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Supplementary Data from YY1 Complex Promotes Quaking Expression via Super-Enhancer Binding during EMT of Hepatocellular Carcinoma

Quaking (QKI) is an alternative splicing factor that can regulate circRNA formation in the progression of epithelial–mesenchymal transition, but the mechanism remains unclear. High expression of QKI is correlated with short survival time, metastasis, and high clinical stage and pathology grade in hepatocellular carcinoma (HCC). Here we report that transcription of the QKI gene was activated by the Yin-Yang 1 (YY1)/p65/p300 complex, in which YY1 bound to the super-enhancer and promoter of QKI, p65 combined with the promoter, and p300 served as a mediator to maintain the stability of the complex. This YY1/p65/p300 complex increased QKI expression to promote the malignancy of HCC as well as an increased circRNA formation in vitro and in vivo. Hyperoside is one of several plant-derived flavonol glycoside compounds. Through virtual screening and antitumor activity analysis, we found that hyperoside inhibited QKI expression by targeting the YY1/p65/p300 complex. Overall, our study suggests that the regulatory mechanism of QKI depends on the YY1/p65/p300 complex and that it may serve as a potential target for treatment of HCC.Significance:These findings identify the YY1/p65/p300 complex as a regulator of QKI expression, identifying several potential therapeutic targets for the treatment of HCC.

Although it is established that the sustained psychologic stress conditions under which patients with tumors often reside accelerates malignant progression of tumors, the molecular mechanism behind this association is unclear. In this work, the effect of psychologic stress on tumor progression was verified using a stress-stimulated tumor-bearing mouse model (Str-tumor). Both D2 dopamine receptor (DRD2) and hypoxia-inducible factor-1α (HIF1α) were highly expressed in the nucleus of Str-tumors. Treatment with trifluoperazine (TFP), a DRD2 inhibitor, elicited better antitumor effects in Str-tumors than the control group. These results indicate that DRD2 may mediate stress-induced malignant tumor progression. DRD2 interacted with von Hippel-Lindau (VHL) in the nucleus, and competitive binding of DRD2 and HIF1α to VHL resulted in reduced ubiquitination-mediated degradation of HIF1α, enhancing the epithelial-mesenchymal transition of tumor cells. TFP acted as an interface inhibitor between DRD2 and VHL to promote the degradation of HIF1α. In conclusion, DRD2 may promote the progression of malignant tumors induced by psychologic stress via activation of the oxygen-independent HIF1α pathway, and TFP may serve as a therapeutic strategy for stress management in patients with cancer. Significance: This work identifies DRD2 regulation of HIF1α as a mechanism underlying the progression of malignant tumors stimulated by psychologic stress and suggests that DRD2 inhibition can mitigate these stress conditions in patients. See related commentary by Bernabé, p. 5144

Long-term ultraviolet (UV) exposure can cause inflammation, pigmentation and photoaging. All-trans retinoic acid (ATRA/tretinoin) is a commonly used retinoic acid receptor (RAR) agonist in the clinical treatment of UV-induced skin problems. However, the use of such drugs is often accompanied by systemic adverse reactions caused by nonspecific activation of RARs. Therefore, this study was designed to screen for a novel RAR-γ-selective agonist with high safety.Molecular docking, dynamic simulation and Biacore were used to screen and identify novel RAR-γ-selective agonists. RT-PCR, ELISA, western blotting, immunofluorescence staining, flow cytometry and proteomic analysis were used to detect the effects of these novel RAR-γ selective agonists on UVA-induced inflammation and photoaging cell models. UVA-induced mouse models were used to evaluate the effects of tectorigenin on skin repair, ageing and inflammation.Tectorigenin is a novel RAR-γ-selective agonist, which inhibits UV-induced oxidative damage, inflammatory factor release and matrix metalloproteinase (MMP) production. Tectorigenin can also reverse the UVA-induced loss of collagen. The results of the signalling pathway research showed that tectorigenin mainly affects the MAPK/JNK/AP-1 pathway. In animal experiments, tectorigenin showed better anti-inflammatory and anti-photoaging effects, and caused less skin irritation than ATRA. Nano-particle loaded tectorigenin significantly improved the utilization of tectorigenin.Tectorignen is a non-retinol RAR-γ-selective agonist that can inhibit UV-induced skin damage and could be developed as a safe pharmaceutical component for the prevention of photoaging and skin inflammation.

