein expression level of GDF10 decreased with the tumorigenesis of LUSC. A prognostic risk model was constructed with GDF10 as the core gene and 5 hub genes (HRASLS, HIST1H2BH, FLRT3, CHEK2, and ALPL) for LUSC. GDF10 showed a significant positive correlation with immune cell infiltration and immune checkpoint expression. GDF10 might serve as a diagnostic biomarker reflecting the tumorigenesis of LUSC and regulating the tumor immune microenvironment to guide more effective treatment for LUSC.

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.

The upregulation of TGM2 is associated with poor prognosis and the shaping of the inflammatory tumor microenvironment in lung squamous cell carcinoma.

Tissue transglutaminase (TGM2) is a member of the glutamine transferase superfamily, located within cells and their membranes. When secreted, it catalyzes the cross-linking of extracellular matrix proteins and promotes the formation of extracellular matrix scaffolds. To determine the function of TGM2 in the tumorigenesis of lung squamous cell carcinoma (LUSC), we conducted a comprehensive bioinformatics analysis of TGM2. Our findings indicate that high expression of TGM2 in LUSC was associated with a poorer prognosis. Additionally, we found that high expression of TGM2 is closely related to tumor-promoting inflammation and may increase sensitivity to immunotherapy. We further confirmed the cancer-promoting effect of TGM2 in LUSC through in vitro overexpression and knockdown experiments and showed that TGM2 primarily affects cancer cell proliferation, apoptosis, and invasion. In summary, TGM2 promoted the progression of LUSC, and targeting TGM2 is expected to become a new therapeutic approach for LUSC treatment.

GPC3 Promotes Lung Squamous Cell Carcinoma Progression and HLA-A2-Restricted GPC3 Antigenic Peptide-Modified Dendritic Cell-Induced Cytotoxic T Lymphocytes to Kill Lung Squamous Cell Carcinoma Cells

Lung squamous cell carcinoma (LUSC) is associated with poor clinical prognosis and lacks available targeted agents. GPC3 is upregulated in LUSC. Our study aimed to explore the roles of GPC3 in LUSC and the antitumor effects of HLA-A2-restricted GPC3 antigenic peptide-sensitized dendritic cell (DC)-induced cytotoxic T lymphocytes (CTLs) on LUSC. LUSC cells with GPC3 knockdown and overexpression were built using lentivirus packaging, and cell viability, clone formation, apoptosis, cycle, migration, and invasion were determined. Western blotting was used to detect the expression of cell cycle-related proteins and PI3K-AKT pathway-associated proteins. Subsequently, HLA-A2-restricted GPC3 antigenic peptides were predicted and synthesized by bioinformatic databases, and DCs were induced and cultured in vitro. Finally, HLA-A2-restricted GPC3 antigenic peptide-modified DCs were co-cultured with T cells to generate specific CTLs, and the killing effects of different CTLs on LUSC cells were studied. A series of cell function experiments showed that GPC3 overexpression promoted the proliferation, migration, and invasion of LUSC cells, inhibited their apoptosis, increased the number of cells in S phase, and reduced the cells in G2/M phase. GPC3 knockdown downregulated cyclin A, c-Myc, and PI3K, upregulated E2F1, and decreased the pAKT/AKT level. Three HLA-A2-restricted GPC3 antigenic peptides were synthesized, with GPC3522-530 FLAELAYDL and GPC3102-110 FLIIQNAAV antigenic peptide-modified DCs inducing CTL production, and exhibiting strong targeted killing ability in LUSC cells at 80 : 1 multiplicity of infection. GPC3 may advance the onset and progression of LUSC, and GPC3522-530 FLAELAYDL and GPC3102-110 FLIIQNAAV antigenic peptide-loaded DC-induced CTLs have a superior killing ability against LUSC cells.

Pre-clinical lung squamous cell carcinoma mouse models to identify novel biomarkers and therapeutic interventions.

