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The targeting protein for Xenopus kinesin-like protein 2 ( TPX2) has been demonstrated to be associated with the tumourigenesis of many cancers. In the present study, we investigated the role and preliminary mechanism of TPX2 in the resistance of lung squamous carcinoma to radiation therapy. The results showed that SK- MES-1R and NCI-H226R cells were more resistant to X-ray irradiation than the parental cells ( SK- MES-1 and NCI-H226). Moreover, TPX2 was upregulated in the radioresistant cells compared with the parental cells. TPX2 knockdown significantly decreased TPX2 expression in SK- MES-1 cells, while TPX2 overexpression increased TPX2 expression in NCI-H226 cells compared with the corresponding control cells. TPX2 knockdown enhanced the radiosensitivity of SK- MES-1 and promoted cell apoptosis following exposure to irradiation, whereas TPX2 overexpression decreased the radiosensitivity of NCI-H226 and inhibited cell apoptosis. In in vivo studies, the combination of TPX2 knockdown and irradiation significantly inhibited tumour growth, decreased tumour weight, downregulated TPX2 expression in tumour tissue and induced cell apoptosis in nude mice, while TPX2 overexpression exerted an opposite effect. Our results indicated that TPX2 was correlated with cell radioresistance and it might be served as a therapeutic target to enhance cell radiosensitivity in the radiation therapy of lung squamous carcinoma. [ABSTRACT FROM AUTHOR]

Recently, tyrosine kinase inhibitors (TKIs) have been recommended as a first-line treatment for advanced non-small cell lung cancer (NSCLC), significantly improving the treatment outcomes of lung adenocarcinoma patients with the EGFR mutation. However, the application of TKIs for lung squamous cell carcinoma (SCC), the second largest pathological subtype of NSCLC, remains controversial because available data for the EGFR mutation profile and frequency in SCC patients are limited. In this study, 89 bronchoscopic--biopsy specimens from Chinese SCC male patients were assayed for EGFR exon 19 mutation, using improved polymerase chain reaction-denature gel gradient electrophoresis. EGFR exon 19 mutations were detected in 77 of 89 (86.5%) patients, and included six kinds of point mutations (11.6%) and two deletions (Del_747-751 [64.9%] and Del_746-751 [23.3%]). We found that the proportion of mutated EGFR varied from 0.98% to 100% in positive specimens and increased with the development of the disease. The difference of proportion between Stage IV patients and Stage II patients or Stage III patients was significant (P,0.001). These results provided valuable clues to explain the reason why patients harboring the same mutation responded distinctly to TKI treatment. Del_747-751 and Del_746-751 were the dominant mutations in the assayed SCC patients (76.4%), and both belong to the EGFR--TKI-sensitive mutation. Recently research demonstrated that Del_746-751 patients have better response to EGFR-TKI than Del_L747-751 patients. However, our study indicated that majority of SCC patients (55.5%) carried Del_ L747-751. We suggest that the unique clinic features of SCC should be further studied to reveal the mechanism of poorer treatment outcome of EGFR--TKI therapy, and that a better treatment plan and more specific, potent targeted drugs for lung SCC need to be developed. [ABSTRACT FROM AUTHOR]

Abnormal expression of claudin (CLDN) subtypes has been reported in various solid cancers. However, it is unknown which subtype plays a key role in the regulation of proliferation in cancer cells. The expression of CLDN3-5, 7, and 18 in human lung squamous carcinoma tissues was lower than that in normal tissue. Here, we examined which combination of exogenous CLDNs expression inhibits proliferation and the molecular mechanism using human lung squamous RERF-LC-AI cells. Real-time polymerase chain reaction and western blotting showed that CLDN3-5, 7, and 18 are little expressed in RERF-LC-AI cells. In the exogenously transfected cells, CLDN5, 7, and 18 were distributed in the cell–cell contact areas concomitant with ZO-1, a tight junctional scaffolding protein, whereas CLDN3 and 4 were not. Cell proliferation was individually and additively suppressed by CLDN5, 7, and 18. The expression of these CLDNs showed no cytotoxicity compared with mock cells. CLDN5, 7, and 18 increased p21 and decreased cyclin D1, resulting in the suppression of cell cycle G1-S transition. The expression of these CLDNs inhibited phosphorylation of Akt without affecting phosphorylated ERK1/2. Furthermore, these CLDNs inhibited the nuclear localization of Akt and its association with 3-phosphoinositide-dependent protein kinase-1 (PDK1). The suppression of G1-S transition caused by CLDN5, 7, and 18 was rescued by the expression of constitutively active-Akt. We suggest that the reduction of CLDN5, 7, and 18 expression loses the suppressive ability of interaction between PDK1 and Akt and causes sustained phosphorylation of Akt, resulting in the disordered proliferation in lung squamous carcinoma cells. [ABSTRACT FROM AUTHOR]

Objective: To investigate the impact that TTN mutation had on the gene heterogeneity expression and prognosis in patients with lung adenocarcinoma., Methods: In this study, the Cancer Genome Atlas (TCGA) dataset was used to analyze the TTN mutations in lung adenocarcinoma. Lung adenocarcinoma data was collected from the TCGA database, clinical information of patients was analyzed, and bioinformatics statistical methods were applied for mutation analysis and prognosis survival analysis. The results were verified using the GEO dataset., Results: The incidence of TTN mutations in lung adenocarcinoma was found to be 73%, and it was related to the prognosis of lung adenocarcinoma. Ten genes were screened with significant contributions to prognosis. A prognosis model was constructed and verified by LASSO COX analysis in the TCGA and GEO datasets based on these ten beneficial factors. The independent prognostic factor H2BC9 for TTN mutation-driven gene heterogeneity expression was screened through multi-factor COX regression analysis., Conclusion: Our data showed that the gene heterogeneity expression, which was driven by TTN mutations, prolonged the survival of lung adenocarcinoma patients and provided valuable clues for the prognosis of TTN gene mutations in lung adenocarcinoma., Competing Interests: ZL, XT and YZ were employed by Biotrans Technology Co., LTD. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Liu, Zhao, Wang, Tang, Zhao, Zhong, Peng and Zhang.)

