Deubiquitination of CDC6 by OTUD6A promotes tumour progression and chemoresistance.

BACKGROUND: CDC6 is an oncogenic protein whose expression level fluctuates during the cell cycle. Although several E3 ubiquitin ligases responsible for the ubiquitin-mediated proteolysis of CDC6 have been identified, the deubiquitination pathway for CDC6 has not been investigated. METHODS: The proteome-wide deubiquitinase (DUB) screening was used to identify the potential regulator of CDC6. Immunofluorescence, protein half-life and deubiquitination assays were performed to determine the protein stability of CDC6. Gain- and loss-of-function experiments were implemented to analyse the impacts of OUTD6A-CDC6 axis on tumour growth and chemosensitivity in vitro. N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced conditional Otud6a knockout (CKO) mouse model and tumour xenograft model were performed to analyse the role of OTUD6A-CDC6 axis in vivo. Tissue specimens were used to determine the association between OTUD6A and CDC6. RESULTS: OTUD6A interacts with, depolyubiquitinates and stabilizes CDC6 by removing K6-, K33-, and K48-linked polyubiquitination. Moreover, OTUD6A promotes cell proliferation and decreases sensitivity to chemotherapy by upregulating CDC6. CKO mice are less prone to BCa tumorigenesis induced by BBN, and knockdown of OTUD6A inhibits tumour progression in vivo. Furthermore, OTUD6A protein level has a positive correlation with CDC6 protein level, and high protein levels of OTUD6A and CDC6 are associated with poor prognosis in patients with bladder cancer. CONCLUSIONS: We reveal an important yet missing piece of novel DUB governing CDC6 stability. In addition, our findings propose a model for the OTUD6A-CDC6 axis that provides novel insights into cell cycle and chemosensitivity regulation, which may become a potential biomarker and promising drug target for cancer treatment.

Cyclin O promotes lung cancer progression and cetuximab resistance via cell cycle regulation and CDK13 interaction.

Background: Cyclin O (CCNO) is a novel cyclin family protein containing a cyclin-like domain, which plays a role in cell cycle regulation. Recent research suggests that inhibition of CCNO leads to cell apoptosis in gastric cancer, cervical squamous cell carcinoma, and post-operative lung cancer. Methods: The protein expression and signal transduction were detected by Western blot (WB) and immunohistochemistry (IHC). Overexpression or lacking CCNO stable cell lines were transfected with lentiviruses and selected with puromycin. The tumor behaviors of lung adenocarcinoma (LUAD) cells were assessed: cell proliferation by 5-Ethynyl-2'-deoxyuridine (EdU) staining and Cell Counting Kit-8 (CCK8) assay, cell cycle and by flow cytometry analysis, and migration and invasion using wound healing and Transwell system. Co-immunoprecipitation was used to detect protein-protein interactions. Xenograft models for evaluating tumor growth and anti-tumor drug efficacy. Results: A higher expression of CCNO was observed in LUAD cancer tissues and predicted the overall survival of LUAD patients. Moreover, CCNO expression was negatively correlated with cancer cell proliferation, migration, and invasion. Co-immunoprecipitation and western blot indicated that CCNO interacted with CDK13 to promote cancer cell proliferation signaling activation. Furthermore, CCNO promoted tumor cell growth and cetuximab resistance in vivo, and a CDK13 inhibitor effectively inhibited the oncological effect of CCNO. Conclusions: The current study suggests that CCNO may be a driver in the development of LUAD and that its function is related to CDK13 interaction that promotes proliferation signaling activation.

EIF4A3 Acts on the PI3K-AKT-ERK1/2-P70S6K Pathway through FLOT1 to Influence the Development of Lung Adenocarcinoma.