Primary lung carcinoma or lung cancer (LC) is classified into small-cell or non-small-cell (NSCLC) lung carcinoma. Lung squamous cell carcinoma (LSCC) is the second most common subtype of NSCLC responsible for 30% of all LCs, and its survival remains low with only 24% of patients living for five years or longer post-diagnosis primarily due to the advanced stage of tumors at the time of diagnosis. The pathogenesis of LSCC is still poorly understood and has hampered the development of effective diagnostics and therapies. This review highlights the known risk factors, genetic and epigenetic alterations, miRNA biomarkers linked to the development and diagnosis of LSCC and the lack of therapeutic strategies to target specifically LSCC. We will also discuss existing animal models of LSCC including carcinogen induced, transgenic and xenograft mouse models, and their advantages and limitations along with the chemopreventive studies and molecular studies conducted using them. The importance of developing new and improved mouse models will also be discussed that will provide further insights into the initiation and progression of LSCC, and enable the identification of new biomarkers and therapeutic targets.

THOC3 interacts with YBX1 to promote lung squamous cell carcinoma progression through PFKFB4 mRNA modification

The THO complex (THOC) is ubiquitously involved in RNA modification and various THOC proteins have been reported to regulate tumor development. However, the role of THOC3 in lung cancer remains unknown. In this study, we identified that THOC3 was highly expressed in lung squamous cell carcinoma (LUSC) and negatively associated with prognosis. THOC3 knockdown inhibited LUSC cell growth, migration, and glycolysis. THOC3 expression was regulated by TRiC proteins, such as CCT8 and CCT6A, which supported protein folding. Furthermore, THOC3 could form a complex with YBX1 to promote PFKFB4 transcription. THOC3 was responsible for exporting PFKFB4 mRNA to the cytoplasm, while YBX1 ensured the stability of PFKFB4 mRNA by recognizing m5C sites in its 3′UTR. Downregulation of PFKFB4 suppressed the biological activities of LUSC. Collectively, these findings suggest that THOC3, folded by CCT proteins can collaborate with YBX1 to maintain PFKFB4 expression and facilitate LUSC development. Therefore, THOC3 could be considered as a novel promising therapeutic target for LUSC.

Downregulation of microRNA‑494 inhibits cell proliferation in lung squamous cell carcinoma via the induction of PUMA‑α‑mediated apoptosis.

Increased evidence has shown that abnormal microRNA (miRNA) plays pivotal roles in numerous types of cancer. However, their expression, function and mechanism in lung squamous cell carcinoma (LSCC) remains to be fully elucidated. The aim of the present study was to investigate the suppressive role of miR-494 in LSCC progression and elucidate its regulatory mechanism. By analyzing expression profiles of miRNAs in LSCC tissues using miRNA microarray, it was revealed that miR-494 was significantly upregulated in 22 pairs of LSCC tissues. Subsequently, reverse transcription-quantitative PCR was performed to determine the expression of miR-494 and p53-upregulated-modulator-of-apoptosis-α (PUMA-α). Western blot analysis was conducted to examine protein levels. Dual-luciferase reporter assay was used to confirm the binding between miR-494 and PUMA-α. Annexin V-fluoresceine isothiocyanate/propidium iodide staining and CCK-8 assays were employed to determine cell apoptosis and cell viability, respectively. It was also revealed that miR-494 was highly expressed in LSCC cell lines compared with that in 16HBE cells. Further experiments confirmed that knockdown of miR-494 reduced cell viability and induced LSCC apoptosis. Bioinformatics analysis predicted that miR-494 could potentially target PUMA-α; also known as Bcl-2-binding component 3, a pro-apoptotic factor, and an inverse correlation between the expression of miR-494 and PUMA-α mRNA levels in LSCC tissues was found. Furthermore, PUMA-α inhibition could reverse the promoting effect of miR-494 knockdown on apoptosis in LSCC cells. Taken together, these findings demonstrated that miR-494 functions as an oncogene by targeting PUMA-α in LSCC, and miR-494 may serve as a novel therapeutic target for treating LSCC.

Reciprocal FGF19-GLI2 signaling induces epithelial-to-mesenchymal transition to promote lung squamous cell carcinoma metastasis.