Objective:To study the effect of tea pigment on the growth and apoptosis of human lung squamous cell carcinoma cell line SK-MES-1. Methods: Tea pigments were extracted by ultrasonic cell disintegration, and the yield was calculated. SK-MES-1 cells were conventionally cultured. MTT assay was used to detect the inhibition ratio of different concentrations of tea pigments (5, 2.5, 1.25, 0.625, 0.3125 mg/mL) for different times (24 h and 48 h) in SK-MES-1 cells, the growth inhibition ratio (IR) and IC50 were calculated. Flow cytometry (FCM) stained with by Annexin V and IP was applied to detect the effects of tea pigments on the apoptosis of SK-MES-1 cells. Results: The extraction rate of tea pigments was 7.35 %. The results of MTT assay demonstrated a dose-time dependent manner. That is, with the increase of concentration and extension of action time, the inhibition rate of SK-MES-1 was gradually increased. IC50 of cells treated for 24 h and 48 h were 2.353 mg/mL and 1.494 mg/mL respectively. FCM technique detected the apoptosis of cells when they were treated for 24 h. The cell apoptosis ratio of control group, low and high concentration of tea pigment were 7.7 %, 20.37 %and 25.25%respectively. Conclusions: Tea pigments could promote the apoptosis of SK-MES-1 cells and inhibit its proliferation. [ABSTRACT FROM AUTHOR]

Background and Aim: Paraneoplastic syndrome is a disease or symptom that is the consequence of cancer in the body but, unlike mass effect, is not due to the local presence of cancer cells. An overlap of two paraneoplastic syndromes secondary to lung cancer in a patient is very rare. In order to improve the diagnosis of two paraneoplastic syndromes secondary to lung cancer, we reported an overlap of two paraneoplastic syndromes secondary to lung squamous carcinoma in a patient. Methods: We reported a case of a 60-year-old male smoker who presentedmultiple erythematous lesions for 6 months with progressive proximal muscular weakness, and developed to dysphonia in 2 months.His chest computed tomography showed a spiculate pulmonary nodule near the hilar in the right upper lobe and mediastinal lymph nodes enlargement. Lung biopsy showed squamous cell carcinoma. The patient's blood test revealed elevated creatine kinase levels. Electromyography results were consistent with moderate myopathy accompanied with a pattern suggestive of Lambert--Eaton myasthenic syndrome. Results: So we made the diagnosis of an overlap of two paraneoplastic syndromes (dermatomyositis and Lambert--Eaton myasthenic syndrome) secondary to lung squamous carcinoma. Conclusion: Recognition of paraneoplastic syndrome is crucial for early diagnosis of lung cancer because up to 80% of patients may present paraneoplastic syndromes before any other direct indication of malignancy. Early diagnosis and treatment of paraneoplastic syndrome is also important because delayed diagnosis may result in rapid disease progression and irreversible neurological damage. [ABSTRACT FROM AUTHOR]

Background: Matrix metalloproteinase 9 (MMP-9) plays an important role in tumor invasion and metastasis, including lung cancer. However, whether variations in serum MMP-9 levels can serve as a biomarker for monitoring chemotherapy curative effect remains unclear. This study was designed to investigate the association between variations in serum MMP-9 levels and chemotherapy curative effect in patients with lung cancer. Patients and methods: A total of 82 patients with advanced lung cancer were included. All newly diagnosed patients were treated with platinum-based doublet chemotherapy. Serial measurements of serum MMP-9 levels were performed by enzyme-linked immunosorbent assay. In this manner, we chose four time points to examine the association, including before chemotherapy, and 3 weeks after the beginning of the first, second, and fourth cycles of chemotherapy. Results: Compared with the serum level of MMP-9 before progressive disease, patients with progressive disease had elevated serum levels of MMP-9. Compared with the previous time point of collecting specimens, the serum levels of MMP-9 in the patients with a complete response/partial response/stable disease decreased or were maintained stable. The differences of variation in serum MMP-9 levels in patients with different chemotherapy curative effects were all statistically significant after one cycle, two cycles, and four cycles (after one cycle: P<0.001; after two cycles: P<0.001; after four cycles: P=0.01). However, patients with smallcell lung cancer did not exhibit similar test results. Conclusion: The variation in serum MMP-9 levels in patients with non-small-cell lung cancer during chemotherapy was closely related to chemotherapy curative effect and could be useful to monitor chemotherapy curative effect for a small portion of patients. [ABSTRACT FROM AUTHOR]

There is still no ideal predictive biomarker for immunotherapy response among patients with non-small cell lung cancer. Costimulatory molecules play a role in anti-tumor immune response. Hence, they can be a potential biomarker for immunotherapy response. The current study comprehensively investigated the expression of costimulatory molecules in lung squamous carcinoma (LUSC) and identified diagnostic biomarkers for immunotherapy response. The costimulatory molecule gene expression profiles of 627 patients were obtained from the The Cancer Genome Atlas, GSE73403, and GSE37745 datasets. Patients were divided into different clusters using the k-means clustering method and were further classified into two discrepant tumor microenvironment (TIME) subclasses (hot and cold tumors) according to the immune score of the ESTIMATE algorithm. A high proportion of activated immune cells, including activated memory CD4 T cells, CD8 T cells, and M1 macrophages. Five CMGs (FAS, TNFRSF14, TNFRSF17, TNFRSF1B, and TNFSF13B) were considered as diagnostic markers using the Least Absolute Shrinkage and Selection Operator and the Support Vector Machine-Recursive Feature Elimination machine learning algorithms. Based on the five CMGs, a diagnostic nomogram for predicting individual tumor immune microenvironment subclasses in the TCGA dataset was developed, and its predictive performance was validated using GSE73403 and GSE37745 datasets. The predictive accuracy of the diagnostic nomogram was satisfactory in all three datasets. Therefore, it can be used to identify patients who may benefit more from immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Duan, Wang, Zhai, Wang, Zhao, Zheng, Rao, Zhou, Long and Lin.)

