[TRIP13 Enhances Radioresistance of Lung Adenocarcinoma Cells 
through the Homologous Recombination Pathway].

BACKGROUND: Radiation therapy is one of the most common treatments for non-small cell lung cancer (NSCLC). However, the insensitivity of some tumor cells to radiation is one of the major reasons for the poor efficacy of radiotherapy and the poor prognosis of patients, and exploring the underlying mechanisms behind radioresistance is the key to solving this clinical challenge. This study aimed to identify the molecules associated with radioresistance in lung adenocarcinoma (LUAD), identified thyroid hormone receptor interactor 13 (TRIP13) as the main target initially, and explored whether TRIP13 is related to radioresistance in LUAD and the specific mechanism, with the aim of providing theoretical basis and potential targets for the combination therapy of LUAD patients receiving radiotherapy in the clinic. METHODS: Three datasets, GSE18842, GSE19188 and GSE33532, were selected from the Gene Expression Omnibus (GEO) database and screened for differentially expressed genes (|log FC|>1.5, P<0.05) in each of the three datasets using the R 4.1.3 software, and then Venn diagram was used to find out the differentially expressed genes common to the three datasets. The screened differential genes were then subjected to protein-protein interaction (PPI) analysis and module analysis with the help of STRING online tool and Cytoscape software, and survival prognosis analysis was performed for each gene with the help of Kaplan-Meier Plotter database, and the TRIP13 gene was identified as the main molecule for subsequent studies. Subsequently, the human LUAD cell line H292 was irradiated with multiple X-rays using a sub-lethal dose irradiation method to construct a radioresistant cell line, H292DR. The radioresistance of H292DR cells was verified using cell counting kit-8 (CCK-8) assay and clone formation assay. The expression levels of TRIP13 in H292 and H292DR cells were measured by Western blot. Small interfering RNA (siRNA) was used to silence the expression of TRIP13 in H292DR cells and Western blot assay was performed. The clone formation ability and migration ability of H292DR cells were observed after TRIP13 silencing, followed by the detection of changes in the expression levels of proteins closely related to homologous recombination, such as ataxia telangiectasia mutated (ATM) protein. RESULTS: Screening of multiple GEO datasets, validation of external datasets and survival analysis revealed that TRIP13 was highly expressed in LUAD and was associated with poor prognosis in LUAD patients who had received radiation therapy. And the results of gene set enrichment analysis (GSEA) of TRIP13 suggested that TRIP13 might be closely associated with LUAD radioresistance by promoting homologous recombination repair after radiation therapy. Experimentally, TRIP13 expression was found to be upregulated in H292DR, and silencing of TRIP13 was able to increase the sensitivity of H292DR cells to radiation. CONCLUSIONS: TRIP13 is associated with poor prognosis in LUAD patients treated with radiation, possibly by promoting a homologous recombination repair pathway to mediate resistance of LUAD cells to radiation.

CTCs Detection and Whole-exome Sequencing Might Be Used to Differentiate Benign and Malignant Pulmonary Nodules.

BACKGROUND: Low-density computed tomography (LDCT) improved early lung cancer diagnosis but introduces an excess of false-positive pulmonary nodules data. Hence, accurate diagnosis of early-stage lung cancer remains challenging. The purpose of the study was to assess the feasibility of using circulating tumour cells (CTCs) to differentiate malignant from benign pulmonary nodules. METHODS: 122 patients with suspected malignant pulmonary nodules detected on chest CT in preparation for surgery were prospectively recruited. Peripheral blood samples were collected before surgery, and CTCs were identified upon isolation by size of epithelial tumour cells and morphological analysis. Laser capture microdissection, MALBAC amplification, and whole-exome sequencing were performed on 8 samples. The diagnostic efficacy of CTCs counting, and the genomic variation profile of benign and malignant CTCs samples were analysed. RESULTS: Using 2.5 cells/5 mL as the cut-off value, the area under the receiver operating characteristic curve was of 0.651 (95% confidence interval: 0.538-0.764), with a sensitivity and specificity of 0.526 and 0.800, respectively, and positive and negative predictive values of 91.1% and 30.3%, respectively. Distinct sequence variations differences in DNA damage repair-related and driver genes were observed in benign and malignant samples. TP53 mutations were identified in CTCs of four malignant cases; in particular, g.7578115T>C, g.7578645C>T, and g.7579472G>C were exclusively detected in all four malignant samples. CONCLUSIONS: CTCs play an ancillary role in the diagnosis of pulmonary nodules. TP53 mutations in CTCs might be used to identify benign and malignant pulmonary nodules.

