Corrigendum: Advanced NSCLC patients with EGFR T790M harbouring TP53 R273C or KRAS G12V cannot benefit from osimertinib based on a clinical multicentre study by tissue and liquid biopsy.

[This corrects the article DOI: 10.3389/fonc.2021.621992.].

Integrin α3/α6 and αV are implicated in ADAM15-activated FAK and EGFR signalling pathway individually and promote non-small-cell lung cancer progression.

Disintegrin-metalloproteinase 15(ADAM15), a member of disintegrin metalloproteinases (ADAMs), plays important roles in various cancer types. However, the underlying ADAM15 functioning in lung cancer is still unclear. In the present study, we find that ADAM15 regulates the epidermal growth factor receptor/focal adhesion kinase (EGFR/FAK) signalling pathway by interactions with integrins. Integrin αV is involved in ADAM15-mediated FAK signalling. Further, we find that ADAM15 and CD151 were co-expressed, and the presence of ADAM15 affected the integrin α3/α6-related EGFR signalling pathway by cooperating with CD151. In addition, we also prove the effect of ADAM15 on proliferation in nude mice. Finally, we show that ADAM15 is a direct target of miR-204-5p by luciferase reporter assays, qRT-PCR and western blot analyses. Our findings provide molecular and cellular evidence that ADAM15 promotes cell proliferation and metastasis in NSCLC, which might provide a potential target for NSCLC treatment.

CPNE1 promotes non-small cell lung cancer progression by interacting with RACK1 via the MET signaling pathway.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and the most lethal tumour worldwide. Copine 1 (CPNE1) was identified as a novel oncogene in NSCLC in our previous study. However, its specific function and relative mechanisms remain poorly understood. METHODS: The biological role of CPNE1 and RACK1 in NSCLC was investigated using gene expression knockdown and overexpression, cell proliferation assays, clonogenic assays, and Transwell assays. The expression levels of CPNE1, RACK1 and other proteins were determined by western blot analysis. The relationship between CPNE1 and RACK1 was predicted and investigated by mass spectrometry analysis, immunofluorescence staining, and coimmunoprecipitation. NSCLC cells were treated with a combination of a MET inhibitor and gefitinib in vitro and in vivo. RESULTS: We found that CPNE1 facilitates tumorigenesis in NSCLC by interacting with RACK1, which further induces activation of MET signaling. CPNE1 overexpression promoted cell proliferation, migration, invasion and MET signaling in NSCLC cells, whereas CPNE1 knockdown produced the opposite effects. In addition, the suppression of the enhancing effect of CPNE1 overexpression on tumorigenesis and MET signaling by knockdown of RACK1 was verified. Moreover, compared to single-agent treatment, dual blockade of MET and EGFR resulted in enhanced reductions in the tumour volume and downstream signaling in vivo. CONCLUSIONS: Our findings show that CPNE1 promotes tumorigenesis by interacting with RACK1 and activating MET signaling. The combination of a MET inhibitor with an EGFR-TKI attenuated tumour growth more significantly than either single-drug treatment. These findings may provide new insights into the biological function of CPNE1 and the development of novel therapeutic strategies for NSCLC. Video Abstract.

The regulation of CPNE1 ubiquitination by the NEDD4L is involved in the pathogenesis of non-small cell lung cancer.

Our previous studies revealed that oncogene CPNE1 is positively correlated with the occurrence, TNM stage, lymph node metastasis, and distant metastasis of non-small-cell lung cancer (NSCLC), and it could be regulated by micro RNAs. But no direct role of post-translational modification of CPNE1 in NSCLC has been reported. This study confirms that CPNE1 is degraded by two pathways: the ubiquitin-proteasome pathway and the autophagy-lysosome pathway. CPNE1 binds with the ubiquitin molecule via its K157 residue. Moreover, we determined that the ubiquitin ligase NEDD4L can mediate the ubiquitination of CPNE1 and promote its degradation. In addition, we find that NEDD4L knockdown promotes the proliferation and metastasis of NSCLC cells by regulating CPNE1 in vitro and vivo. This study aims to further investigate the mechanism of CPNE1 ubiquitination in the occurrence and development of NSCLC and provide a new potential target for NSCLC treatment.

ALCAP2 inhibits lung adenocarcinoma cell proliferation, migration and invasion via the ubiquitination of ß-catenin by upregulating the E3 ligase NEDD4L.

Lung cancer is recognized as the leading cause of cancer-related death worldwide, with non-small cell lung cancer (NSCLC) being the predominant subtype, accounting for approximately 85% of lung cancer cases. Although great efforts have been made to treat lung cancer, no proven method has been found thus far. Considering ß, ß-dimethyl-acryl-alkannin (ALCAP2), a natural small-molecule compound isolated from the root of Lithospermum erythrorhizon. We found that lung adenocarcinoma (LUAD) cell proliferation and metastasis can be significantly inhibited after treatment with ALCAP2 in vitro, as it can induce cell apoptosis and arrest the cell cycle. ALCAP2 also significantly suppressed the volume of tumours in mice without inducing obvious toxicity in vivo. Mechanistically, we revealed that ALCAP2-treated cells can suppress the nuclear translocation of ß-catenin by upregulating the E3 ligase NEDD4L, facilitating the binding of ubiquitin to ß-catenin and eventually affecting the wnt-triggered transcription of genes such as survivin, cyclin D1, and MMP9. As a result, our findings suggest that targeting the oncogene ß-catenin with ALCAP2 can inhibit the proliferation and metastasis of LUAD cells, and therefore, ALCAP2 may be a new drug candidate for use in LUAD therapeutics.