Background: At present, immunotherapy is a very promising treatment method for lung cancer patients, while the factors affecting response are still controversial. It is crucial to predict the efficacy of lung squamous carcinoma patients who received immunotherapy., Methods: In our retrospective study, we enrolled lung squamous carcinoma patients who received immunotherapy at Beijing Chest Hospital from January 2017 to November 2021. All patients were grouped into two cohorts randomly, the training cohort (80% of the total) and the test cohort (20% of the total). The training cohort was used to build neural network models to assess the efficacy and outcome of immunotherapy in lung squamous carcinoma based on clinical information. The main outcome was the disease control rate (DCR), and then the secondary outcomes were objective response rate (ORR), progression-free survival (PFS), and overall survival (OS)., Results: A total of 289 patients were included in this study. The DCR model had area under the receiver operating characteristic curve (AUC) value of 0.9526 (95%CI, 0.9088-0.9879) in internal validation and 0.9491 (95%CI, 0.8704-1.0000) in external validation. The ORR model had AUC of 0.8030 (95%CI, 0.7437-0.8545) in internal validation and 0.7040 (95%CI, 0.5457-0.8379) in external validation. The PFS model had AUC of 0.8531 (95%CI, 0.8024-0.8975) in internal validation and 0.7602 (95%CI, 0.6236-0.8733) in external validation. The OS model had AUC of 0.8006 (95%CI, 0.7995-0.8017) in internal validation and 0.7382 (95%CI, 0.7366-0.7398) in external validation., Conclusions: The neural network models show benefits in the efficacy evaluation of immunotherapy to lung squamous carcinoma patients, especially the DCR and ORR models. In our retrospective study, we found that neoadjuvant and adjuvant immunotherapy may bring greater efficacy benefits to patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Li, Ma, Fu, Gao, Qin, Wu, Zhang, Wang, Pan and Liu.)

Tumor angiogenesis inhibitors such as Bevacizumab, Ramucirumab and Endostar have been applied to the therapy of non-small cell lung carcinoma (NSCLC) patients, especially for lung adenocarcinoma (LUAD). However, several safe concerns such as neutropenia, febrile neutropenia and hypertension pulmonary hemorrhage limit their further development. And they often showed poor efficacy and serious side effect for lung squamous cell carcinoma (LUSC) patient. Thus, identification of effective and safe tumor angiogenesis inhibitor for NSCLC therapy is warranted. Apigenin is a bioflavonoid with potential anti-tumor effect and perfect safety, but its effect on tumor angiogenesis and underlying mechanism are still unclear. Herein, we found that apigenin not merely suppressed endothelial cells related motilities but also reduced pericyte coverage. Further research showed that apigenin had strong suppressive activity against HIF-1α expression and its downstream VEGF-A/VEGFR2 and PDGF-BB/PDGFβR signaling pathway. Apigenin also reduced microvessel density and pericyte coverage on the xengraft model of NCI–H1299 cells, leading to suppression of tumor growth. Moreover, apigenein showed perfect anti-angiogenic effect in xengraft model of LUSC cell NCI–H1703 cells, indicating it may be developed into a potential angiogenesis inhibitor for LUSC patient. Collectively, our study provides new insights into the anti-tumor mechanism of apigenin and suggests that apigenin is a safe and effective angiogenesis inhibitor for NSCLC therapy. • Apigenin suppresses endothelial cells- and pericyte-motility during angiogenesis. • Apigenin inhibits HIF-1α synthesis and accelerates its degradation. • Apigenin may serve as a safe and effective anti-angiogenic agent for LUAD and LUSC. [ABSTRACT FROM AUTHOR]

Annexin A5 is a Ca-dependent phospholipid-binding protein and protein kinase C inhibitory protein. It has a potential role in cellular signal transduction, inflammation, growth and differentiation. In this study, we evaluated the expression of this protein in lung tumor tissues and subsequently established a NCI-H520 cell line that stably expresses the wild-type ANXA5 gene to determine the effects of annexin A5 upregulation on the cell morphology, proliferation and metastasis potential in vitro. The effects of annexin A5 on NCI-H520 cells were tested by crystal violet staining, CCK-8 assay, scratch wound assay, and Transwell assay. The expressions of Akt, PCNA, vimentin, and E-cadherin were examined by Western blot assay. In this study, we demonstrated that annexin A5 is expressed at lower levels in tumor tissues compared with normal tissues. Additionally, the upregulation of this protein may inhibit the proliferation, migration, and invasion abilities of NCI-H520 cells in vitro. The transfected cells were arrested in the G/S phase of the cell cycle, and the expression levels of Akt, PCNA and Vimentin were downregulated, while E-cadherin was upregulated. [ABSTRACT FROM AUTHOR]

Abstract: Background: Novel vascular-independent conduits have been observed in some cancers. These have been variously described as vasculogenic mimicry, mosaic vessel formation, vascular co-option and intratumour embryonic-like vasculogenesis. Despite lung cancer being the most common cancer worldwide, there is little information on its neovascularisation or the pathways involved. Methods: An in vitro model involving co-cultures of microvascular lung endothelial cells and squamous or adenocarcinoma lung cancer cells was developed to assess their angiogenic interaction. Cells were incubated and examined by phase contrast microscopy and by immunocytochemistry in both mono- and co-cultures. Cultured cells and lung cancer tissue sections were assessed for new tumour vessel formation, expression of the endothelial marker CD31 and morphology. Results: Lung tumour cells and endothelial cells interacted morphologically via pseudopodia and used alternative pathways to generate new vessels. Co-culturing microvascular endothelial and squamous carcinoma cells led to endothelial cells surrounding tumour cells and the tumour cells being incorporated into vessel walls. Co-culturing endothelial and adenocarcinoma cells resulted in cellular contact and the formation of tumour cell bridges around clusters of endothelial cells. These adencocarcinoma cells became strongly positive for CD31. Tumour tissue section studies supported the in vitro findings. Conclusion: Lung carcinoma cells when co-cultured with lung endothelial cells modify their cellular and molecular features that encourage alternative means of providing blood supply. The mechanisms underpinning these non-angiogenic processes need to be further investigated and should be considered when anti-tumour therapeutic interventions are being considered. [Copyright &y& Elsevier]