Lung adenocarcinoma (LUAD) is a major lung cancer subtype. In this study, we discovered that the eukaryotic translation initiation factor EIF4A3 expression was significantly higher in LUAD tissues and that this higher expression was closely linked to a poor prognosis for LUAD. In addition, we demonstrated that the knockdown of EIF4A3 significantly inhibited the proliferation, invasion, and migration of LUAD cells in vitro and in vivo. The findings of mass spectrometry analysis revealed that EIF4A3 could interact with Flotillin-1 in LUAD cells and that EIF4A3 could positively regulate the expression of FLOT1 at the protein level. Meanwhile, transcriptome sequencing showed that EIF4A3 could influence the development of LUAD by affecting PI3K-AKT-ERK1/2-P70S6K and PI3K class III-mediated autophagy in the Apelin pathway. In addition, we confirmed that Flotillin-1 expression was upregulated in LUAD based on the existing literature, and knockdown of FLOT1 could inhibit the proliferation and migration of LUAD cells. In addition, the knockdown of Flotillin-1 reversed the increase of cell proliferation and migration caused by EIF4A3 overexpression. Furthermore, we found that the activation of PI3K-AKT-ERK1/2-P70S6K signaling pathway and PI3K class III-mediated autophagy caused by EIF4A3 overexpression was rescued by the knockdown of FLOT1. In a word, we proved that EIF4A3 positively regulates the expression of FLOT1 and plays a procancer role in LUAD. IMPLICATIONS: Our study revealed the role of EIF4A3 in prognosis and tumor progression in LUAD, indicating that EIF4A3 could be used as the molecular diagnostic and prognostic therapeutic target.

Circular RNA circTRPS1-2 inhibits the proliferation and migration of esophageal squamous cell carcinoma by reducing the production of ribosomes.

Circular RNAs play important roles in many cancers, including esophageal squamous cell carcinoma (ESCC), but the precise functions of most circular RNAs are poorly understood. Here we detected significant downregulation of circTRPS1-2 in ESCC based on high-throughput sequencing of three pairs of ESCC tissue and adjacent normal tissue, followed by PCR validation with another 30 tissue pairs. Patients with ESCC whose circTRPS1-2 expression was below the median level for the sample showed significantly shorter median overall survival (13 months) than patients whose circTRPS1-2 expression was above the median (36 months). Overexpressing circTRPS1-2 in the human ESCC cell lines K150 and E109, which express low endogenous levels of circTRPS1-2, inhibited cell proliferation and migration. Conversely, knocking down circTRPS1-2 using short interfering RNA promoted cell proliferation and migration. Similar results were observed in mice bearing K150 xenografts in which circTRPS1-2 was overexpressed or knocked down. Several ribosomal proteins co-immunoprecipitated with circTRPS1-2 from K150 cells in culture, and K150 cells overexpressing circTRPS1-2 showed reduced numbers of ribosomes by A260 absorbance measure and electron microscopy. Our results suggest that circTRPS1-2 can inhibit ESCC proliferation and migration by reducing the production of ribosomes, establishing its potential usefulness in ESCC treatment and prediction of prognosis.

Extrachromosomal circular DNAs are common and functional in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is the leading cause of cancer-related mortality. While recent studies have documented the presence of extrachromosomal circular DNAs (eccDNAs) in various tumors, to date, there have been no studies examining the distribution and function of eccDNAs in ESCC. METHODS: The eccDNAs from three surgically matched ESCC tissue samples were extracted and amplified by rolling circle amplification after removal of linear DNA and mitochondrial circular DNA. High-throughput eccDNA sequencing and bioinformatics analysis was performed to study the distribution pattern and the level of eccDNA expression. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed on the genes associated with the differentially expressed eccDNAs. Five up-regulated and five down-regulated candidate eccDNAs were validated by routine polymerase chain reaction (PCR), TOPO-TA cloning and Sanger sequencing. The nucleotides flanking the eccDNA junctions were analyzed to explore the mechanisms of eccDNA formation. RESULTS: A total of 184,557 eccDNAs was identified. The overall length distribution ranged from 33 to 968,842 base pairs (bp), with the peak at approximately 360 bp. These eccDNAs mainly originated from 5'- and 3'-untranslated regions (UTRs), and rarely from exons, introns, LINE, or Alu repeat regions. The chromosome distribution, length distribution, and genomic annotation of the eccDNAs were comparable between ESCC samples and matched normal epithelium. Nevertheless, 16,031 eccDNAs were found to be differentially expressed between ESCC and matched normal epithelium, including 10,126 up-regulated eccDNAs and 5,905 down-regulated eccDNAs. GO analysis and KEGG pathway analysis showed enriched in cancer pathways, mitogen-activated protein kinase (MAPK) pathway, GTPase-related activity, and cytoskeleton function. PCR, TOPO-TA cloning, and Sanger sequencing validated the junctional sites of five up-regulated candidate eccDNAs and four other unexpected eccDNAs. A repeat nucleotide pattern between the position flanking the start site and that flanking the end site was detected. CONCLUSIONS: This study demonstrated the genome-wide presence of eccDNAs, explored the differential expression of eccDNAs, and revealed the potential mechanisms of eccDNAs in ESCC. This work provides further insights into our understanding of genome plasticity, the role of eccDNAs in ESCC, and may contribute to the development of potential clinical therapies.