Metastatic lung squamous cell carcinoma (LUSC) is one of the most common causes of cancer death worldwide. As yet, however, the molecular mechanism underlying LUSC metastasis remains elusive. In this study, we report a novel mechanism involving signaling interactions between FGF19 and GLI2 that could drive the progression of LUSC. The expression of FGF19 in human LUSC samples was assessed by immunohistochemistry. The concentration of FGF19 in serum samples was assessed by ELISA. RNA sequencing, scratch wound-healing, trans-well, GO analysis, GSEA, luciferase reporter, Western blotting, immunofluorescence and immunohistochemistry assays, as well as an animal model were used to investigate the molecular mechanism underlying FGF19 driven LUSC progression. The therapeutic effect of a GLI2 inhibitor was determined using both in vitro cellular and in vivo animal experiments. We found that FGF19, a member of the fibroblast growth factor family, plays a crucial role in the invasion and metastasis of LUSC, and identified GLI2 as an important downstream effector of FGF19 involved in metastasis. Surprisingly, we found that FGF19 and GLI2 could reciprocally induce the expression of each other, and form a positive feedback loop to promote LUSC cell invasion and metastasis. These findings were corroborated by an association between a poor prognosis of LUSC patients and FGF19/GLI2 co-expression. In addition, we found that the GLI inhibitor GANT61 could effectively reduce FGF19-mediated LUSC invasion and metastasis. Our data suggest that FGF19 may serve as a novel biomarker for predicting metastatic LUSC. Intervening with the FGF19-GLI2 feedback loop may be a strategy for the treatment of FGF19-driven LUSC metastasis.

The Mechanism of Quercetin in the Treatment of Lung Squamous Cell Carcinoma Based on a Protein-Protein Interaction Network.

Lung squamous cell carcinoma (LUSC) is characterized by poor prognosis and obvious limitations of therapeutic methods. The molecular target and mechanism of quercetin (QR), a natural anticancer product with extensive pharmacological activities, on lung squamous cell carcinoma is still unclear. The effects of QR on LUSC were examined using cell proliferation, migration, and invasion tests. Key target genes were screened using The Cancer Genome Atlas (TCGA) database, Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) database, STRING website, topology, and prognosis analysis, molecular docking, and other bioinformatics methods for further analysis. Finally, the effects of QR on the expression of key targets in LUSC cells were detected using a cell cycle assay and western blotting. Our study demonstrates that QR not only inhibits the proliferation of LUSC but also affects the invasion and metastasis of LUSC. After downloading and analyzing the TCGA database, 2150 differentially expressed genes were identified. PLK1, CDC20, and BUB1B were identified using enrichment analysis, topological network analysis, cluster analysis, and molecular docking screening. Subsequent experiments showed that QR could interfere with the cell cycle and downregulate the expression of the target gene PLK1 at the protein level. We found that QR not only inhibits the proliferation, migration, and invasion but also blocks the cell cycle progression of LUSC. QR downregulated the expression of the LUSC target gene PLK1 at the protein level.

LINC01232 promotes lung squamous cell carcinoma progression through modulating miR-181a-5p/SMAD2 axis

BackgroundLINC01232 has been implicated in the progression of multiple malignancies. Yet, the function of LINC01232 in the carcinogenesis of lung squamous cell carcinoma (LUSC) remains unclear. This study aims to examine the role LINC01232 plays in LUSC progression. MethodsmRNA and protein levels were assessed using qRT-PCR and western blot, respectively. Cell proliferation was assessed by CCK-8 and colony formation assays. Cell migration and invasion were evaluated by transwell assay. The interactions between LINC01232, miR-181a-5p, and SMAD2 were assessed using luciferase reporter, RNA pull-down, and RNA immunoprecipitation (RIP) assays. The subcellular distribution of LINC01232 was examined by cytosolic/nuclear fractionation assay ResultsLINC01232 was upregulated in both LUSC tissues and cell lines. Knockdown of LINC01232 impaired cell proliferation, migration and invasion capability in H1229 and A549 cells, a phenotype that could be reversed by miR-181a-5p silencing. In addition, LINC01232 silencing reduced levels of N-cadherin, Vimentin, and Snail in H1229 and A549 cells, but increased the level of E-cadherin, which can be abrogated by miR-181a-5p inhibitors. ConclusionsIn summary, our study demonstrates that LINC01232 expression increases in LUSC tissues and cell lines and promotes LUSC progression by modulating the miR-181a-5p/SMAD2 signaling, providing new potential drug targets for LUSC treatment.

Downregulation of T-cell cytotoxic marker IL18R1 promotes cancer proliferation and migration and is associated with dismal prognosis and immunity in lung squamous cell carcinoma.