Background: DNA methylation controls the transcription of genes and is involved in the development of lung cancer. Our preliminary bioinformatics prediction revealed that sperm associated antigen 6 (SPAG6) was considerably hypermethylated in lung squamous cell carcinoma (LUSC). Thus, this study aimed to probe the mechanism underlying its hypermethylation., Methods: The effect of DNA methylation of SPAG6 on its expression in LUSC was analyzed. The contributors to SPAG6 DNA hypermethylation were sought. CCK-8, EdU, and Transwell assays were carried out to assess the malignant phenotype of LUSC cells. KEGG pathway enrichment analysis was used to screen for pathways affected by SPAG6, which were confirmed by dual-luciferase assays. Bioinformatics analysis was conducted to dissect the impact of SPAG6 on the immune response and cancer cell stemness in LUSC., Results: DNA methyltransferase 3b (DNMT3b)-mediated hypermethylation of the SPAG6 promoter in LUSC led to SPAG6 downregulation. SPAG6 reverted the malignant phenotype of LUSC cells. SPAG6 regulated the JAK/STAT pathway by inhibiting the transcription of STAT1 and STAT3. The expression of SPAG6 was positively related to immune infiltration in LUSC and inversely related to the expressions of the immunosuppressive genes CTLA4 and PDCD1. SPAG6 expression was negatively correlated with cancer cell stemness in LUSC, and its expression inhibited the expressions of Nanog, ALDH1, and Sox2, markers of cancer cell stemness., Conclusions: DNMT3b-mediated SPAG6 promoter hypermethylation activates the JAK/STAT pathway to promote LUSC progression., Competing Interests: None., (AJTR Copyright © 2022.)

Cancer immunotherapy is an increasingly successful strategy for treating patients with advanced or conventionally drug-resistant cancers. T cells have been proved to play important roles in anti-tumor and tumor microenvironment shaping, while these roles have not been explained in lung squamous cell carcinoma (LUSC). In this study, we first performed a comprehensive analysis of single-cell RNA sequencing (scRNA-seq) data from the gene expression omnibus (GEO) database to identify 72 T-cell marker genes. Subsequently, we constructed a 5-gene prognostic signature in the training cohort based on the T-cell marker genes from the cancer genome atlas (TCGA) database, which was further validated in the testing cohort and GEO cohort. The areas under the receiver operating characteristic curve at 1-, 3-, and 5-years were 0.614, 0.713 and 0.702 in the training cohort, 0.669, 0.603 and 0.645 in the testing cohort, 0.661, 0.628 and 0.590 in the GEO cohort, respectively. Furthermore, we created a highly reliable nomogram to facilitate clinical application. Gene set enrichment analysis showed that immune-related pathways were mainly enriched in the high-risk group. Tumor immune microenvironment indicated that high-risk group exhibited higher immune score, stromal score, and immune cell infiltration levels. Moreover, genes of the immune checkpoints and human leukocyte antigen family were all overexpressed in high-risk group. Drug sensitivity revealed that low-risk group was sensitive to 8 chemotherapeutic drugs and high-risk group to 4 chemotherapeutic drugs. In short, our study reveals a novel prognostic signature based on T-cell marker genes, which provides a new target and theoretical support for LUSC patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shi, Dong, Jia, Zheng, Wang, Wang, Yang, Lu and Yang.)

Background: Non-small cell lung cancer (NSCLC) is a malignant tumor with high mortality. Lung squamous carcinoma (LUSC) and lung adenocarcinoma (LUAD) are the common subtypes of NSCLC. However, how LUSC and LUAD are compatible remains to be elucidated., Methods: We used a network approach to find highly interconnected genes shared with LUSC and LUAD, and we then built modules to assess the degree of preservation between them. To quantify this result, Z -scores were used to summarize the interrelationships between LUSC and LUAD. Furthermore, we correlated network hub genes with patient survival time to identify risk factors., Results: Our findings provided a look at the regulatory pattern for LUSC and LUAD. For LUSC, several genes, such as AKR1C1 , AKR1C2 , and AKR1C3 , play key roles in regulating network modules of cell growth pathways. In addition, CCL19 , CCR7 , CCL21 , and LY9 are enriched in LUAD network modules of T lymphocyte-related pathways. LUSC and LUAD have similar expressed gene expression patterns. Their networks share 46 hub genes with connectivity greater than 0.9. These genes are correlated with patient survival time. Among them, the expression level of COL5A2 in LUSC and LUAD is higher than that in normal tissues, which is closely related to the poor prognosis of LUSC and LUAD patients., Conclusion: LUSC and LUAD share a network pattern. COL5A2 may be a risk factor in poor prognosis in LUSC and LUAD. The common landscape of LUSC and LUAD will help better define the regulation of NSCLC candidate genes and achieve the goals of precision medicine., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2022 Li, Kuang, Zhang and Ma.)