N6-methyladenosine demethylase ALKBH5 suppresses malignancy of esophageal cancer by regulating microRNA biogenesis and RAI1 expression.

N6-Methyladenosine (m6A) is the most prevalent epigenetic RNA modification and is vital in regulating malignancies. The roles of m6A modifiers on noncoding RNAs have not been fully investigated in esophageal cancer. By screening all m6A modifiers, ALKBH5 was the most potent member related to patient outcomes and suppressing esophageal cancer malignancy in cell and animal models. It demethylated pri-miR-194-2 and inhibited miR-194-2 biogenesis through an m6A/DGCR8-dependent manner. RAI1, previously considered as a circadian clock transcriptional regulator, was the main target of miR-194-2. It enhanced transcription of Hippo pathway upstream genes by binding to their 3'UTR and suppressed YAP/TAZ nuclear translocation. The ALKBH5/miR-194-2/RAI1 axis was also validated in clinical samples. In addition, the increased malignancy by low ALKBH5 was abolished by the YAP inhibitor verteporfin. Our findings uncover a critical role of ALKBH5 in miRNAs biogenesis and provide novel insight for developing treatment strategies in esophageal cancer.

Let-7b-3p inhibits tumor growth and metastasis by targeting the BRF2-mediated MAPK/ERK pathway in human lung adenocarcinoma.

BACKGROUND: Lung cancer is a malignant tumor with the highest morbidity and mortality rates worldwide, of which lung adenocarcinoma (LUAD) is the most common subtype. Overall, current treatments of LUAD are not satisfactory; therefore, novel targets need to be explored. Let-7b-3p is an important member of the let-7 family of microRNAs (miRNAs), and has not been studied separately in LUAD. This study aimed to investigate the role and molecular mechanism of let-7b-3p in LUAD. METHODS: Herein, let-7b-3p expression was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and fluorescence in situ hybridization (FISH) assays. MTT, colony formation assay, flow cytometry analysis, wound-healing, Transwell and in vivo experiments were conducted to assess let-7b-3p's function in LUAD. The downstream target TFIIB-related factor 2 (BRF2) was predicted using bioinformatics analyses and confirmed by dual-luciferase reporter assay and rescue experiments. Additionally, BRF2 was found to affect the MAPK/ERK pathway through transcriptome sequencing analysis and western blot (WB) assay. RESULTS: Let-7b-3p is downregulated in LUAD cells and tissue samples and low let-7b-3p expression is correlated with a poor prognosis in LUAD patients. Let-7b-3p suppresses the proliferation and metastasis of LUAD cells both in vivo and in vitro by directly targeting the BRF2-mediated MAPK/ERK pathway. CONCLUSIONS: Let-7b-3p inhibits the development of LUAD and is an ideal novel therapeutic target for the treatment of LUAD.

Circular RNA hsa-circ-000881 suppresses the progression of lung adenocarcinoma in vitro via a miR-665/PRICKLE2 axis.