Sepsis is a disease characterized by multiple organ failure caused by immune hyperactivation and cytokine storms. Studies have shown that the incidence of sepsis in melanoma patients is substantially lower compared to the general population. It is also observed that experimental tumor-bearing animals have high survival rates after sepsis induction, suggesting that tumors may suppress sepsis-associated immune overactivation, thereby alleviating sepsis. Based on the above-described findings, this work assesses whether tumor cells play an antisepsis role in mice through the secretion of exosomes. Analysis of exosome activity reveals that the induced exosomes (iExo) secreted by tumor cells following lipopolysaccharide (LPS) treatment improve sepsis to a greater extent than normal secretory exosomes. Further analysis reveals that iExo exert their protective effects mainly through seven key miRNAs. In vitro bionic simulation of exosomes is carried out using exosome mimics generated by loading the aforementioned microRNAs into hyaluronic acid-polyethylenimine nanoparticles. Exosome mimics at specific miRNA ratios alleviate sepsis in mice and cynomolgus monkeys, indicating that biomimetic simulation of tumor-suppressive exosomes may represent a promising therapeutic method for the treatment of sepsis and cytokine-storm-related conditions.

Rationale: Many external factors can induce the melanogenesis and inflammation of the skin. Salidroside (SAL) is the main active ingredient of Rhodiola, which is a perennial grass plant of the Family Crassulaceae. This study evaluated the effect and molecular mechanism of SAL on skin inflammation and melanin production. It then explored the molecular mechanism of melanin production under ultraviolet (UV) and inflammatory stimulation. Methods: VISIA skin analysis imaging system and DermaLab instruments were used to detect the melanin reduction and skin brightness improvement rate of the volunteers. UV-treated Kunming mice were used to detect the effect of SAL on skin inflammation and melanin production. Molecular docking and Biacore were used to verify the target of SAL. Immunofluorescence, luciferase reporter assay, CO-IP, pull-down, Western blot, proximity ligation assay (PLA), and qPCR were used to investigate the molecular mechanism by which SAL regulates skin inflammation and melanin production. Results: SAL can inhibit the inflammation and melanin production of the volunteers. SAL also exerted a protective effect on the UV-treated Kunming mice. SAL can inhibit the tyrosinase (TYR) activity and TYR mRNA expression in A375 cells. SAL can also regulate the ubiquitination degradation of interferon regulatory factor 1 (IRF1) by targeting prolyl 4-hydroxylase beta polypeptide (P4HB) to mediate inflammation and melanin production. This study also revealed that IRF1 and upstream stimulatory factor 1 (USF1) can form a transcription complex to regulate TYR mRNA expression. IRF1 also mediated inflammatory reaction and TYR expression under UV- and lipopolysaccharide-induced conditions. Moreover, SAL derivative SAL-plus (1-(3,5-dihydroxyphenyl) ethyl-β-d-glucoside) showed better effect on inflammation and melanin production than SAL. Conclusion: SAL can inhibit the inflammation and melanogenesis of the skin by targeting P4HB and regulating the formation of the IRF1/USF1 transcription complex. In addition, SAL-plus may be a new melanin production and inflammatory inhibitor.

Summary Multi-targeted kinase inhibitors, such as sorafenib, have been used in various malignancies, but their efficacy in clinical applications varies among individuals and lacks pretherapeutic prediction measures. We applied the concept of “click chemistry” to pathological staining and established a drug-loaded probe staining assay. We stained the cells and different types of pathological sections and demonstrated that the assay was reliable. We further verified in cells, cell-derived xenograft model, and clinical level that the staining intensity of the probe could reflect drug sensitivity. The stained samples from 300 patients who suffered from hepatocellular carcinoma and used the sorafenib probe also indicated that staining intensity was closely related to clinical information and could be used as an independent marker without undergoing sorafenib therapy for prognosis. This assay provided new ideas for multi-target drug clinical trials, pre-medication prediction, and pathological research., Graphical Abstract, Highlights • The assay could be used for the rapid sensitivity detection of multi-target drugs • The assay could serve as an auxiliary technology for NGS and PDX • Sorafenib had multiple unknown primary contribution targets • Drug-probe staining could be used as an independent diagnostic and prognostic marker, Drugs; Medical Biochemistry; Biochemical Assay

Quaking (QKI) is an alternative splicing factor that can regulate circRNA formation in the progression of epithelial–mesenchymal transition, but the mechanism remains unclear. High expression of QKI is correlated with short survival time, metastasis, and high clinical stage and pathology grade in hepatocellular carcinoma (HCC). Here we report that transcription of the QKI gene was activated by the Yin-Yang 1 (YY1)/p65/p300 complex, in which YY1 bound to the super-enhancer and promoter of QKI, p65 combined with the promoter, and p300 served as a mediator to maintain the stability of the complex. This YY1/p65/p300 complex increased QKI expression to promote the malignancy of HCC as well as an increased circRNA formation in vitro and in vivo. Hyperoside is one of several plant-derived flavonol glycoside compounds. Through virtual screening and antitumor activity analysis, we found that hyperoside inhibited QKI expression by targeting the YY1/p65/p300 complex. Overall, our study suggests that the regulatory mechanism of QKI depends on the YY1/p65/p300 complex and that it may serve as a potential target for treatment of HCC. Significance: These findings identify the YY1/p65/p300 complex as a regulator of QKI expression, identifying several potential therapeutic targets for the treatment of HCC.