High Expression of Cancer-IgG Is Associated With Poor Prognosis and Radioresistance via PI3K/AKT/DNA-PKcs Pathway Regulation in Lung Adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) is the dominant type of lung neoplasms, and radiotherapy is its mainstay treatment, yet poor prognosis caused by radioresistance remains problematic. Cancer-derived immunoglobulin G (cancer-IgG) has been detected in multiple cancers and plays important roles in carcinogenesis. This study aimed to demonstrate that cancer-IgG is associated with poor prognosis of LUAD and to identify its role in radioresistance. METHODS: Cancer-IgG expression was detected by immunohistochemistry from 56 patients with stage III LUAD and by western blot and immunofluorescence in LUAD cell lines and in a human bronchial epithelial cell line. The effects of cancer-IgG silencing on the proliferation and apoptosis of PC9 and H292 cells were evaluated by plate cloning and apoptosis assay; the effects of cancer-IgG silencing on DNA damage repair ability and radiosensitivity were evaluated by colony-forming assay, γH2AX immunofluorescence, and neutral comet assay. Finally, we used the protein phosphorylation microarray and western blot to explore mechanisms involving cancer-IgG that increased radioresistance. RESULTS: Cancer-IgG is widely expressed in stage III LUAD, and the overall survival and disease-free survival of patients with positive expression are notably lower than those of patients with negative expression, indicating the associations between cancer-IgG and poor prognosis as well as radioresistance. The expression of cancer-IgG in the four LUAD cell lines was located mainly on the cell membrane and cytoplasm and not in the normal lung epithelial cell. Knockdown of cancer-IgG in PC9 and H292 cells resulted in increased apoptosis and negatively affected cancer cell proliferation. After irradiation, silencing of cancer-IgG showed a decrease in colonies as well as increases in the Olive tail moment and γH2AX foci in nucleus, indicating that the knockdown of cancer-IgG resulted in a decrease in the damage repair ability of DNA double-strand breaks in LUAD cells and an enhanced radiosensitivity. The expression of p-AKT, p-GSK3ß, and p-DNA-PKcs decreased in the knockdown group after radiotherapy, suggesting that cancer-IgG could affect radiotherapy resistance by mediating double-strand breaks damage repair in LUAD cells through the PI3K/AKT/DNA-PKcs pathway. CONCLUSIONS: This study revealed that cancer-IgG regulates PI3K/AKT/DNA-PKcs signaling pathways to affect radioresistance of LUAD and associated with poor prognosis.

A clinicopathologic analysis of microscopic extension in small cell lung cancer and lung adenocarcinoma: Determination of clinical target volume with precise radiotherapy.