Lung squamous carcinoma (LUSC) is a malignant tumor of the respiratory system with highly heterogeneous characteristics. Lactate is the main product of aerobic glycolysis during the metabolic reprogramming of tumors. There is growing evidence that lactate metabolic processes have a broad and sophisticated impact on tumor phenotypic plasticity and tumor microenvironment (TME). However, the pattern of lactate metabolism in patients with LUSC and its impact on TME, phenotype, prognosis, and treatment have not been fully elucidated. In this study, we identified two subtypes with different lactate metabolism patterns in LUSC by non-negative matrix factorization and explored their multi-omics features. We observed that lactate metabolism levels in LUSC extensively influenced tumor immune infiltration patterns, adaptation to the hypoxia environment, and energy metabolic reprogramming. Subsequently, we constructed the lactate metabolism-related prognostic index (LMRPI) using Cox stepwise regression analysis. LMRPI showed excellent stability and accuracy, and based on the median value of LMRPI, LUAD were divided into two subgroups. The two subgroups have different patterns of immune infiltration and somatic mutations. Meanwhile, the two subgroups had different responsiveness to immune checkpoint inhibitor (ICI) therapies and different sensitivity to various chemotherapeutic and molecular targeting agents. In conclusion, we defined two subtypes with different lactate metabolism patterns in LUSC and extensively characterized their multi-omics profile. Furthermore, we developed LMRPI that predicts the prognosis of LUSC patients while also predicting their response to various adjuvant therapies, including immunotherapy, to guide their individualized treatment., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Background: Globally, non-small-cell lung cancer (NSCLC) has a high incidence, and NSCLC patients have poor prognoses. Lung squamous carcinoma (LUSC) is a major pathological type of NSCLC. LncRNAs play important roles in tumor progression and immune system functions. The aim of this study was to construct a predictive model with immune-related lncRNAs and to assess the immune microenvironment in middle- or advanced-stage LUSC patients., Methods: RNA sequencing data and corresponding clinical LUSC data were downloaded from The Cancer Genome Atlas. Immune genes were obtained from the Molecular Signatures Database. Immune-related lncRNAs were identified by Pearson correlation analysis in R. The model was constructed using univariate and multivariate Cox regression analyses. Finally, we validated the prognostic immune-related lncRNA model in a cohort from the Fudan University Shanghai Cancer Center., Results: Our risk model included four immune-related lncRNAs (LINC00944, AL034550.2, AC020907.1 and AC027682.6). Survival analysis revealed that overall and disease-free survival were shorter in the high-risk group than in the low-risk group. Independent prognostic analysis showed that our model could be used as an independent prognostic predictor. The high-risk group was positively associated with CD8+ T cells, B cells, myeloid dendritic cells, macrophages, regulatory T cells (Tregs) and cancer-associated fibroblasts and high expression of PD1 and CTLA4. Additionally, a low-risk score was correlated with lower half maximal inhibitory concentrations (IC 50 s) of cisplatin, docetaxel, vinorelbine and paclitaxel and a higher IC 50 of gemcitabine. Gene set enrichment analysis suggested that these lncRNAs may participate in tumor progression and immune processes. Validation with the clinical cancer cohort demonstrated that higher risk scores were associated with a higher, but not statistically significant, likelihood of recurrence., Conclusion: We established a risk score model including four immune-related lncRNAs. The model accurately predicts the prognosis of middle- or advanced-stage LUSC patients and provides an important reference for individualized treatment., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. Published by Elsevier Ltd.)

Abstract: A quantitative proteomic approach was used to discover potential protein markers associated with lymph node metastasis (LNM) in human lung squamous carcinoma (LSC). Laser capture microdissection was performed to purify LSC cells with LNM (LNM LSC) and LSC without LNM (non-LNM LSC). The differentially expressed proteins between pooled microdissected non-LNM LSC and LNM LSC cells were identified by two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry (MS). 14 proteins were found to be differentially expressed between non-LNM LSC and LNM LSC. Among these proteins, ten proteins were overexpressed in LNM LSC compared with non-LNM LSC, and four proteins were downregulated in LNM LSC. Some of these identified proteins (Annexin A2, HSP27, CK19, and 14-3-3σ) were further confirmed by Western blotting and immunohistochemical analysis. These results show the value of LCM coupled with 2D-DIGE in identifying potential markers for lymph node metastasis of LSC, and also provide further insights into the prognosis of LSC. [Copyright &y& Elsevier]

Abstract: In this study, we aim to screen metastasis-related proteins in human lung squamous carcinoma (LSC) using laser capture microdissection and a proteomic approach. Twenty two differential proteins were identified from pooled microdissected primary LSC and matched lymph node (LN) metastatic tissues. Expression of the differential protein 14-3-3 sigma was determined by Western blotting and immunohistochemistry. In cell invasion assay, down-regulated 14-3-3 sigma by siRNA increased in vitro invasive ability of HTB-182 and A549 cells, up-regulation of 14-3-3 sigma by pcDNA3.0/14-3-3 sigma decreased in vitro invasive ability of HTB-182 and A549 cells. The data suggest that 14-3-3 sigma is a potential LN metastasis-related protein in LSC, and its dysregulation might play an important role in the LN metastatic process of LSC. [Copyright &y& Elsevier]

Objective To construct a database of human lung squamous carcinoma cell line NCI-H226 and to facilitate discovery of novel subtypes markers of lung cancer. Method Proteomic technique was used to analyze human lung squamous carcinoma cell line NCI-H226. The proteins of the NCI-H226 cells were separated by two-dimensional gel electrophoresis and identified by mass spectrometry. Results The results showed that a good reproducibility of the 2-D gel pattern was attained. The position deviation of matched spots among three 2-D gels was 1.95±0.53 mm in the isoelectric focusing direction, and 1.73±0.45 mm in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis direction. One hundred and twenty-seven proteins, including enzymes, signal transduction proteins, structure proteins, transport proteins, etc. were characterized, of which, 29 identified proteins in NCI-H226 cells were reported for the first time to be involved in lung cancer carcinogenesis. Conclusion The information obtained from this study could provide some valuable clues for further study on the carcinogenetic mechanism of different types of lung cancer, and may help us to discover some potential subtype-specific biomarkers of lung cancer. [ABSTRACT FROM AUTHOR]