Immunotherapy can improve the survival of patients with advanced lung squamous cell carcinoma (LUSC). T cytotoxic cells are one of the main members of the immune microenvironment. Herein, we aimed to identify the roles of T-cell cytotoxic markers interleukin 18 (IL18) receptor 1 (IL18R1) in the LUSC progression using bioinformatics, clinical tissue specimen, and cell experiment. We assessed the association between the IL18R1 expression and immune infiltration and IL18R1-related competing RNA network. The IL18R1 expression was downregulated in the LUSC tissues. The IL18R1 expression downregulation was associated with diagnosis and short overall survival and disease-specific survival, and it was also an independent risk factor for dismal survival time in LUSC. IL18R1-related nomograms predicted the survival time of patients with LUSC. IL18R1 overexpression inhibited the proliferation, migration, and invasion of LUSC cells. The IL18R1 expression was significantly associated with the microenvironment (stromal, immune, and estimate scores), immune cells (such as the T cells, cytotoxic cells, CD8 T cells), and immune cell markers (such as the CD8A, PD-1, and CTLA4) in LUSC. AC091563.1 and RBPMS-AS1 downregulation was positively associated with the IL18R1 expression, negatively associated with the miR-128-3p expression, and associated with short disease-specific survival and progression in LUSC. In conclusion, IL18R1 was significantly downregulated and associated with the prognosis and immune microenvironment. IL18R1 overexpression inhibits the growth and migration of cancer cells in LUSC. Furthermore, AC091563.1 and RBPMS-AS1 might compete with IL18R1 to bind miR-128-3p for participating in LUSC progression. These results showed that IL18R1 is a biomarker for evaluating the prognosis of patients with LUSC.

Chromium (VI)-induced ALDH1A1/EGF axis promotes lung cancer progression.

Cr(VI) is broadly applied in industry. Cr(VI) exposure places a big burden on public health, thereby increasing the risk of lung squamous cell carcinoma (LUSC). The mechanisms underlying Cr(VI)-induced LUSC remain largely elusive. Here, we report that the cancer stem cell (CSC)/tumour-initiating cell (TIC)-like subgroup within Cr(VI)-transformed bronchial epithelial cells (CrT) promotes lung cancer tumourigenesis. Mechanistically, Cr(VI) exposure specifically increases the expression levels of aldehyde dehydrogenase 1A1 (ALDH1A1), a CSC marker, through KLF4-mediated transcription. ALDH1A1 maintains self-renewal of CrT/TICs and facilitates the expression and secretion of EGF from CrT/TICs, which subsequently promotes the activation of EGFR signalling in differentiated cancer cells and tumour growth of LUSC. In addition, the ALDH1A1 inhibitor A37 and gemcitabine synergistically suppress LUSC progression. Importantly, high ALDH1A1 expression levels are positively correlated with advanced clinical stages and predict poor survival in LUSC patients. These findings elucidate how ALDH1A1 modulates EGF secretion from TICs to facilitate LUSC tumourigenesis, highlighting new therapeutic strategies for malignant lung cancers.

Identification of tumour-infiltrating myeloid subsets associated with overall survival in lung squamous cell carcinoma.

Lung squamous cell carcinoma (LUSC) is a primary subtype of lung cancer with limited therapeutic options and poor prognosis, and tumour-infiltrating myeloid cells (TIMs) are key regulators of LUSC. However, the correlation between the abundance of TIM subtypes and clinical outcomes of LUSC remains unexplored. This study aimed to develop and validate a prognostic model for low- and high-risk patients with LUSC based on myeloid cell microenvironments. TIM markers in the tumoural (T) and stromal (S) regions were quantified using immunohistochemistry for 502 LUSC patients. L1-penalized Cox regression was used to develop a myeloid survival score (MSS) model based on the training cohort, followed by validation in distinct cohorts from multiple centres. RNA sequencing and immunostaining were used to examine the mechanisms of myeloid cells in LUSC progression and predict potential drug targets and therapeutic agents. Of the 12 myeloid markers, CD163T, CD163S, and S100A12T were highly associated with overall survival (OS) in LUSC patients. The MSS of the three myeloid signatures accurately categorized LUSC patients into risk categories, with an observable difference in OS between the training and validation cohorts. Tumours with high MSS were associated with enhanced antioxidative ability and hedgehog signalling and a shift to a more pro-tumorigenic microenvironment, accompanied by a reduced tumour cell immunogenicity and increased CD8+ T cell exhaustion patterns. Additionally, in high-risk patients, potential drug targets and compounds regulating hedgehog signalling were identified. Our study provides the first prognostic myeloid signature for LUSC, which may help advance precision medicine. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Long Intergenic Nonprotein Coding RNA 00174 Aggravates Lung Squamous Cell Carcinoma Progression via MicroRNA-185-5p/Nuclear Factor IX axis.

