EGR3 Inhibits Tumor Progression by Inducing Schwann Cell-Like Differentiation.

The mechanism and function of the expression of Schwann characteristics by nevus cells in the mature zone of the dermis are unknown. Early growth response 3 (EGR3) induces Schwann cell-like differentiation of melanoma cells by simulating the process of nevus maturation, which leads to a strong phenotypic transformation of the cells, including the formation of long protrusions and a decrease in cell motility, proliferation, and melanin production. Meanwhile, EGR3 regulates the levels of myelin protein zero (MPZ) and collagen type I alpha 1 chain (COL1A1) through SRY-box transcription factor 10 (SOX10)-dependent and independent mechanisms, by binding to non-strictly conserved motifs, respectively. Schwann cell-like differentiation demonstrates significant benefits in both in vivo and clinical studies. Finally, a CD86-P2A-EGR3 recombinant mRNA vaccine is developed which leads to tumor control through forced cell differentiation and enhanced immune infiltration. Together, these data support further development of the recombinant mRNA as a treatment for cancer.

MSC microvesicles loaded G-quadruplex-enhanced circular single-stranded DNA-9 inhibits tumor growth by targeting MDSCs.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) promote tumor growth, metastasis, and lead to immunotherapy resistance. Studies revealed that miRNAs are also expressed in MDSCs and promote the immunosuppressive function of MDSCs. Currently, few studies have been reported on inducible cellular microvesicle delivery of nucleic acid drugs targeting miRNA in MDSCs for the treatment of malignant tumors. RESULTS AND CONCLUSION: In this study, we designed an artificial DNA named G-quadruplex-enhanced circular single-stranded DNA-9 (G4-CSSD9), that specifically adsorbs the miR-9 sequence. Its advanced DNA folding structure, rich in tandem repeat guanine (G-quadruplex), also provides good stability. Mesenchymal stem cells (MSCs) were prepared into nanostructured vesicles by membrane extrusion. The MSC microvesicles-encapsulated G4-CSSD9 (MVs@G4-CSSD9) was delivered into MDSCs, which affected the downstream transcription and translation process, and reduced the immunosuppressive function of MDSCs, so as to achieve the purpose of treating melanoma. In particular, it provides an idea for the malignant tumor treatment.

Intratumor Mycoplasma promotes the initiation and progression of hepatocellular carcinoma.

The carcinogenesis and progression of hepatocellular carcinoma (HCC) are closely related to viral infection and intestinal bacteria. However, little is known about bacteria within the HCC tumor microenvironment. Here, we showed that intratumoral Mycoplasma hyorhinis (M. hyorhinis) promoted the initiation and progression of HCC by enhancing nuclear ploidy. We quantified M. hyorhinis in clinical tissue specimens of HCC and observed that patients with high M. hyorhinis load had poor prognosis. We found that gastrointestinal M. hyorhinis can retrogradely infect the liver through the oral-duodenal-hepatopancreatic ampulla route. We further found that the increases in mononuclear polyploidy and cancer stemness resulted from mitochondrial fission caused by intracellular M. hyorhinis. Mechanistically, M. hyorhinis infection promoted the decay of mitochondrial fusion protein (MFN) 1 mRNA in an m6A-dependent manner. Our findings indicated that M. hyorhinis infection promoted pathological polyploidization and suggested that Mycoplasma clearance with antibiotics or regulating mitochondrial dynamics might have the potential for HCC therapy.

Polydopamine-loaded prunetin nanomaterials activate DRD2 to reduce UV-induced inflammation by stabilizing and promoting Nrf2 nuclear translocation.

Skin damage caused by exposure to ultraviolet (UV) light has been well documented clinically and histologically. Dopamine receptor D2 (DRD2) possesses various biological functions. However, no study has reported the possible association of DRD2 with UV-induced skin damage. We established DRD2 conditional knockout and UV damage models in this work. The results showed that DRD2 played an important role in the treatment of UV-induced skin damage. The findings of the molecular mechanism study revealed that the internalization of DRD2 after activation can stabilize nuclear factor erythroid 2-related factor 2 (Nrf2). However, the entry of Nrf2 into the nucleus did not increase. We prepared and characterized hyaluronic acid (HA)-coated mesoporous polydopamine (MPDA) nanoparticles (H@P@M). HA facilitated skin epidermal penetration of the nanoparticles to reach the site of inflammation smoothly. Meanwhile, MPDA activated DRD2 internalization to stabilize Nrf2. The release of prunetin inhibited the interaction of Kelch-like ECH-associated protein 1 with Nrf2 and promoted the nuclear translocation of Nrf2. In summary, this study unveiled that in skin inflammation, H@P@M activated and internalized DRD2, which subsequently formed a protein complex with arrestin beta 1-ubiquitin specific protease 8 (USP8)-Nrf2. Deubiquitination was performed to stabilize Nrf2 while promoting the nuclear translocation of Nrf2 to exert anti-inflammatory and antioxidant functions. STATEMENT OF SIGNIFICANCE: Skin is the body's largest physical barrier, always protecting the body from the interference of the external environment. However, excessive exposure to ultraviolet rays in the sun can cause skin inflammation, leading to skin erythema, itching, edema and pain, which can be troublesome in our daily lives. The complex mechanism of skin inflammation caused by ultraviolet radiation has not been fully clarified. In this study, the role of DRD2 in UV-induced skin inflammation was explored, and nano-composite particles HA@Prunetin@MPDA, which act on multiple targets in the anti-inflammatory pathway of DRD2, were developed to maximize the effect of the drug. It provides a new way to treat skin inflammation caused by UV.