Abstract: Background: To investigate the relationship between the expression of glucose-regulated protein94 (GRP94) and GRP78 at the level of mRNA and protein in vivo and in human lung cancer. Methods: RT-PCR, real-time PCR, immunohistochemistry and/or Western blot were used in 54 cases of lung cancer and corresponding normal lung tissue. Results: The expression pattern of GRP94 and GRP78 was similar. There was a significant overexpression of GRP94 and GRP78 at both mRNA and protein levels in cancer tissues as compared to normal tissues. The relative levels of GRP94 and GRP78 mRNA evaluated by RT-PCR in cancer and normal lung tissue were: GRP94: 3.48±2.06 versus 2.01±1.83; GRP78: 3.64±1.87 versus 2.21±1.54; by real-time PCR were: GRP94: 2.89±0.64 versus 1.12±0.54; GRP78: 2.56±0.82 versus 0.96±0.42. The relative level of GRP94 and GRP78 protein by Western blot in cancer and normal lung tissue were: GRP94: 3.46±1.72 versus 1.81±0.92; GRP78: 4.84±2.55 versus 1.91±1.15, indicating an approximate 2-fold and a 3-fold increase in GRP94 and GRP78 protein in cancer tissue as compared with normal tissue. Immunohistochemistry result for GRP94 and GRP78 in cancer and normal tissue was similar, that is: a stronger stain was observed in cancer tissue (main intensity of staining ++ to +++) compared to normal tissue (main intensity of staining + to ++). All the difference for GRP94 and GRP78 between the two tissues were significant (p <0.05). Furthermore, the overexpression of GRP94 and GRP78 in the cancer tissue correlated with grade of differentiation and stage of tumors. There was stronger expression in poorly differentiated tumors than in well-moderately differentiated tumors (p <0.05). There was also stronger expression in stage III than in stages I and II tumors (p <0.05). No statistically significant differences were found among various pathologic types of tumors. Correlation analysis showed that there is a positive correlation between GRP94 and GRP78. Conclusion: The expression pattern of GRP94 and GRP78 was similar in human lung cancer. They both were related with the differentiation and progression of the cancer. The expression at mRNA and protein level may be valuable in evaluating the grade of differentiation and clinical stage of human lung cancer. [Copyright &y& Elsevier]

Processes involved in malignant transformation of the lung from preneoplasia are poorly understood. To better understand this process, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) profiles of proteins from the normal, metaplasia, dysplasia and carcinoma tissues of human bronchial epithelia were examined by differential proteomic analysis. The selected differential protein-spots were identified by peptide mass fingerprint based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and database searching. The average spots for normal epithelium, metaplasia, dysplasia and invasive carcinoma were 1189.50 ± 39.89, 1227.00 ± 37.90, 1273.00 ± 43.31 and 1326.00 ± 66.63, respectively. Well-resolved, reproducible 2-D PAGE patterns of the normal-metaplasia-dysplasia-carcinoma tissues of bronchial epithelia were obtained. After matching, the number of spots of differential proteins between normal tissue and metaplasia, metaplasia and dysplasia, and dysplasia and invasive cancer tissues were 31.50 ± 7.67, 41.00 ± 9.07 and 56.00 ± 8.96, respectively. In total, 35 differential proteins, expressed only at the later stage of a two-stage comparison, were identified, some of which are known to be involved in regulating the processes of proliferation, differentiation and signal transduction. Current data in this study, for the first time, provide the basis for identification of potential tumor markers of human lung squamous carcinoma and their involvement in the progression of malignant transformation of bronchial epithelium. [ABSTRACT FROM AUTHOR]

• LUSC patients could be divided into 2 TME clusters based on the relative abundance of 24 immune cells. • The immune-related genes, cytokines, and miRNA were higher expressed in higher immune cells infiltration cluster. • The number of somatic mutations may play an important role in immune infiltration. • The TME cluster was a valuable predictive factor in LUSC patients. To compare the landscape of tumor microenvironment (TME) of lung squamous carcinoma (LUSC) in different immune pattern and explore potential factors on immune therapy and prognosis. We have obtained the LUSC data from TCGA, GEO, and our department and classified them into 2 TME clusters by random forest model based on the infiltration pattern of 24 immune cell populations. We systemically compared the genomic significance, clinical characteristics, and immune infiltration pattern in 2 TME clusters. Samples were divided into 2 TME clusters based on the relative abundance of 24 immune cells, and a random forest classifier model was constructed. TME cluster B was a higher immune infiltration group with lower mutation load, richer co-infiltrate immune cells, upregulated immune-related cytokines, immune checkpoint molecules, and higher active immune cells. TME cluster was also an independent predictor in prognosis (B vs. A, p < 0.05) in patients from TCGA, GEO, and our department. Our study has described the microenvironment landscape of LUSC in different immune infiltration patterns and systemically analyzed genomic and clinical characteristics with distinct immunophenotypes, thus partly revealed the interaction between tumors and the immune microenvironment, which may guide a more precise and personalized immune therapeutic strategy for LUSC patients. [ABSTRACT FROM AUTHOR]

Synchronous double primary lung squamous carcinoma (sDPLSCC) is rare and difficult to distinguish from metastatic disease, histopathologically. Owing to the heterogeneity of cancer, it is also difficult to select the optimal therapeutic strategy for patients with multiple primary lung cancer (MPLC). The present study reports a rare case of a 61-year-old male patient with sDPLSCC diagnosed using histology and genetic profiling. LSCC-related driver mutations were detected in this patient, and we reported the TP53 c.475G>C mutation, which has been detected in both breast cancer and hepatocellular carcinoma, but not previously in lung squamous carcinoma. Our findings provide further evidence supporting the necessity of genetic testing for primary tumor diagnosis.