Extensive studies have presented that long noncoding RNAs (lncRNAs) are closely implicated in the pathogenesis of various human malignancies, including lung squamous cell carcinoma (LUSC). This study explored the biological role and the underlying mechanism of long intergenic nonprotein coding RNA 00174 (LINC00174) in LUSC. LINC00174 expression was measured by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Both in vitro and in vivo experiments were conducted to determine LINC00174 function in LUSC. Mechanical assays were performed to investigate the molecular mechanism involving LINC00174 and related genes. LINC00174 expression was high in LUSC cells. Silencing of LINC00174 could restrain LUSC cells proliferation, migration, and invasion while promoting cell apoptosis. Mechanically, LINC00174 could interact with miR-185-5p to upregulate nuclear factor IX (NFIX), which was the direct target gene of miR-185-5p. Notably, NFIX elevation could rescue the repressing effect of LINC00174 silence on LUSC cell malignant behaviors. Our data suggested that LINC00174 aggravated LUSC progression via serving as a competing endogenous RNA (ceRNA) to sponge miR-185-5p and ultimately upregulate NFIX, which offered a promising novel target for LUSC therapy.

TM4SF19-AS1 facilitates the proliferation of lung squamous cell carcinoma by recruiting WDR5 to mediate TM4SF19

BackgroundAs reported, long non-coding RNAs are a pivotal player in lung squamous cell carcinoma (LSCC) progression. We noticed the remarkably upregulated transmembrane-4-l-six-family-19 antisense RNA 1 (TM4SF19-AS1) in LSCC and further demonstrated the function it played in LSCC and the possible molecular mechanism. MethodsVia bioinformatics approach, we evaluated TM4SF19-AS1 and TM4SF19 levels in LSCC tissue, and real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot revealed their mRNA and protein levels in LSCC cells. Cell Counting Kit-8 and colony formation assays analyzed the proliferation ability of LSCC cells, and cell adhesion ability was detected via cell adhesion assay. RNA immunoprecipitation and chromatin immunoprecipitation analyzed the underlying mechanism of TM4SF19-AS1 regulating its target, while methylation-specific PCR indicated the methylation level of TM4SF19-AS1. ResultsTM4SF19-AS1 was markedly upregulated in LSCC. Functional assays revealed that TM4SF19-AS1 could facilitate the proliferation and adhesion of LSCC. Besides, we revealed the mechanism of TM4SF19-AS1 regulation that it directly bound to WD repeat-containing protein 5 (WDR5), and was then recruited to TM4SF19 promoter region, which activated DNA demethylation, thereby suppressing malignant LSCC progression. ConclusionOur research demonstrated that TM4SF19-AS1 affected LSCC cell proliferation by recruiting WDR5 to manipulate transmembrane-4-lsix-family-member-19 (TM4SF19), which offers a new observation on LSCC pathogenesis, indicating that TM4SF19-AS1 is able to be a promising target for LSCC treatment.

CircCNN2 Accelerates Cell Proliferation and Invasion in Lung Squamous Cell Carcinoma via Regulating miR-184/E2F1 and Activating MAPK Signaling Pathway.