BACKGROUND: Circular RNA (circRNA) has become a new focus in the field of tumor biology research in recent years. Many circRNAs have been showed to play an important role in the progression of lung adenocarcinoma (LUAD). In this work, we studied the oncological role of hsa-circ-000881 in LUAD and attempted to explore the related mechanism. METHODS: The relative expressions of hsa-circ-000881, miR-665, and PRICKLE2 were detected by RT-qPCR or western blot. Functional assays were conducted to analyze the role of hsa-circ-000881 in the proliferation, migration, and invasion of LUAD cells. A luciferase reporter assay was performed to verify whether hsa-circ-000881, miR-665, and PRICKLE2 interact with each other. RESULTS: Circ-000881 was remarkably downregulated in LUAD. Overexpression of circ-000881 attenuated cell growth, migration, and invasion, whereas its knockdown enhanced the malignancy of LUAD cells. The results of luciferase reporter assay and bioinformatics analysis confirmed that circ-000881 served as a sponge for miR-665, and PRICKLE2 was a direct target of miR-665.Overexpression of miR-665 or silencing of PRICKLE2 abolished circ-000881-mediated inhibition of malignant tumor behavior in LUAD cells. CONCLUSIONS: Circ-000881 has inhibitory effects on LUAD via a miR-665/PRICKLE2 axis, suggesting that circ-000881 may be an underlying therapeutic target for LUAD.

Oncogenic role of abnormal spindle­like microcephaly­associated protein in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is a common malignant cancer worldwide. It is urgent to explore its underlying molecular mechanism and identify novel diagnostic biomarkers. Abnormal spindle­like microcephaly (ASPM) has recently received considerable attention due to its function in tumor progression. However, its role in LUAD is unclear. The present study aimed to explore the clinical role of ASPM in LUAD. Seven pairs of LUAD and adjacent normal tissues were collected to identify potential LUAD biomarkers using transcriptome sequencing. The association between ASPM expression and LUAD progression was evaluated using bioinformatics analysis and data obtained from clinical specimens. Using small interfering RNA technology, the function of ASPM was analyzed in the LUAD H1299 and A549 cell lines. Transcriptional profiling of ASPM­deficient H1299 cells was then performed to determine the downstream targets of ASPM. Using databases and clinical specimens, it was revealed that ASPM expression was frequently elevated in LUAD tissues, and this upregulation was highly associated with LUAD progression. ASPM served as an oncogenic regulator of LUAD cell proliferation and metastasis. Mechanistically, ASPM facilitated epithelial­mesenchymal transition (EMT) via the PI3K/AKT signaling pathway and 740 Y­P, an activator of this pathway, restored the migratory ability of ASPM­knockdown LUAD cells. The current study identified ASPM as an independent prognostic biomarker of LUAD that served an important oncogenic role in regulating LUAD cell metastasis by promoting EMT via the PI3K/AKT signaling pathway. Targeting ASPM may therefore be a therapeutic strategy for treating LUAD.

Mex3a interacts with LAMA2 to promote lung adenocarcinoma metastasis via PI3K/AKT pathway.

Lung adenocarcinoma (LUAD) is the main subtype of lung cancer. In this study, we found that RBP Mex3a was significantly upregulated in LUAD tissues and elevated Mex3a expression was associated with poor LUAD prognosis and metastasis. Furthermore, we demonstrated that Mex3a knockdown significantly inhibited LUAD cell migration and invasion in vitro and metastasis in nude mice. Transcriptome sequencing indicated that Mex3a affected gene expression linked to ECM-receptor interactions, including laminin subunit alpha 2(LAMA2). RNA immunoprecipitation (RIP) assay revealed Mex3a directly bound to LAMA2 mRNA and Mex3a increased the instability of LAMA2 mRNA in LUAD cells. Furthermore, we discovered that LAMA2 was surprisingly downregulated in LUAD and inhibited LUAD metastasis. LAMA2 knockdown partially reverse the decrease of cell migration and invasion caused by Mex3a knockdown. In addition, we found that both Mex3a and LAMA2 could influence PI3K-AKT pathway, which are downstream effectors of the ECM-receptor pathway. Moreover, the reduced activation of PI3K-AKT pathway in caused by Mex3a depletion was rescued by LAMA2 knockdown. In conclusion, we demonstrated that Mex3a downregulates LAMA2 expression to exert a prometastatic role in LUAD. Our study revealed the prognostic and prometastatic effects of Mex3a in LUAD, suggesting that Mex3a can serve as a prognostic biomarker and a target for metastatic therapy.