HSF4/COIL complex-dependent R-loop mediates ultraviolet-induced inflammatory skin injury.

Intense ultraviolet (UV) exposure can cause phototoxic reactions, such as skin inflammation, resulting in injury. UV is a direct cause of DNA damage, but the mechanisms underlying transcriptional regulation within cells after DNA damage are unclear. The bioinformatics analysis of transcriptome sequencing data from UV-irradiated and non-UV-irradiated skin showed that transcription-related proteins, such as HSF4 and COIL, mediate cellular response to UV irradiation. HSF4 and COIL can form a complex under UV irradiation, and the preference for binding target genes changed because of the presence of a large number of R-loops in cells under UV irradiation and the ability of COIL to recognize R-loops. The regulation of target genes was altered by the HSF4-COIL complex, and the expression of inflammation and ageing-related genes, such as Atg7, Tfpi, and Lims1, was enhanced. A drug screen was performed for the recognition sites of COIL and R-loop. N6-(2-hydroxyethyl)-adenosine can competitively bind COIL and inhibit the binding of COIL to the R-loop. Thus, the activation of downstream inflammation-related genes and inflammatory skin injury was inhibited.

Isorhamnetin and anti-PD-L1 antibody dual-functional mesoporous silica nanoparticles improve tumor immune microenvironment and inhibit YY1-mediated tumor progression.

BACKGROUND: The immune checkpoint inhibitor (ICI) anti-PD-L1 monoclonal antibody can inhibit the progress of hepatocellular carcinoma (HCC). Epithelial-mesenchymal transformation (EMT) can promote tumor migration and the formation of immune-suppression microenvironment, which affects the therapeutic effect of ICI. Yin-yang-1 (YY1) is an important transcription factor regulating proliferation, migration and EMT of tumor cells. This work proposed a drug-development strategy that combined the regulation of YY1-mediated tumor progression with ICIs for the treatment of HCC. METHODS: We first studied the proteins that regulated YY1 expression by using pull-down, co-immunoprecipitation, and duo-link assay. The active compound regulating YY1 content was screened by virtual screening and cell-function assay. Isorhamnetin (ISO) and anti-PD-L1 antibody dual-functional mesoporous silica nanoparticles (HMSN-ISO@ProA-PD-L1 Ab) were prepared as an antitumor drug to play a synergistic anti-tumor role. RESULTS: YY1 can specifically bind with the deubiquitination enzyme USP7. USP7 can prevent YY1 from ubiquitin-dependent degradation and stabilize YY1 expression, which can promote the proliferation, migration and EMT of HCC cells. Isorhamnetin (ISO) were screened out, which can target USP7 and promote YY1 ubiquitin-dependent degradation. The cell experiments revealed that the HMSN-ISO@ProA-PD-L1 Ab nanoparticles can specifically target tumor cells and play a role in the controlled release of ISO. HMSN-ISO@ProA-PD-L1 Ab nanoparticles inhibited the growth of Hepa1-6 transplanted tumors and the effect was better than that of PD-L1 Ab treatment group and ISO treatment group. HMSN-ISO@ProA-PD-L1 Ab nanoparticles also exerted a promising effect on reducing MDSC content in the tumor microenvironment and promoting T-cell infiltration in tumors. CONCLUSIONS: The isorhamnetin and anti-PD-L1 antibody dual-functional nanoparticles can improve tumor immune microenvironment and inhibit YY1-mediated tumor progression. This study demonstrated the possibility of HCC treatment strategies based on inhibiting USP7-mediated YY1 deubiquitination combined with anti-PD-L1 monoclonal Ab.

Live-cell imaging-based dynamic vascular formation assay for antivascular drug evaluation and screening.

New vessel formation (angiogenesis) is an essential physiological process for embryologic development, normal growth, and tissue repair. Angiogenesis is tightly regulated at the molecular level. Dysregulation of angiogenesis occurs in various pathologies and is one of the hallmarks of cancer. However, most existing methods for evaluating cell vascular formation are limited to static analysis and prone to bias due to time, field of vision, and parameter selection. Code scripts, such as AngiogenesisAnalyzer.ijm, AutomaticMeasure.ijm, and VM.R., were developed to study the dynamic angiogenesis process. This method was used to screen drugs that could affect the time, maximum value, tilt, and decline rate of cell vascular formation and angiogenesis. Animal experiments have confirmed that these drugs could inhibit the formation of blood vessels. This work provides a new perspective for the research of angiogenesis process and is helpful to the development of drugs related to angiogenesis.

