Disrupting of IGF2BP3-stabilized HK2 mRNA by MYO16-AS1 competitively binding impairs LUAD migration and invasion.

Since invasive cancer is associated with poor clinical outcomes, exploring the molecular mechanism underlying LUAD progression is crucial to improve the prognosis of patients with advanced disease. Herein, we found that MYO16-AS1 is expressed mainly in lung tissue but is notably downregulated in LUAD tissues. Overexpression of MYO16-AS1 inhibited the migration and invasion of LUAD cells. Mechanistic studies indicated that H3K27Ac modification mediated MYO16-AS1 transcription. Furthermore, we found that MYO16-AS1 competitively bound to the IGF2BP3 protein and in turn reduced IGF2BP3 protein binding to HK2 mRNA, decreasing HK2 mRNA stability and inhibiting glucose metabolism reprogramming and LUAD cell invasion in vitro and in vivo. The finding that the MYO16-AS1/IGF2BP3-mediated glucose metabolism reprogramming mechanism regulates HK2 expression provides novel insight into the process of LUAD invasion and suggests that MYO16-AS1 may be a therapeutic target for LUAD.

Circ_16601 facilitates Hippo pathway signaling via the miR-5580-5p/FGB axis to promote my-CAF recruitment in the TME and LUAD progression.

BACKGROUND: Lung cancer represents a significant public health issue in China, given its high incidence and mortality rates. Circular RNAs (circRNAs) have been recently proposed to participate in the development and progression of tumors. Nevertheless, their particular roles in the pathogenesis of lung adenocarcinoma (LUAD), the tumor microenvironment (TME), and the underlying molecular mechanisms are still not well understood. METHODS: High-throughput sequencing was used to analyze the circRNAs expression profiles in 7 pairs of human LUAD tissues. shRNA was used to knockdown the YAP1 and FGB genes. RNA sequencing and RT-qPCR were performed to classify the regulatory effects of circ_16601 in LUAD cells. The progression effect of circ_16601 on lung cancer was investigated in vitro and in vivo. RESULTS: The circ_16601 is significantly elevated in LUAD tissues compared to adjacent normal lung tissues, and its high expression is positively associated with poor prognosis in LUAD patients. Additionally, circ_16601 overexpression promotes LUAD cell proliferation in vitro and increases xenograft tissue growth in mice in vivo; circ_16601 also could recruit fibroblasts to cancer associate fibroblasts. Mechanistically, circ_16601 can directly bind to miR-5580-5p, preventing its ability to degrade FGB mRNA and enhancing its stability. Subsequently, circ_16601 promotes the activation of the Hippo pathway in a YAP1-dependent manner, leading to LUAD progression. CONCLUSIONS: Our findings shed valuable insights into the regulatory role of circ_16601 in LUAD progression and highlight its potential as a diagnostic and therapeutic target in LUAD. Overall, this study provides theoretical support to improve the prognosis and quality of life of patients suffering from this devastating disease.

JSI-124 inhibits cell proliferation and tumor growth by inducing autophagy and apoptosis in murine malignant mesothelioma.

Malignant pleural mesothelioma (MPM), mainly caused by asbestos exposure, has a poor prognosis and lacks effective treatment compared with other cancer types. The intracellular transcription factor signal transducer and activator of transcription 3 (STAT3) is overexpressed and hyperactivated in most human cancers. In this study, the role of STAT3 in murine MPM was examined. Inhibition of the Janus kinase 2 (JAK2)/STAT3 pathway with the selective inhibitor JSI-124 has an antitumor effect in murine MPM. Specifically, we demonstrated that JSI-124 inhibits murine MPM cell growth and induces apoptotic and autophagic cell death. Exposure of RN5 and AB12 cells to JSI-124 resulted in apoptosis via the Bcl-2 family of proteins. JSI-124 triggered autophagosome formation, accumulation, and conversion of LC3I to LC3II. Autophagy inhibitors, Chloroquine (CQ) and Bafilomycin A1 (Baf-A1), inhibited autophagy and sensitized RN5 and AB12 cells to JSI-124-induced apoptosis. Our data indicate that JSI-124 is a promising therapeutic agent for MPM treatment.