Circular RNAs (circRNAs) have been demonstrated as potential biomarkers for the diagnosis and treatment of human diseases. Previous studies have unveiled the carcinogenic role of circRNA_102399 (circCNN2) in lung cancer. Through the UALCAN database, it was found that CNN2, the parent gene of circCNN2, was specifically highly expressed in human lung squamous cell carcinoma (LUSC) cells, but the regulatory mechanism of circCNN2 in LUSC is unclear. In this study, circCNN2 expression in LUSC cells was examined via RT-qPCR, and its effects on LUSC progression was verified through functional assays. The subcellular localization of circCNN2 was identified via FISH assay, and the underlying mechanism of circCNN2 on the activation of MAPK signaling pathway was determined through mechanism experiments. Results demonstrated that circCNN2 was upregulated in LUSC cells, and it promoted LUSC cell proliferation and invasion. Besides, circCNN2 acted as a competing endogenous RNA (ceRNA) to sponge miR-184 to upregulate E2F transcription factor 1 (E2F1) expression. Furthermore, it was verified that circCNN2 activated the generic mitogen-activated protein kinases (MAPK) signaling pathway through E2F1 and thus promoting LUSC progression. In a word, this study indicated that circCNN2 activated MAPK signaling pathway through the miR-184/E2F1 axis to promote proliferation and invasion of LUSC cells.

Identification of a dysregulated ceRNA network modulated by copy number variation-driven lncRNAs in lung squamous cell carcinoma.

Lung cancer is primarily responsive for cancer death, and its progression is aggressively affected by copy number variation (CNV). Through bioinformatics approach, a ceRNA network of CNV-driven lncRNAs in lung squamous cell carcinoma (LUSC) patients was constructed. Data on normal and LUSC tumor tissue from The Cancer Genome Atlas (TCGA)-LUSC dataset were subjected to differential analysis, and differentially expressed lncRNAs (DElncRNAs), DEmiRNAs, and DEmRNAs were obtained. Based on TCGA-LUSC, CNVs of normal and tumor tissue samples were then compared using a Chi-square test, and lncRNAs were intersected based on their CNVs and expression alternation. In combination with the Kruskal-Wallis test, CNV-driven lncRNAs were acquired. Afterwards, miRNAs and mRNAs that interacted with CNV-driven lncRNAs were obtained based on databases (LncBase, starBase, miRDB, mirDIP and TargetScan), DElncRNAs, DEmiRNAs and DEmRNAs, and correlation analysis. The acquired lncRNAs, miRNAs and mRNAs were subjected to Cytoscape software to construct a CNV-driven ceRNA network, which involved 5 lncRNAs (MIR143HG, LINC00702, MIR22HG, RP11-180 N14.1, RP11-473 M20.9), 6 miRNAs (miR-3200-3p, miR-1301-3p, miR-93-3p, miR-96-5p, miR-96-5p, miR-130b-5p, miR-205-5p) and 80 mRNAs. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analyses indicated that downstream mRNAs were mainly correlated with blood vessel development and T cell-mediated immunity. In summary, we devoted to analyzing CNV-related lncRNAs, mRNAs, and miRNAs in LUSC, thus clarifying 5 lncRNAs that may influence the malignant progression of LUSC. The ceRNA network regulated by these lncRNAs may be the novel pathogenesis of LUSC.

Abstract 5948: Modeling lung squamous cell carcinoma progression using primary human bronchial basal cells

Abstract Background: Lung squamous cell carcinoma (LUSC) arises from the accumulation of genetic driver alterations in bronchial basal cells. LUSC genomes lack obvious actionable mutations and present with a high degree of inter-tumor heterogeneity. Tractable, patient relevant models are needed to study LUSC pathogenesis and investigate the role of recurrent genomic alterations in driving cancer progression. Therefore, we are developing a tractable human model of LUSC progression to increase our understanding of this disease. Methods: To investigate the contribution and co-operation of frequently mutated genes (TP53 and CDKN2A) and commonly deregulated pathways (SOX2/P63, PI3K/AKT and KEAP1/NRF2 pathways) in driving LUSC progression, we genetically engineered primary human bronchial basal cells. This culminated in eight basal cell mutants harboring ubiquitous LOF alterations in TP53 and CDKN2A combined with additional pathway alterations. To assess the function of the genetic alterations in LUSC progression, we investigated proliferation using cell cycle analysis and colony forming assays. Air-liquid interface organotypic cultures were used to model histological changes in the bronchial epithelium driven by different combinations of mutations. The resultant cultures were analyzed for changes in histology indicative of LUSC progression using immunostaining to evaluate changes to epithelial cell proliferation, and expression of LUSC biomarkers. Results: Increases in proliferation were driven by inactivation of TP53, CDKN2A and PTEN, whereas SOX2 overexpression inhibited mutant basal cell proliferation. LUSC alterations also drove striking phenotypic changes in 3D organotypic cultures of the bronchial epithelium. All mutant cultures displayed thickening of the bronchial epithelium and significantly increased cellularity (p<0.001), indicating hyper-proliferation. SOX2 overexpression inhibited mucociliary differentiation and drove the appearance of a disordered epithelium with clear cellular and nuclear pleomorphism, indicative of high-grade premalignant lesions. Conclusions: We developed a basal cell model of recurrent LUSC genetic alterations. This approach is unique due to [1] the use of primary human cells and [2] the representation of interpatient heterogeneity and developmental stages by introducing different combinations of pathway alterations. Early results indicate that basal cells carrying cumulative alterations display increasingly complex phenotypes with features of low and high-grade premalignant lesions. Further work is required to assess the tumorigenicity of mutant cells. Citation Format: Julia Ogden, Caroline Dive, Carlos Lopez Garcia. Modeling lung squamous cell carcinoma progression using primary human bronchial basal cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5948.

