Novel exosomal circEGFR facilitates triple negative breast cancer autophagy via promoting TFEB nuclear trafficking and modulating miR-224-5p/ATG13/ULK1 feedback loop.

Triple-negative breast cancer (TNBC) cells are in a more hypoxic and starved state than non-TNBC cells, which makes TNBC cells always maintain high autophagy levels. Emerging evidence has demonstrated that circular RNAs (circRNAs) are involved in the progress of tumorigenesis. However, the regulation and functions of autophagy-induced circRNAs in TNBC remain unclear. In our study, autophagy-responsive circRNA candidates in TNBC cells under amino acid starved were identified by RNA sequencing. The results showed that circEGFR expression was significantly upregulated in autophagic cells. Knockdown of circEGFR inhibited autophagy in TNBC cells, and circEGFR derived from exosomes induced autophagy in recipient cells in the tumor microenvironment. In vitro and in vivo functional assays identified circEGFR as an oncogenic circRNA in TNBC. Clinically, circEGFR was significantly upregulated in TNBC and was positively associated with lymph node metastasis. CircEGFR in plasma-derived exosomes was upregulated in breast cancer patients compared with healthy people. Mechanistically, circEGFR facilitated the translocation of Annexin A2 (ANXA2) toward the plasma membrane in TNBC cells, which led to the release of Transcription Factor EB (a transcription factor of autophagy-related proteins, TFEB) from ANXA2-TFEB complex, causing nuclear translocation of TFEB, thereby promoting autophagy in TNBC cells. Meanwhile, circEGFR acted as ceRNA by directly binding to miR-224-5p and inhibited the expression of miR-224-5p, which weakened the suppressive role of miR-224-5p/ATG13/ULK1 axis on autophagy. Overall, our study demonstrates the key role of circEGFR in autophagy, malignant progression, and metastasis of TNBC. These indicate circEGFR is a potential diagnosis biomarker and therapeutic target for TNBC.

MiR-3653 blocks autophagy to inhibit epithelial-mesenchymal transition in breast cancer cells by targeting the autophagy-regulatory genes ATG12 and AMBRA1.

BACKGROUND: Metastasis is the main cause of tumor-associated death and mainly responsible for treatment failure of breast cancer. Autophagy accelerates tumor metastasis. In our work, we aimed to investigate the possibility of microRNAs (miRNAs) which participate in the regulation of autophagy to inhibit tumor metastasis. METHODS: MiRNA array and comprehensive analysis were performed to identify miRNAs which participated in the regulation of autophagy to inhibit tumor metastasis. The expression levels of miR-3653 in breast cancer tissues and cells were detected by quantitative real-time polymerase chain reaction. In vivo and in vitro assays were conducted to determine the function of miR-3653. The target genes of miR-3653 were detected by a dual luciferase reporter activity assay and Western blot. The relationship between miR-3653 and epithelial-mesenchymal transition (EMT) was assessed by Western blot. Student's t -test was used to analyze the difference between any two groups, and the difference among multiple groups was analyzed with one-way analysis of variance and a Bonferroni post hoc test. RESULTS: miR-3653 was downregulated in breast cancer cells with high metastatic ability, and high expression of miR-3653 blocked autophagic flux in breast cancer cells. Clinically, low expression of miR-3653 in breast cancer tissues (0.054 ±â€Š0.013 vs . 0.131 ±â€Š0.028, t  = 2.475, P  = 0.014) was positively correlated with lymph node metastasis (0.015 ±â€Š0.004 vs . 0.078 ±â€Š0.020, t  = 2.319, P  = 0.023) and poor prognosis ( P  < 0.001). miR-3653 ameliorated the malignant phenotypes of breast cancer cells, including proliferation, migration (MDA-MB-231: 0.353 ±â€Š0.013 vs . 1.000 ±â€Š0.038, t  = 16.290, P  < 0.001; MDA-MB-468: 0.200 ±â€Š0.014 vs . 1.000 ±â€Š0.043, t  = 17.530, P  < 0.001), invasion (MDA-MB-231: 0.723 ±â€Š0.056 vs . 1.000 ±â€Š0.035, t  = 4.223, P  = 0.013; MDA-MB-468: 0.222 ±â€Š0.016 vs . 1.000 ±â€Š0.019, t  = 31.050, P  < 0.001), and colony formation (MDA-MB-231: 0.472 ±â€Š0.022 vs . 1.000 ±â€Š0.022, t  = 16.620, P  < 0.001; MDA-MB-468: 0.650 ±â€Š0.040 vs . 1.000 ±â€Š0.098, t  = 3.297, P  = 0.030). The autophagy-associated genes autophagy-related gene 12 ( ATG12 ) and activating molecule in beclin 1-regulated autophagy protein 1 ( AMBRA1 ) are target genes of miR-3653. Further studies showed that miR-3653 inhibited EMT by targeting ATG12 and AMBRA1 . CONCLUSIONS: Our findings suggested that miR-3653 inhibits the autophagy process by targeting ATG12 and AMBRA1 , thereby inhibiting EMT, and provided a new idea and target for the metastasis of breast cancer.