Circular RNA circ-FIRRE interacts with HNRNPC to promote esophageal squamous cell carcinoma progression by stabilizing GLI2 mRNA.

Increasing evidence has shown that circular RNAs (circRNAs) interact with RNA-binding proteins (RBPs) and promote cancer progression. However, the function and mechanism of the circRNA/RBP complex in esophageal squamous cell carcinoma (ESCC) are still largely unknown. Herein, we first characterized a novel oncogenic circRNA, circ-FIRRE, by RNA sequencing (Ribo-free) profiling of ESCC samples. Furthermore, we observed marked circ-FIRRE overexpression in ESCC patients with high TNM stage and poor overall survival. Mechanistic studies indicated that circ-FIRRE, as a platform, interacts with the heterogeneous nuclear ribonucleoprotein C (HNRNPC) protein to stabilize GLI2 mRNA by directly binding to its 3'-UTR in the cytoplasm, thereby resulting in elevated GLI2 protein expression and subsequent transcription of its target genes MYC, CCNE1, and CCNE2, ultimately contributing to ESCC progression. Moreover, HNRNPC overexpression in circ-FIRRE knockdown cells notably abolished circ-FIRRE knockdown-mediated Hedgehog pathway inhibition and ESCC progression impairment in vitro and in vivo. Clinical specimen results showed that circ-FIRRE and HNRNPC expression was positively correlated with GLI2 expression, which reveals the clear significance of the circ-FIRRE/HNRNPC-GLI2 axis in ESCC. In summary, our results indicate that circ-FIRRE could serve as a valuable biomarker and potential therapeutic target for ESCC and highlight a novel mechanism of the circ-FIRRE/HNRNPC complex in ESCC progression regulation.

CircABCA13 acts as a miR-4429 sponge to facilitate esophageal squamous cell carcinoma development by stabilizing SRXN1.

Circular RNAs (circRNAs) play a pivotal role in the tumorigenesis and progression of various cancers. However, the role and mechanisms of circABCA13 in esophageal squamous cell carcinoma (ESCC) are largely unknown. Here, we reported that circABCA13, a novel circular RNA generated by back-splicing of the intron of the ABCA13 gene, is highly expressed in ESCC tumor tissues and cell lines. Upregulation of circABCA13 correlated with TNM stage and a poor prognosis in ESCC patients. While knockdown of circABCA13 in ESCC cells significantly reduced cell proliferation, migration, invasion, and anchorage-independent growth, overexpression of circABCA13 facilitated tumor growth both in vitro and in vivo. In addition, circABCA13 directly binds to miR-4429 and sequesters miR-4429 from its endogenous target, SRXN1 mRNA, which subsequently upregulates SRXN1 and promotes ESCC progression. Consistently, overexpression of miR-4429 or knockdown of SRXN1 abolished malignant behavior promotion of ESCC results from circABCA13 overexpression in vitro and in vivo. Collectively, our study uncovered the oncogenic role of circABCA13 and its mechanism in ESCC, suggesting that circABCA13 could be a potential therapeutic target and a predictive biomarker for ESCC patients.

Exosomal miR-10527-5p Inhibits Migration, Invasion, Lymphangiogenesis and Lymphatic Metastasis by Affecting Wnt/ß-Catenin Signaling via Rab10 in Esophageal Squamous Cell Carcinoma.