This research aims to illustrate the effect of lncRNA StAR Related Lipid Transfer Domain Containing 13 antisense RN (STARD13-AS)/miR-1248/C3A on lung squamous carcinoma cells growth and metastasis. Bioinformatics analysis was applied to detect the expression of STARD13-AS/miR-1248/C3A in lung cancer samples and establish the ceRNA network. Transfection was performed to construct over-expression or knockdown models. PCR was implemented to examine the transfection efficiency. The biological function including growth, invasion and migration of LUSC cells were estimated by CCK-8 analysis, colony formation assay and transwell assay. Luciferase assay was executed to analyze the relationship between C3A and miR-1248, as well as miR-1248 and STARD13-AS. By consulting the TCGA database and GEPIA website, we found that C3A expression was significantly reduced in LUSC samples. Additionally, we also discovered that miR-1248, which was a downstream target of STARD13-AS, was presented as an upstream regulator of C3A. Moreover, STARD13-AS was under expressed in LUSC cells and has a negative effect on LUSC cells growth ability. C3A expression was co-regulated by miR-1248 and STARD13-AS. Importantly, the inhibitory effect of C3A or the promoting effect of miR-1248 on LUSC cells growth, invasion and migration abilities can be regulated by STARD13-AS. Our findings revealed that overexpression of STARD13-AS restricted the growth and aggressiveness of LUSC cells via regulating miR-1248/C3A. [ABSTRACT FROM AUTHOR]

Introduction: Unlike lung adenocarcinoma patients, there is no FDA-approved targeted-therapy likely to benefit lung squamous cell carcinoma patients., Materials and Methods: We performed survival analyses of lung squamous cell carcinoma patients harboring therapeutically relevant alterations identified by whole exome sequencing and mass spectrometry-based validation across 430 lung squamous tumors., Results: We report a mean of 11.6 mutations/Mb with a characteristic smoking signature along with mutations in TP53 (65%), CDKN2A (20%), NFE2L2 (20%), FAT1 (15%), KMT2C (15%), LRP1B (15%), FGFR1 (14%), PTEN (10%) and PREX2 (5%) among lung squamous cell carcinoma patients of Indian descent. In addition, therapeutically relevant EGFR mutations occur in 5.8% patients, significantly higher than as reported among Caucasians. In overall, our data suggests 13.5% lung squamous patients harboring druggable mutations have lower median overall survival, and 19% patients with a mutation in at least one gene, known to be associated with cancer, result in significantly shorter median overall survival compared to those without mutations., Conclusions: We present the first comprehensive landscape of genetic alterations underlying Indian lung squamous cell carcinoma patients and identify EGFR, PIK3CA, KRAS and FGFR1 as potentially important therapeutic and prognostic target., Competing Interests: CONFLICTS OF INTEREST Authors have no conflicts of interest to declare., (Copyright: © 2021 Joshi et al.)

In this study, we performed bioinformatics analyses to identify hub genes that regulate tumor infiltration by immune cells and antitumor immunity in the lung squamous cell carcinoma (LUSC). We identified 1738 robust and stable differentially expressed genes (DEGs) in the LUSC tissues based on robust rank aggregation (RRA) analysis of RNA-sequencing data from 5 GEO-LUSC datasets. We then classified TCGA-LUSC patients based on ssGSEA and ESTIMATE analyses of LUSC tissues into high, medium and low immunity subgroups showing significant differences in tumor purity. Weighted gene co-expression network analysis of the robust DEGs revealed five immunity-related modules, including the brown module with 762 DEGs and 30 hub genes showing the highest correlation with the immunity-related LUSC patient subgroups and their clinicopathological characteristics. We selected four hub genes, LAPTM5, C1QC, CSF1R and SLCO2B1 , for validation of the immunity status and prognosis of LUSC patients. High expression of these four genes correlated with increased infiltration of immune cell types, upregulation of the immunosuppressive TOX pathway genes, CD8 + T cell exhaustion, and shorter overall survival of LUSC patients. These findings demonstrate that four hub genes regulate tumor infiltration of immune cells, anti-tumor immunity, and survival outcomes in LUSC patients.

Lung squamous cell carcinoma (LUSC) is often diagnosed at the advanced stage with poor prognosis. The mechanisms of its pathogenesis and prognosis require urgent elucidation. This study was performed to screen potential biomarkers related to the occurrence, development and prognosis of LUSC to reveal unknown physiological and pathological processes. Using bioinformatics analysis, the lung squamous cell carcinoma microarray datasets from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases were analyzed to identify differentially expressed genes (DEGs). Furthermore, PPI and WGCNA network analysis were integrated to identify the key genes closely related to the process of LUSC development. In addition, survival analysis was performed to achieve a prognostic model that accomplished good prediction accuracy. Three hundred and thirty-seven up-regulated and 119 down-regulated genes were identified, in which four genes have been found to play vital roles in LUSC development, namely CCNA2, AURKA, AURKB, and FEN1. The prognostic model contained 5 genes, which were all detrimental to prognosis. The AUC of the established prognostic model for predicting the survival of patients at 1, 3, and 5 years was 0.692, 0.722, and 0.651 in the test data, respectively. In conclusion, this study identified several biomarkers of significant interest for additional investigation of the therapies and methods of prognosis of lung squamous cell carcinoma., Competing Interests: The authors declare no conflict of interest.