HLJ2 Effectively Ameliorates Colitis-Associated Cancer via Inhibition of NF-κB and Epithelial-Mesenchymal Transition.

INTRODUCTION: Colitis-associated cancer (CAC) accounts for approximately 15% of IBD patient mortalities. However, currently available anti-CAC drugs possess many disadvantages including safety, specificity and side effects. Therefore, the development of novel anti-CAC compounds is imperative. HLJ2 was a monomeric compound synthesized by our institute and reported to have an effect on ulcer colitis. METHODS: In vivo the AOM/DSS-induced CAC model was used to evaluate the effects of HLJ2 on ameliorating CAC symptoms, immunohistochemical analysis was used to analyze the pathological damage to colons and epithelial-mesenchymal transition was for changes of cytokines. In vitro, flow cytometric analysis, immunofluorescence and Western blot were used to detect the inhibition effect of HLJ2 on nuclear factor-κB and epithelial-mesenchymal transition in TGF-ß1-stimulated SW480 cells. RESULTS: In the AOM/DSS animal model, HLJ2 was demonstrated to inhibit the secretion of inflammatory cytokines and nuclear factor-κB, levels of tumorigenesis-related proteins including snail, and finally inhibited a key step in metastasis, epithelial-mesenchymal transition. In vitro, HLJ2 was also shown to inhibit nuclear factor-κB and epithelial-mesenchymal transition in TGF-ß1-stimulated SW480 cells in accordance with in vivo results. Meanwhile, the nuclear factor-κB inhibitor could interrupt the effect of HLJ2 on epithelial-mesenchymal transition. DISCUSSION: HLJ2 may ameliorate CAC through inhibiting nuclear factor-κB and then downstream epithelial-mesenchymal transition. The combination of the obvious improvement in effects on CAC without obvious side effects suggests that HLJ2 could be developed as a potential CAC therapeutic candidate.

Colitis Is Effectively Ameliorated by (±)-8-Acetonyl-dihydrocoptisine via the XBP1-NF-κB Pathway.

Ulcerative colitis (UC) is a recurrent, chronic intestinal disease. Available treatments for UC are poor effective and/or cause severe adverse events. X-box binding protein 1 (XBP1) and nuclear factor-κB (NF-κB) have been reported to play important roles in UC. Specifically, deletion or downregulation of XBP1 leads to spontaneous enteritis and results in imbalanced secretion of NF-κB and other proinflammatory cytokines. (±)-8-acetonyl-dihydrocoptisine, i.e., (±)-8-ADC, is a monomer semi-synthesized from coptisine. In vitro, (±)-8-ADC activated the transcriptional activity of XBP1, inhibited expression of NF-κB, and reduced production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1ß), in lipopolysaccharide-stimulated IEC6 cells. Therefore, silencing XBP1 would reduce the inhibition effect of (±)-8-ADC on NF-κB expression and the cytokines secretion in vitro. In a dextran sulfate sodium-induced colitis mouse model, oral administration of (±)-8-ADC ameliorated weight loss and colon contracture, and decreased the average disease activity index score and pathological damage. Simultaneously, (±)-8-ADC also increased XBP1 expression, and decreased NF-κB expression and secretion of myeloperoxidase, TNF-α, IL-6 and IL-1ß in the colon. Therefore, (±)-8-ADC may ameliorate UC via the XBP1-NF-κB pathway and should be considered as a therapeutic candidate for UC.

Development of an XBP1 agonist, HLJ2, as a potential therapeutic agent for ulcerative colitis.

There is a severe lack of effective treatments for ulcerative colitis (UC), a recurrent and intractable inflammatory bowel disease. The identification of valid targets and new drugs is an urgent need. In this study, we identified the XBP-1 agonist HLJ2 as a promising treatment candidate. In an in vivo mouse model of DSS-induced colitis, HLJ2 decreased weight loss, colon contracture, disease activity index (DAI), colon mucosa damage index (CMDI) and histopathological index (HI). HLJ2 also decreased myeloperoxidase (MPO) activity and reduced production of the inflammatory cytokines TNF-α, IL-1ß, and IL-6. HLJ2 improved intestinal mucosa damage induced by dextran sodium sulfate (DSS) and increased the expression of ZO-1 and claudin-1. Fecal 16s rRNA high-throughput sequencing demonstrated a significant improvement in UC intestinal dysbacteriosis in mice treated with HLJ2, including increased abundance of probiotics such as Lachnospiraceae, Prevotellaceae, and Lactobacillaceae. At the same time there was a reduction in the abundance of pathogenic or conditional pathogenic microorganisms such as Bacteroidaceae, Porphyromonadaceae, Deferribacteraceae, and Pseudomonadaceae in HLJ2-treated mice compared with untreated mice. Our results demonstrated that the XBP1 agonist HLJ2 inhibits inflammation, regulates the intestinal flora, and protects the intestinal mucosa. It is thus a potential therapeutic agent for ulcerative colitis.