Rationale: The role of SLUG in epithelial-mesenchymal transition during tumor progression has been thoroughly studied, but its precise regulation remains poorly explored. Methods: The affinity purification, mass spectrometry and CO-IP were performed to identify the interaction between SLUG and ubiquitin-specific protease 5 (USP5). Cycloheximide chase assays and deubiquitination assays confirmed that the effect of USP5 on the deubiquitin of SLUG. The dual-luciferase reporter and chromatin immunoprecipitation assays were employed to observe the direct transcriptional regulation of E-cadherin by SLUG effected by USP5. EMT related markers was detected by western blotting and immunofluorescence. Molecular docking, SPR sensor (biacore) and co-location were detected to prove Formononetin targets USP5. Bioinformatics analysis was used to study the relation of USP5 and SLUG to malignancy degree of HCC. Cell migration, invasion in HCC cells and xenografts model in nude mouse were conducted to detect the promotion of USP5 and the inhibition of Formononetin on EMT. Results: USP5 interacts with and stabilizes SLUG to regulate its abundance through USP5 deubiquitination activities in epithelial-mesenchymal transition (EMT) of hepatocellular carcinoma (HCC). USP5 is highly expressed and positively correlated with SLUG expression in HCC with high malignancy. Knockdown of USP5 inhibits SLUG deubiquitination and inhibits HCC cells proliferation, metastasis, and invasion, while overexpression of USP5 promotes SLUG stability and EMT in vitro and in vivo. Through virtual screening, we found that Formononetin exhibits excellent binding to USP5. Moreover, Formononetin inhibits deubiquitinating activities of USP5 to SLUG and consequently impedes the EMT and malignant progression of HCC. Conclusion: Our findings reveal that USP5 serve as a potential target for tumor intervention and provide a preliminary antitumor therapy for inhibit EMT by targeting USP5 or its interaction with SLUG in HCC.

Hepatocellular carcinoma (HCC) is a typical hypervascular solid tumor. Vasculogenic mimicry (VM) formed by aggressive tumor cells to mimic vasculogenic networks plays an important role in the tumor malignancy of HCC. Hsp90β promotes endothelial cell-dependent angiogenesis in HCC. However, the relationship between Hsp90β and VM formation is unclear. In this study, we found that Hsp90β is positively correlated with VM and EMT marker proteins in HCC tissues and promotes tube formation, cell migration, and invasion in vitro. Hsp90β interacts with Twist1 and promotes its deubiquitination and stabilization to nuclear translocation and enhances the VE-cadherin promoter activity. Results of in vitro analysis indicate that Hsp90β enhances the tumor VM in tumor-burdened mice, and the Hsp90 inhibitor NVP-BEP800 suppresses VM formation by releasing Hsp90β and Twist1 interaction. This study provides a potential antitumor therapy for inhibiting VM by targeting Hsp90β in HCC.

Twist is a critical epithelial–mesenchymal transition (EMT)–inducing transcription factor that increases expression of vimentin. How Twist1 regulates this expression remains unclear. Here, we report that Twist1 regulates Cullin2 (Cul2) circular RNA to increase expression of vimentin in EMT. Twist1 bound the Cul2 promoter to activate its transcription and to selectively promote expression of Cul2 circular RNA (circ-10720), but not mRNA. circ-10720 positively correlated with Twist1, tumor malignance, and poor prognosis in hepatocellular carcinoma (HCC). Twist1 promoted vimentin expression by increasing levels of circ-10720, which can absorb miRNAs that target vimentin. circ-10720 knockdown counteracted the tumor-promoting activity of Twist1 in vitro and in patient-derived xenograft and diethylnitrosamine-induced TetOn-Twist1 transgenic mouse HCC models. These data unveil a mechanism by which Twist1 regulates vimentin during EMT. They also provide potential therapeutic targets for HCC treatment and provide new insight for circular RNA (circRNA)-based diagnostic and therapeutic strategies.Significance: A circRNA-based mechanism drives Twist1-mediated regulation of vimentin during EMT and provides potential therapeutic targets for treatment of HCC.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/15/4150/F1.large.jpg. Cancer Res; 78(15); 4150–62. ©2018 AACR.