CD151 drives cancer progression depending on integrin α3ß1 through EGFR signaling in non-small cell lung cancer.

BACKGROUND: Tetraspanins CD151, a transmembrane 4 superfamily protein, has been identified participating in the initiation of a variety of cancers. However, the precise function of CD151 in non-small cell lung cancer (NSCLC) remains unclear. Here, we addressed the pro-tumoral role of CD151 in NSCLC by targeting EGFR/ErbB2 which favors tumor proliferation, migration and invasion. METHODS: First, the mRNA expression levels of CD151 in NSCLC tissues and cell lines were measured by RT-PCR. Meanwhile, CD151 and its associated proteins were analyzed by western blotting. The expression levels of CD151 in NSCLC samples and its paired adjacent lung tissues were then verified by Immunohistochemistry. The protein interactions are evaluated by co-immunoprecipitation. Flow cytometry was applied to cell cycle analysis. CCK-8, EdU Incorporation, and clonogenic assays were used to analyze cell viability. Wound healing, transwell migration, and matrigel invasion assays were utilized to assess the motility of tumor cells. To investigate the role of CD151 in vivo, lung carcinoma xenograft mouse model was applied. RESULTS: High CD151 expression was identified in NSCLC tissues and cell lines, and its high expression was significantly associated with poor prognosis of NSCLC patients. Further, knockdown of CD151 in vitro inhibited tumor proliferation, migration, and invasion. Besides, inoculation of nude mice with CD151-overexpressing tumor cells exhibited substantial tumor proliferation compared to that in control mice which inoculated with vector-transfected tumor cells. Noteworthy, we found that overexpression of CD151 conferred cell migration and invasion by interacting with integrins. We next sought to demonstrate that CD151 regulated downstream signaling pathways via activation of EGFR/ErbB2 in NSCLC cells. Therefore, we infer that CD151 probably affects the sensitivity of NSCLC in response to anti-cancer drugs. CONCLUSIONS: Based on these results, we demonstrated a new mechanism of CD151-mediated tumor progression by targeting EGFR/ErbB2 signaling pathway, by which CD151 promotes NSCLC proliferation, migration, and invasion, which may considered as a potential target of NSCLC treatment.

Construction of a Prognostic Immune-Related LncRNA Risk Model for Lung Adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) originates mainly from the mucous epithelium and glandular epithelium of the bronchi. It is the most common pathologic subtype of non-small cell lung cancer (NSCLC). At present, there is still a lack of clear criteria to predict the efficacy of immunotherapy. The 5-year survival rate for LUAD patients remains low. METHODS: All data were downloaded from The Cancer Genome Atlas (TCGA) database. We used Gene Set Enrichment Analysis (GSEA) database to obtain immune-related mRNAs. Immune-related lncRNAs were acquired by using the correlation test of the immune-related genes with R version 3.6.3 (Pearson correlation coefficient cor = 0.5, P < 0.05). The TCGA-LUAD dataset was divided into the testing set and the training set randomly. Based on the training set to perform univariate and multivariate Cox regression analyses, we screened prognostic immune-related lncRNAs and given a risk score to each sample. Samples were divided into the high-risk group and the low-risk group according to the median risk score. By the combination of Kaplan-Meier (KM) survival curve, the receiver operating characteristic (ROC) (AUC) curve, the independent risk factor analysis, and the clinical data of the samples, we assessed the accuracy of the risk model. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed on the differentially expressed mRNAs between the high-risk group and the low-risk group. The differentially expressed genes related to immune response between two risk groups were analyzed to evaluate the role of the model in predicting the efficacy and effects of immunotherapy. In order to explain the internal mechanism of the risk model in predicting the efficacy of immunotherapy, we analyzed the differentially expressed genes related to epithelial-mesenchymal transition (EMT) between two risk groups. We extracted RNA from normal bronchial epithelial cell and LUAD cells and verified the expression level of lncRNAs in the risk model by a quantitative real-time polymerase chain reaction (qRT-PCR) test. We compared our risk model with other published prognostic signatures with data from an independent cohort. We transfected LUAD cell with siRNA-LINC0253. Western blot analysis was performed to observed change of EMT-related marker in protein level. RESULTS: Through univariate Cox regression analysis, 24 immune-related lncRNAs were found to be strongly associated with the survival of the TCGA-LUAD dataset. Utilizing multivariate Cox regression analysis, 10 lncRNAs were selected to establish the risk model. The K-M survival curves and the ROC (AUC) curves proved that the risk model has a fine predictive effect. The GO enrichment analysis indicated that the effect of the differentially expressed genes between high-risk and low-risk groups is mainly involved in immune response and intercellular interaction. The KEGG enrichment analysis indicated that the differentially expressed genes between high-risk and low-risk groups are mainly involved in endocytosis and the MAPK signaling pathway. The expression of genes related to the efficacy of immunotherapy was significantly different between the two groups. A qRT-PCR test verified the expression level of lncRNAs in LUAD cells in the risk model. The AUC of ROC of 5 years in the independent validation dataset showed that this model had superior accuracy. Western blot analysis verified the change of EMT-related marker in protein level. CONCLUSION: The immune lncRNA risk model established by us could better predict the prognosis of patients with LUAD.