Background: Cancer cell-derived exosomal microRNAs (miRNAs) play critical role in orchestrating intercellular communication between tumor cells and tumor microenvironmental factors, including lymphatic endothelial cells (LECs). Nevertheless, the functions and underlying mechanisms of exosomal miRNAs in lymphatic metastasis and lymphangiogenesis in esophageal squamous cell carcinoma (ESCC) remain unclear. Methods: Small RNA sequencing, Gene Expression Omnibus (GEO) analysis and qRT‒PCR were performed to identify the candidate exosomal miRNAs involved in ESCC metastasis. Receiver operating characteristic curve analysis was conducted to evaluate the diagnostic potential of exosomal miR-10527-5p in predicting lymph node metastasis (LNM) status. An in vitro coculture system was used to investigate the effects of exosomal miR-10527-5p on ESCC cells and human LECs (HLECs), followed by a popliteal LNM assay in vivo. The relationship between miR-10527-5p and Rab10 was identified by dual-luciferase reporter, fluorescence in situ hybridization and qRT‒PCR assays. Then, a series of rescue assays were performed to further investigate whether Rab10 is involved in exosomal miR-10527-5p mediated ESCC metastasis. Results: MiR-10527-5p was found to be notably reduced in both the plasma exosomes and tumor tissues of ESCC patients with LNM, and plasma exosomal miR-10527-5p had a high sensitivity and specificity for discrimination of LNM status. Moreover, exosome-shuttled miR-10527-5p suppressed the migration, invasion and epithelial-to-mesenchymal transition (EMT) of ESCC cells as well as the migration and tube formation of HLECs via Wnt/ß-catenin signaling in vitro and in vivo. Further investigation revealed that Rab10 was a direct target of miR-10527-5p, and re-expression of Rab10 neutralized the inhibitory effects of exosomal miR-10527-5p. Conclusion: Our study demonstrated that exosomal miR-10527-5p had a strong capability to predict preoperative LNM status and anti-lymphangiogenic effect. Exosomal miR-10527-5p inhibited lymphangiogenesis and lymphatic metastasis of ESCC in a vascular endothelial growth factor-C (VEGF-C)-independent manner, showing potential as a therapeutic target for ESCC patients.

USP36 facilitates esophageal squamous carcinoma progression via stabilizing YAP.

Esophageal squamous carcinoma (ESCC) is the major subtype of esophageal cancer in China, accounting for 90% of cases. Recent studies revealed that abnormalities in the Hippo/YAP axis are pervasive in ESCC and are recognized as the important driver of ESCC progression. Since the activity of Hippo signaling is controlled by phosphorylation cascade, it is a mystery why the major effector YAP is still over-activated when the cascade is inhibited. Several studies suggested that in addition to phosphorylation, other protein modifications such as ubiquitination also play important roles in manipulating Hippo/YAP signaling activity. Since YAP protein stability is controlled via an appropriate balance between E3 ubiquitin ligases and deubiquitinases, we performed deubiquitinase siRNA screening and identified USP36 as a deubiquitinase significantly related to Hippo/YAP signaling activity and ESCC progression. USP36 expression was elevated in ESCC samples and correlated with poor differentiation. USP36 expression was correlated with YAP protein levels in ESCC samples. Molecular studies demonstrated that USP36 associated with the YAP protein and enhanced YAP protein stability by blocking the K48-linked polyubiquitination of YAP. In conclusion, our study revealed a novel deubiquitinase in regulating Hippo signaling in ESCC, which could be an encouraging drug target for Hippo-driven ESCC.

Triptolide promotes degradation of the unfolded gain-of-function Tp53R175H/Y220C mutant protein by initiating heat shock protein 70 transcription in non-small cell lung cancer.

Background: The mutation rate of the tumor protein P53 (TP53) has been reported to be greater than 50% in non-small cell lung cancer (NSCLC), and gain-of-function (GOF) mutations in unfolded P53 (TP53R175H and TP53Y220C) have been associated with poor prognosis. However, the best treatment for patients with NSCLC harboring unfolded mutant P53 (mutp53) remains unclear. Triptolide is a natural compound derived from Tripterygium wilfordii that has shown a strong antitumor effect in a variety of cancers. Our study aimed to explore the GOF mutations in unfolded mutp53 (TP53R175H and TP53Y220C) and to clarify the molecular mechanisms by which triptolide regulates the degradation of unfolded mutp53 proteins in NSCLC. Methods: Two unfolded proteins harboring TP53R175H and TP53Y220C mutations were selected to explore their functions in NSCLC progression. NCI-H1299 cells (TP53-null) were transfected with wild-type TP53 (TP53WT), TP53R175H, or TP53Y220C genes and treated with triptolide or a vehicle. Wound healing and transwell assays were performed to measure cell migration and invasion in vitro. Lung metastasis models were constructed through tail vein injection of mutant cells into BALB/c nude mice to evaluate the effect of triptolide on metastasis in vivo. Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), immunoprecipitation, and dual-luciferase reporter assays were performed to explore the relevant molecular mechanisms. Results: Our study revealed that triptolide treatment reduced TP53R175H levels and that the TP53Y220C mutation enhanced the invasion and migration of NCI-H1299 cells. Mechanistically, triptolide promoted TP53R175H and TP53Y220C protein proteasomal degradation mediated through the E3 ligase murine double minute 2 (MDM2) by directly interacting with heat shock protein 70 (HSP70). Moreover, by upregulating HSP70 transcription, triptolide contributed to the protein degradation of the GOF mutp53. Conclusions: Our study reports, for the first time, the mechanism underlying triptolide-regulated protein degradation of TP53R175H or TP53Y220C, which offers new insight into developing a better therapeutic strategy for patients with NSCLC who express the unfolded mutp53 GOF protein.