Twist1-YY1-p300 complex promotes the malignant progression of HCC through activation of miR-9 by forming phase-separated condensates at super-enhancers and relieved by metformin.

Hepatocellular carcinoma (HCC) is one of the leading causes of death, which deserves further study to reveal the underlying molecular mechanisms. Studies have shown that miR-9 in associated with poor prognosis in HCC patients. However, the mechanisms of transcriptional activation regulation of miR-9 and its role in the malignant progression of HCC have been rarely investigated. Some transcriptional coactivators can form phase-separated condensates at super-enhancers that compartmentalize and concentrate the transcription apparatus to drive robust gene expression. Here, we demonstrate that Twist1 and YY1 could form a transcriptional complex with p300, creating local high-concentration phase-separated interaction hubs at the super-enhancers of miR-9 and activate its expression to promote the malignant progression of HCC by stimulating the migration and invasion of hepatocellular carcinoma cells. Twist1-YY1-p300 phase-separated condensates were disrupted by metformin (Met) and thus reduce miR-9 expression, thereby inhibiting the malignant progression of HCC. Our study demonstrates that the Twist1 transcriptional factor complex involved in the malignant progression of HCC can form phase separation condensates at super-enhancers of miR-9 to promote the expression of oncogenes in HCC cells. It provides a potential target for the therapy of HCC and offers insights into the mechanism of Met in HCC inhibition.

Exosome-like nanovesicles derived from Phellinus linteus inhibit Mical2 expression through cross-kingdom regulation and inhibit ultraviolet-induced skin aging.

BACKGROUND: Phellinus linteus (PL), which is a typical medicinal fungus, has been shown to have antitumor and anti-inflammatory activities. However, studies on the effect of anti-photoaging are limited. Studies have shown that exosome-like nanovesicles are functional components of many medicinal plants, and miRNAs in exosome-like nanovesicles play a cross-kingdom regulatory role. At present, research on fungi exosome-like nanovesicles (FELNVs) is few. RESULTS: We systematically evaluated the anti-aging effects of PL. FELNVs of PL were isolated, and the functional molecular mechanisms were evaluated. The results of volunteer testing showed that PL had anti-aging activity. The results of component analysis showed that FELNVs were the important components of PL function. FELNVs are nanoparticles (100-260 nm) with a double shell structure. Molecular mechanism research results showed that miR-CM1 in FELNVs could inhibit Mical2 expression in HaCaT cells through cross-kingdom regulation, thereby promoting COL1A2 expression; inhibiting MMP1 expression in skin cells; decreasing the levels of ROS, MDA, and SA-ß-Gal; and increasing SOD activity induced by ultraviolet (UV) rays. The above results indicated that miR-CM1 derived from PL inhibited the expression of Mical2 through cross-kingdom regulation and inhibited UV-induced skin aging. CONCLUSION: miR-CM1 plays an anti-aging role by inhibiting the expression of Mical2 in human skin cells through cross-species regulation.

A non-retinol retinoic acid receptor-γ (RAR-γ/NR1B3) selective agonist, tectorigenin, can effectively inhibit the ultraviolet A-induced skin damage.

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY RESULTS: 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. CONCLUSIONS AND IMPLICATIONS: 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.

Biomimetic Immunosuppressive Exosomes that Inhibit Cytokine Storms Contribute to the Alleviation of Sepsis.

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.

Psychologic Stress Drives Progression of Malignant Tumors via DRD2/HIF1α Signaling.

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.

Salidroside can target both P4HB-mediated inflammation and melanogenesis of the skin.

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-ß-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.

Targeted Drug-Loaded Chemical Probe Staining Assay to Predict Therapy Response and Function as an Independent Pathological Marker.

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.

USP5 promotes epithelial-mesenchymal transition by stabilizing SLUG in hepatocellular carcinoma.

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.

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.

Hsp90ß promotes aggressive vasculogenic mimicry via epithelial-mesenchymal transition in hepatocellular carcinoma.

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. Hsp90ß promotes endothelial cell-dependent angiogenesis in HCC. However, the relationship between Hsp90ß and VM formation is unclear. In this study, we found that Hsp90ß is positively correlated with VM and EMT marker proteins in HCC tissues and promotes tube formation, cell migration, and invasion in vitro. Hsp90ß 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 Hsp90ß enhances the tumor VM in tumor-burdened mice, and the Hsp90 inhibitor NVP-BEP800 suppresses VM formation by releasing Hsp90ß and Twist1 interaction. This study provides a potential antitumor therapy for inhibiting VM by targeting Hsp90ß in HCC.