Advanced NSCLC Patients With EGFR T790M Harboring TP53 R273C or KRAS G12V Cannot Benefit From Osimertinib Based on a Clinical Multicentre Study by Tissue and Liquid Biopsy.

BACKGROUND: Non-small cell lung cancer (NSCLC) patients treated with first-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) almost always acquire resistance, and the development of novel techniques analyzing circulating tumor DNA (ctDNA) have made it possible for liquid biopsy to detect genetic alterations from limited amount of DNA with less invasiveness. While a large amount of patients with EGFR exon 21 p.Thr790 Met (T790M) benefited from osimertinib treatment, acquired resistance to osimertinb has subsequently become a growing challenge. METHODS: We performed tissue and liquid rebiopsy on 50 patients with EGFR-mutant NSCLC who acquired resistance to first-generation EGFR-TKIs. Plasma samples underwent droplet digital PCR (ddPCR) and next-generation sequencing (NGS) examinations. Corresponding tissue samples underwent NGS and Cobas® EGFR Mutation Test v2 (Cobas) examinations. RESULTS: Of the 50 patients evaluated, the mutation detection rates of liquid biopsy group and tissue biopsy group demonstrated no significant differences (41/48, 85.4% vs. 44/48, 91.7%; OR=0.53, 95% CI=0.15 to 1.95). Overall concordance, defined as the proportion of patients for whom at least one identical genomic alteration was identified in both tissue and plasma, was 78.3% (36/46, 95% CI=0.39 to 2.69). Moreover, our results showed that almost half of the patients (46%, 23/50) resistant to first-generation EGFR-TKI harbored p.Thr790 Met (T790M) mutation. 82.6% (19/23) of the T790M positive patients were analyzed by liquid biopsy and 60.9% (14/23) by tumor tissue sequencing. Meanwhile, a wide range of uncommon mutations was detected, and novel mechanisms of osimertinib resistance were discovered. In addition, 16.7% (2/12) of the T790M positive patients with either TP53 R237C or KRAS G12V failed to benefit from the subsequent osimertinib treatment. CONCLUSION: Our results emphasized that liquid biopsy is applicable to analyze the drug resistance mechanisms of NSCLC patients treated with EGFR-TKIs. Moreover, we discovered two uncommon mutations, TP53 R273C and KRAS G12V, which attenuates the effectiveness of osimertinib.

EVI5 is an oncogene that regulates the proliferation and metastasis of NSCLC cells.

BACKGROUND: The Ecotropic viral integration site 5 (EVI5), an important protein in regulating cell cycle, cytokinesis and cellular membrane traffic, functions as a stabilizing factor maintaining anaphase-promoting complex/cyclosome (APC/C) inhibitor Emi1 in S/G2 phase. However, the mechanism by which EVI5 promotes malignant transformation of non-small cell lung cancer (NSCLC) remains unknown. In the present study, we addressed the role of EVI5 in NSCLC by regulating tumor growth, migration and invasion. METHODS: The expression levels of EVI5 and miR-486-5p in NSCLC tissues and cells were measured by real-time PCR. Meanwhile, EVI5 and its associated protein expression were analyzed by western blot and co-immunoprecipitation assay. Flow cytometry was performed to determine cell proliferation and apoptosis. CCK-8 and clonogenic assays were used to analyze cell viability. Wound healing, transwell migration and matrigel invasion assays were utilized to assess the motility of tumor cells. To investigate the role of EVI5 in vivo, lung carcinoma xenograft mouse model was applied.. RESULTS: EVI5 was upregulated in NSCLC tissues and cell lines when compared with that in normal tissues and cell line. Knockdown of EVI5 in vitro inhibited tumor cell proliferation, migration and invasion in NSCLC cells. Further, inoculation of EVI5-deficient tumor cells into nude mice suppressed tumor proliferation and metastasis compared to control mice inoculated with unmanipulated tumor cells. These data indicated that EVI5 promote the proliferation of NSCLC cells which was consistent with our previous results. Additionally, we showed that EVI5 was directly regulated by miR-486-5p, and miR-486-5p-EVI5 axis affected the NSCLC migration and invasion through TGF-ß/Smad signaling pathway by interacting with TGF-ß receptor II and TGF-ß receptor I. CONCLUSIONS: Based on these results, we demonstrated a new post-transcriptional mechanism of EVI5 regulation via miR-486-5p and the protumoral function of EVI5 in NSCLC by interacting with Emi1 and/or TGF-ß receptors, which provides a new insight into the targeted therapy of NSCLC.