Abstract 3783: GSTO2, a novel tumor suppressor gene of lung squamous cell carcinoma, regulates mitochondria function via the p38/β-catenin signaling pathway

Abstract Glutathione S-transferase omega 2 (GSTO2) is one of regulators of GSH/GSSG balance, and its polymorphism showed strong associations with lung functions as well as the risk of chronic obstructive pulmonary disease. Recently, we found that GSTO2 was exclusively expressed in airway basal cells, Clara cells and type II alveolar cells, which have self-renewal capacity in the lungs; however, its expression was lost in lung squamous cell carcinoma (LSCC). In the present study, we restored GSTO2 expression in LSCC cell lines (LK-2 and H520) to clarify the significance of GSTO2 loss in LSCC. In both LSCC cell lines used, GSTO2 overexpression significantly inhibited cell growth and colony formation in vitro. In a subcutaneous xenograft model, GSTO2-transfected LK-2 cells formed smaller tumors in nude mice than mock-transfected cells. Upon intravenous injection into nude mice, the incidence of liver metastasis was lower in mice injected with GSTO2-transfected LK-2 cells than in those injected with mock-transfected cells. Metabolomic analyses using the XF96 extracellular flux analyzer revealed that GSTO2 overexpression suppressed mitochondrial oxidative phosphorylation but did not affect glycolysis. Upon JC-1 dye staining, GSTO2-transfected cells showed decreased mitochondrial membrane potential compared with mock-transfected cells. Since β-catenin has been reported as a novel regulator of the OXPHOS in hepatocytes, we next examined the effect of GSTO2 expression on β-catenin expression in LSCC and found that GSTO2 overexpression suppressed the expression of β-catenin. Because p38 phosphorylation was accelerated in both GSTO2-transfected cells, we examined the involvement of the p38 signaling pathway in the GSTO2-mediated downregulation of β-catenin as well as mitochondrial membrane potential in LSCC.When GSTO2-transfected cells were treated with SB203580, a specific inhibitor of p38 MAPK, β-catenin expression and mitochondrial membrane potentialwere restored. Finally, we examine whether DNA methylation of the GSTO2 could explain the loss of GSTO2 expression in LSCC. When human LSCC cell lines were treated with 5-aza-2′-deoxycytidine, a DNA-methyltransferase inhibitor, GSTO2 transcription was induced. Bisulfite sequencing showed that the promoter region of the GSTO2 was frequently methylated in LSCC tissues than that of normal tissue. Our study indicated that the loss of GSTO2 via DNA hypermethylation contributes to the cell growth and progression of LSCC, probably by modulating oxidative phosphorylation in mitochondria via the p38/β-catenin signaling pathway. Citation Format: Ryusuke Sumiya, Masayoshi Terayama, Teruki Hagiwara, Kazuaki Nakata, Satoshi Nagasaka, Kazuhiko Yamada, Norihiro Kokudo, Hiromu Suzuki, Kawamura I. Yuki. GSTO2, a novel tumor suppressor gene of lung squamous cell carcinoma, regulates mitochondria function via the p38/β-catenin signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3783.