Synergic effect of PD-1 blockade and endostar on the PI3K/AKT/mTOR-mediated autophagy and angiogenesis in Lewis lung carcinoma mouse model.

BACKGROUND: Immunotherapy has been shown to be effective as a first-line treatment option for non-small cell lung cancer (NSCLC) patients. Unfortunately, it has failed to acquire an anticipant anti-tumour effect for relatively lower clinical benefit rates. It is therefore important to identify novel strategies for improving immunotherapy. Endostar is a novel recombinant human endostatin that exerts its anti-angiogenic effects via vascular endothelial growth factor (VEGF)-related signalling pathways. Anti-programmed death receptor 1 (PD-1) antibody is an immune checkpoint inhibitor that was developed to stimulate the immune system. In this study, the synergy of PD-1 blockade and endostar was assessed in a lung carcinoma mouse model. METHODS: Lewis lung carcinoma (LLC)-bearing mice were randomly assigned into three groups: controls, anti-PD-1 and anti-PD-1+endostar. The levels of cytokines such as interleukin (IL)-17, transforming growth factor-ß1 (TGF-ß1) and interferon-γ (IFN-γ) were measured with enzyme-linked immune sorbent assay (ELISA). The expression of VEGF, CD34 and CD31 was assessed with immunohistochemistry (IHC). The proportion of mature dendritic cells (mDC) and myeloid-derived suppressor cells (MDSC) was analysed with flow cytometry. The major proteins in PI3K/AKT/mTOR and autophagy were quantified with Western blot. RESULTS: Anti-PD-1 combined with endostar dramatically suppressed tumour growth in LLC mouse models. This synergistic effect resulted in decreased pro-inflammatory cytokine IL-17 and immunosuppressive factor TGF-ß1 levels, increased IFN-γ secretion, reduced myeloid-derived suppressor cell (MDSC) accumulation, and reversed CD8 + T cell suppression. The expression of VEGF, CD34 and CD31 was significantly down-regulated, while tumour cell apoptosis and PI3K/AKT/mTOR-mediated autophagy was up-regulated. CONCLUSION: The combination of anti-PD-1 and endostar has a remarkably synergic effect on LLC tumour growth by means of improving the tumour microenvironment and activating autophagy.

Salivary Microbial Dysbiosis is Associated with Systemic Inflammatory Markers and Predicted Oral Metabolites in Non-Small Cell Lung Cancer Patients.