PURPOSE: The identification of the clinical target volume (CTV) is particularly important in the precise radiotherapy of lung cancer. The purpose of this study was to determine the extension margin from gross tumor volume (GTV) to CTV in primary small cell lung cancer (SCLC) and lung adenocarcinoma (ADC) by microscopic extension (ME). MATERIAL AND METHODS: The data of 25 cases of SCLC and 29 cases of ADC from August 2015 to August 2020 were analyzed. The measurement of tumor size between preoperative thoracic computed tomography (CT) and postoperative macroscopic specimens was compared, and the ME range of tumor cells was measured under a microscope to determine its correlation with clinical features and pathological manifestations. RESULTS: A total of 217 slides were examined, corresponding to 103 slides for SCLC and 114 slides for ADC. The radiologic sizes of the tumors in SCLC and ADC were 12.8 and 7.9 mm, respectively (p = 0.09), and the macroscopic sizes were 12.5 and 8.5 mm, respectively (p = 0.07). There was a significant correlation between the radiologic and macroscopic size of the same tumor sample (r = 0.886). Compared with ADC, more SCLC tumor cells infiltrated through vascular or lymphatic dissemination (16% vs. 9%, p = 0.047). The mean ME value was 2.81 mm for SCLC and 2.02 mm for ADC (p = 0.012). To take into account 95% of the ME, a margin of 8 and 7.7 mm must be expanded for SCLC and ADC, respectively. The ME value of the tumor was related to the presence of atelectasis, the location of the tumor, and the Ki-67 cell proliferation index. CONCLUSION: The GTV of the tumor was contoured according to CT images, which was basically consistent with the actual tumor size. The GTVs of SCLC and ADC should be expanded by 8 and 7.7 mm, respectively, to fully cover the subclinical lesions in 95% of cases.

A signature of tumor DNA repair genes associated with the prognosis of surgically-resected lung adenocarcinoma.

BACKGROUND: Lung cancer has the highest morbidity and mortality of cancers worldwide. Lung adenocarcinoma (LUAD) is the most common pathological subtype of lung cancer and surgery is its most common treatment. The dysregulated expression of DNA repair genes is found in a variety of cancers and has been shown to affect the origin and progression of these diseases. However, the function of DNA repair genes in surgically-treated LUAD is unclear. METHODS: We sought to determine the association between the signature of DNA repair genes for patients with surgical LUAD and their overall prognosis. We obtained gene expression data and corresponding clinical information of LUAD from The Cancer Genome Atlas (TCGA) database. The differently expressed DNA repair genes of surgically-treated LUAD and normal tissues were identified using the Wilcoxon rank-sum test. We used uni- and multivariate Cox regression analyses to shrink the aberrantly expressed genes, which were then used to construct the prognostic signature and the risk score formula associated with the independent prognosis of surgically-treated LUAD. We used Kaplan-Meier and Cox hazard ratio analyses to confirm the diagnostic and prognostic roles. Two validation sets (GSE31210 and GSE37745) were downloaded from the Gene Expression Omnibus (GEO) and were used to externally verify the prognostic value of the signature. OSluca online database verifies the hazard ratio for the DNA repair genes by which the signature was constructed. We investigated the correlation between the signature of the DNA repair genes and the clinical parameters. The potential molecular mechanisms and pathways of the prognostic signature were explored using Gene Set Enrichment Analysis (GSEA). RESULTS: We determined the prognostic signature based on six DNA repair genes (PLK1, FOXM1, PTTG1, CCNO, HIST3H2A, and BLM) and calculated the risk score based on this formula. Patients with surgically-treated LUAD were divided into high-risk and low-risk groups according to the median risk score. The high-risk group showed poorer overall survival than the low-risk group; the signature was used as an independent prognostic indicator and had a greater prognostic value in surgically-treated LUAD. The prognostic value was replicated in GSE31210 and GSE37745. OSluca online database analysis shows that six DNA repair genes were associated with poor prognosis in most lung cancer datasets. The prognostic signature risk score correlated with the pathological stage and smoking status in surgically-treated LUAD. The GSEA of the risk signature in high-risk patients showed pathways associated with the cell cycle, oocyte meiosis, mismatch repair, homologous recombination, and nucleotide excision repair. CONCLUSIONS: A six-DNA repair gene signature was determined using TCGA data mining and GEO data verification. The gene signature may serve as a novel prognostic biomarker and therapeutic target for surgically-treated LUAD.