Background: To investigate the mechanism of microRNA9 in inhibiting proliferation and migration of lung squamous cell carcinoma cells via neuron-restricted silencing factor/epidermal growth factor receptor., Material and Methods: Detection of microRNA9, neuron-restricted silencing factor, and epidermal growth factor receptor expression levels in lung cancer patients' tissues and lung cancer cells by Western blotting and quantitative polymerase chain reaction. Detection of cell proliferation by colony formation assay and cell counting kit-8 assay. Detection of cell migration by wound-healing assay and Transwell assay. And detection of the regulatory effect between neuron-restricted silencing factor and epidermal growth factor receptor by Luciferase reporter gene system. Subcutaneous implantation mouse models of NCI-H520 cells were constructed to detect cell proliferation in vivo , and Kaplan-Meier method calculated patient survival., Results: The expression of microRNA9 and epidermal growth factor receptor was higher in lung cancer tissues than in normal lung tissues, while the expression of neuron-restricted silencing factor was lower in lung cancer tissues than in normal lung tissues. MicroRNA9 higher expression was strongly related to tumor size, and TNM stage and predicted showed reduced overall survival in patients with lung cancer. Further loss of function and enhancement experiments revealed that inhibition of microRNA9 could significantly inhibit lung squamous carcinoma cell proliferation and migration. Luciferase reporter assay demonstrated that microRNA9 could bind to NRSF messenger RNA and inhibit its expression, neuron-restricted silencing factor overexpression also exerted inhibitory effects on cell proliferation and migration. Moreover, Luciferase reporter assay showed that neuron-restricted silencing factor downregulate epidermal growth factor receptor expression levels by binding to epidermal growth factor receptor promoter regions, and Pearson's correlation analysis indicated that the levels of microRNA9 in lung cancer tissues were correlated with neuron-restricted silencing factor and epidermal growth factor receptor. Combined microRNA9 with neuron-restricted silencing factor or epidermal growth factor receptor to predict the prognosis of patients with lung cancer may be more accurate., Conclusion: MicroRNA9 inhibits proliferation and migration of lung squamous cell carcinoma cells by inhibiting neuron-restricted silencing factor/epidermal growth factor receptor axis. MicroRNA9 can be a new prognostic marker and therapeutic target for lung squamous cell carcinoma.

Background: P38MAPK has been investigated as a tumor-related signaling molecule because of its apparent association with tumorigenesis. This study aimed to investigate P38MAPK expression and its role in lung squamous carcinoma (LSCC)., Methods: The expression of P38MAPK and phosphorylated P38 (P-P38) in LSCC tissues and cells was examined by Western blot, real-time PCR, and immunohistochemistry. The influence of P38MAPK inhibitor SB203580 on the proliferation of LSCC cells was detected by MTT and flow cytometry., Results: The expression of P-P38 in LSCC tissues and cells was lower than that in cancer-adjacent normal tissues and normal bronchial epithelial cells (P<0.05). In addition, the expression of P-P38 was downregulated in LSCC tissues of poor differentiation, stages III and IV, and with lymph node metastasis compared with the LSCC tissues of well differentiation, stages I and II, and without lymph node metastasis (P<0.05). Moreover, the cell proliferation of LSCC SK-MES-1 cells treated by P38MAPK inhibitor SB203580 significantly increased in a concentration-dependent manner compared with that of SK-MES-1 cells without SB203580 (P<0.05). The inhibition of P38MAPK promoted the transition of the S phase to the G2 phase., Conclusions: P-P38 was poorly expressed in LSCC tissues and cells. Its low expression was correlated with low-grade differentiation, lymph node metastasis, and advanced stage of LSCC. Inhibition of P38MAPK expression could significantly increase the proliferation of LSCC cells by promoting the transition of the S phase to the G2 phase., Competing Interests: None.

Background: Matrix metalloproteinase 9 (MMP-9) plays an important role in tumor invasion and metastasis, including lung cancer. However, whether variations in serum MMP-9 levels can serve as a biomarker for monitoring chemotherapy curative effect remains unclear. This study was designed to investigate the association between variations in serum MMP-9 levels and chemotherapy curative effect in patients with lung cancer., Patients and Methods: A total of 82 patients with advanced lung cancer were included. All newly diagnosed patients were treated with platinum-based doublet chemotherapy. Serial measurements of serum MMP-9 levels were performed by enzyme-linked immunosorbent assay. In this manner, we chose four time points to examine the association, including before chemotherapy, and 3 weeks after the beginning of the first, second, and fourth cycles of chemotherapy., Results: Compared with the serum level of MMP-9 before progressive disease, patients with progressive disease had elevated serum levels of MMP-9. Compared with the previous time point of collecting specimens, the serum levels of MMP-9 in the patients with a complete response/partial response/stable disease decreased or were maintained stable. The differences of variation in serum MMP-9 levels in patients with different chemotherapy curative effects were all statistically significant after one cycle, two cycles, and four cycles (after one cycle: P<0.001; after two cycles: P<0.001; after four cycles: P=0.01). However, patients with small-cell lung cancer did not exhibit similar test results., Conclusion: The variation in serum MMP-9 levels in patients with non-small-cell lung cancer during chemotherapy was closely related to chemotherapy curative effect and could be useful to monitor chemotherapy curative effect for a small portion of patients.

Similarities in gene expression between both developing embryonic and precancerous tissues and cancer tissues may help identify much-needed biomarkers and therapeutic targets in lung squamous carcinoma. In this study, human lung samples representing ten successive time points, from embryonic development to carcinogenesis, were used to construct global gene expression profiles. Differentially expressed genes with similar expression in precancerous and cancer samples were identified. Using a network-based greedy searching algorithm to analyze the training cohort (n = 69) and three independent testing cohorts, we successfully identified a significant 22-gene module in which expression levels were correlated with overall survival in lung squamous carcinoma patients.

Background: Cactus polysaccharides are the active components of Opuntia dillenii which have been used extensively in folk medicine. In this study, we investigate the anti-tumor effect of cactus polysaccharides on lung squamous carcinoma cells SK-MES-1., Materials and Methods: The inhibitory effect of Cactus polysaccharides on lung squamous carcinoma cells were detected by MTT assay. Cell cycle was determined by flow cytometry and cell apoptosis was determined by AnnexinV assay. Western-blotting was applied to detect P53 and PTEN protein expression in the cells treated with cactus polysaccharides., Results: Results showed that different concentrations of wild cactus polysaccharides prevent SK-MES-1 cells growth and induces S phase arrest. The data also revealed that cactus polysaccharides cause apoptosis in SK-MES-1 cells determined by Annexin-V assay. Furthermore, cactus polysaccharides induced growth arrest and apoptosis may be due to the increase of P53 and phosphatase and tension homolog deleted on chromosome ten (PTEN) protein., Conclusion: Cactus polysaccharides have anti-tumor activity on lung squamous carcinoma cells.