An increasing number of studies have suggested the dysbiosis of salivary microbiome has been linked to the advancement of multiple diseases and proved to be helpful for the diagnosis of them. Although epidemiological studies of salivary microbiota in carcinogenesis are mounting, no systemic study exists regarding the oral microbiota of non-small cell lung cancer (NSCLC) patients. In this study, we presented the characteristics of the salivary microbiota in patients from NSCLC and healthy controls by sequencing of the 16S rRNA microbial genes. Our result revealed distinct salivary microbiota composition in patients from NSCLC compared to the healthy controls. As principal co-ordinates analysis (PCoA) showed, saliva samples clearly differed between the two groups, considering the weighted (p = 0.001, R2 = 0.17), and unweighted (p = 0.001, R2 = 0.25) UniFrac distance. Phylum Firmicutes (31.69% vs 24.25%, p < 0.05) and its two genera Veillonella (15.51%% vs 9.35%, p < 0.05) and Streptococcus (9.96% vs 6.83%, p < 0.05) were strongly increased in NSCLC group compared to the controls. Additionally, the relative abundances of Fusobacterium (3.06% vs 4.92%, p = 0.08), Prevotella (1.45% vs 3.52%, p < 0.001), Bacteroides (0.56% vs 2.24%, p < 0.001), and Faecalibacterium (0.21% vs 1.00%, p < 0.001) in NSCLC group were generally decreased. Furthermore, we investigated the correlations between systemic inflammation markers and salivary microbiota. Neutrophil-lymphocyte ratio (NLR) positively correlated with the Veillonella (r =0.350, p = 0.007) and lymphocyte-monocyte ratio (LMR) negatively correlated with Streptococcus (r =-0.340, p = 0.008). Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways inferred by phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) showed that pathways related to xenobiotics biodegradation and metabolism (p < 0.05) and amino acid metabolism (p < 0.05) were enriched in the NSCLC group. Folate biosynthesis (p < 0.05) significantly decreased in NSCLC group. The specific correlations of clinical systemic inflammation markers and predicted KEGG pathways also could pronounce a broad understanding of salivary microbiota in patients with NSCLC. Moreover, our study extended the new sight into salivary microbiota-targeted interventions to clinically improve the therapeutic strategies for salivary dysbiosis in NSCLC patients. Further investigations of the potential mechanism of salivary microbiota in the progression of NSCLC are still in demand.

Alterations of fecal bacterial communities in patients with lung cancer.

Emerging evidence suggests the microbiome may affect a number of diseases, including lung cancer. However, the direct relationship between gut bacteria and lung cancer remains uncharacterized. In this study, we directly sequenced the hypervariable V1-V2 regions of the 16S rRNA gene in fecal samples from patients with lung cancer and healthy volunteers. Unweighted principal coordinate analysis (PCoA) revealed a clear difference in the bacterial community membership between the lung cancer group and the healthy control group. The lung cancer group had remarkably higher levels of Bacteroidetes, Fusobacteria, Cyanobacteria, Spirochaetes, and Lentisphaerae but dramatically lower levels of Firmicutes and Verrucomicrobia than the healthy control group (P < 0.05). Despite significant interindividual variation, eight predominant genera were significantly different between the two groups. The lung cancer group had higher levels of Bacteroides, Veillonella, and Fusobacterium but lower levels of Escherichia-Shigella, Kluyvera, Fecalibacterium, Enterobacter, and Dialister than the healthy control group (P < 0.05). Most notably, correlations between certain specific bacteria and serum inflammatory biomarkers were identified. Our findings demonstrated an altered bacterial community in patients with lung cancer, providing a significant step in understanding the relationship between gut bacteria and lung cancer. To our knowledge, this is the first study to evaluate the correlations between certain specific bacteria and inflammatory indicators. To better understand this relationship, further studies should investigate the underlying mechanisms of gut bacteria in lung cancer animal models.

Oxytocin evokes a pulsatile PGE2 release from ileum mucosa and is required for repair of intestinal epithelium after injury.

We measured the short-circuit current (Isc) in rat ileum mucosa to identify the effect of oxytocin (OT) on mucosal secretion in small intestine. We identified a COX-2-derived pulsatile PGE2 release triggered by OT in rat ileum mucosa. OT receptors (OTR) are expressed in intestine crypt epithelial cells. Notably, OT evoked a dynamic change of [Ca(2+)]i in ileum crypts, which was responsible for this pulsatile release of PGE2. OT ameliorated 5-FU-, radiation- or DSS- induced injury in vivo, including the improvement of weight loss, reduced villus height and impaired survival of crypt transit-amplifying cells as well as crypt. Moreover, these protective effects of OT against intestinal injury were eliminated by coadministration of a selective inhibitor of PGE2, AH6809. Our findings strongly suggest that OT, a novel and important regulator of intestine mucosa barrier, is required for repair of intestinal epithelium after injury. Considering that OT is an FDA-approved drug, this work reveals a potential novel and safe way to combat or prevent chemo-radiotherapy induced intestine injury or to treat IBD.