Doxycycline displays high efficiency for cancer therapy. However, the molecular mechanism is poorly understood. In our previous study, doxycycline was found to suppress tumor progression by directly targeting proteinase-activated receptor 1 (PAR1). In this study, microRNAs were found to be involved in PAR1-mediated anti-tumor effects of doxycycline. Among these miRNAs, miR-17 was found to promote breast cancer cell metastasis both in vivo and in vitro. Moreover, miR-17 could reverse partial doxycycline inhibition effects on breast cancer. Employing luciferase and chromatin immunoprecipitation assays, nuclear factor-kappaB (NF-κB) was found to bind miR-17 promoters. Furthermore, E-cadherin was identified as the target gene of miR-17. These results showed that miR-17 can resist the inhibitory effects of doxycycline on breast cancer epithelial-mesenchymal transformation (EMT) by targeting E-cadherin.

The co-silencing of multiple tumor suppressor genes can lead to escalated malignancy in cancer cells. Given the limited efficacy of anticancer therapies targeting single tumor suppressor genes, we developed small circular single-stranded DNA (CSSD) that can up-regulate the expression of co-silenced tumor suppressor genes by sequestering microRNAs (miRNAs) that negatively regulate these genes. We found that cancer patients with low tumor expression of the tumor suppressor genes KLF17, CDH1, and LASS2 had shortened survival times. The up-regulation of these genes upon transfection of artificial CSSD-9 inhibited tumor proliferation and metastasis and promoted apoptosis in vitro as well as in ex vivo and patient-derived xenograft models. In addition, CSSD is more stable and effective than current miRNA inhibitors, and transfecting CSSDs via nanoparticles substantially improved delivery efficiency. The use of a single CSSD can promote the inhibition of multiple tumor suppressor genes. This study provides evidence for the possibility of using CSSDs as therapeutic miRNA inhibitors to target the co-silencing of multiple tumor suppressor genes.

Background Vascular endothelial growth factor receptors (VEGFRs) are the major receptors involved in endothelial cell-dependent tumor angiogenesis. There are studies account for the effects of Hsp90 on angiogenesis, but the role and mechanism of Hsp90β isoforms and NVP-BEP800, a specific inhibitor of Hsp90β, in tumor angiogenesis is rarely mentioned. Methods Immunohistochemistry and statistical analysis was used to evaluate the correlation between Hsp90β expression, CD31 endothelial cell-dependent vessel density, and VEGFRs expression in tissue samples of 96 HCCs. Kaplan-Meier survival analysis and COX proportional hazards analysis the relation of Hsp90β and prognosis. HUVEC cells were transfected with Hsp90β or treated with NVP-BEP800, and then cell proliferation, migration, invasion and tube formation were investigated. The VEGFR1 and VEGFR2 expression was determined by Western blot and immunofluorescence. The VEGFR1 and VEGFR2 promoter activities were detected by dual luciferase report system. In vivo, the angiogenesis promotion of Hsp90β and anti-angiogenesis efficacy of NVP-BEP800 was tested in HCC xenograft models. Histological analysis was performed on tumor samples to evaluate Hsp90β, VEGFRs expression and MVD. Results This study investigated the correlation between Hsp90β expression and CD31+ endothelial cell-dependent vessel density. Hsp90β promoted VEGFRs expression by increasing their promoter activities. The proliferation, migration, invasion, and tube formation activities of human endothelial cells significantly increased when Hsp90β was overexpressed. NVP-BEP800 down-regulated VEGFRs expression to significantly reduce tubular differentiation, as well as endothelial cell proliferation, migration, and invasion. Furthermore, NVP-BEP800 decreased VEGFR1 and VEGFR2 promoter activities. In vivo, Hsp90β promoted VEGFRs and CD31 expression in human hepatocellular carcinoma tumor xenografts and was associated with increased tumor microvessel density. After 18 days of treatment with 30 mg/kg/day NVP-BEP800, VEGFRs and CD31 expression significantly decreased. Conclusion Hsp90β induced endothelial cell-dependent tumor angiogenesis by activating VEGFRs transcription. NVP-BEP800 has potential as a therapeutic strategy for inhibiting tumor angiogenesis by decreasing endothelial cell progression and metastasis. It can help develop a therapeutic strategy for tumor treatment through the inhibition of endothelial cell progression and metastasis. Electronic supplementary material The online version of this article (doi:10.1186/s12943-017-0640-9) contains supplementary material, which is available to authorized users.