KDM4C represses liver fibrosis by regulating H3K9me3 methylation of ALKBH5 and m6A methylation of snail1 mRNA.

OBJECTIVE: We aimed to disclose the molecular mechanism of snail1 in liver fibrosis. METHODS: Carbon tetrachloride (CCl4) was used to induce a liver fibrosis model in mice whereby serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were evaluated, and liver pathological alternations were assessed. Rat hepatic stellate cells (HSC-T6) were irritated with transforming growth factor (TGF)-ß1, followed by assessment of cell viability and migration. The levels of snail1, ALKBH5, and lysine specific demethylase 4C (KDM4C) were quantified by immunohistochemistry, western blot, or reverse transcription-quantitative polymerase chain reaction, in addition to α-smooth muscle actin (SMA), anti-collagen type I α1 (COL1A1), vimentin, and E-cadherin. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation and RNA stability were evaluated to determine the relationship between ALKBH5 and snail1. Changes in KDM4C-bound ALKBH5 promoter and enrichment of histone H3 lysine 9 trimethylation (H3K9me3) at the ALKBH5 promoter were determined using chromatin immunoprecipitation. RESULTS: In fibrosis mice, snail1 was upregulated while ALKBH5 and KDM4C were downregulated. KDM4C overexpression reduced serum ALT and AST levels, liver injury, and α-SMA, COL1A1 and VIMENTIN expressions but increased E-cadherin expression. However, the aforementioned trends were reversed by concurrent overexpression of snail1. In HSC-T6 cells exposed to TGF-ß1, ALKBH5 overexpression weakened cell viability and migration, downregulated α-SMA, COL1A1 and VIMENTIN, upregulated E-CADHERIN, and decreased m6A modification of snail1 and its mRNA stability. KDM4C increased ALKBH5 expression by lowering H3K9me3 level, but inhibited HSC-T6 cell activation by regulating the ALKBH5/snail1 axis. CONCLUSION: KDM4C decreases H3K9me3 methylation to upregulate ALKBH5 and subsequently inhibits snail1, ultimately impeding liver fibrosis.

SIRT1 Stabilizes ß-TrCP1 to Inhibit Snail1 Expression in Maintaining Intestinal Epithelial Integrity to Alleviate Colitis.

BACKGROUND & AIMS: Dysfunction of the intestinal epithelial barrier comprising the junctional complex of tight junctions and adherent junctions leads to increased intestinal permeability, which is a major cause of uncontrolled inflammation related to inflammatory bowel disease (IBD). The NAD+-dependent deacetylase SIRT1 is implicated in inflammation and the pathologic process of IBD. We aimed to elucidate the protective role and underlying mechanism of SIRT1 in cell-cell junction and intestinal epithelial integrity. METHODS: The correlation of SIRT1 expression and human IBD was analyzed by GEO or immunohistochemical analyses. BK5.mSIRT1 transgenic mice and wild-type mice were given dextran sodium sulfate (DSS) and the manifestation of colitis-related phenotypes was analyzed. Intestinal permeability was measured by FITC-dextran and cytokines expression was analyzed by quantitative polymerase chain reaction. The expression of the cell junction-related proteins in DSS-treated or SIRT1-knockdown Caco2 or HCT116 cells was analyzed by Western blotting. The effects of nicotinamide mononucleotide in DSS-induced mice colitis were investigated. Correlations of the SIRT1-ß-TrCP1-Snail1-Occludin/Claudin-1/E-cadherin pathway with human IBD samples were analyzed. RESULTS: Reduced SIRT1 expression is associated with human IBD specimens. SIRT1 transgenic mice exhibit much-reduced manifestations of DSS-induced colitis. The activation of SIRT1 by nicotinamide mononucleotide bolsters intestinal epithelial barrier function and ameliorates DSS-induced colitis in mice. Mechanistically, DSS downregulates SiRT1 expression, leading to destabilization of ß-TrCP1 and upregulation of Snail1, accompanied by reduced expression of E-cadherin, Occludin, and Claudin-1, consequently resulting in increased epithelial permeability and inflammation. The deregulated SIRT1-ß-TrCP1-Snail1-Occludin/Claudin-1/E-cadherin pathway correlates with human IBD. CONCLUSIONS: SIRT1 is pivotal in maintaining the intestinal epithelial barrier integrity via modulation of the ß-TrCP1-Snail1-E-cadhein/Occludin/Claudin-1 pathway.

Energy stress-activated AMPK phosphorylates Snail1 and suppresses its stability and oncogenic function.

Triple-negative breast cancer (TNBC) is a highly lethal malignancy with limited therapy options. Aberrant metabolism, a key hallmark of human cancers, plays a crucial role in tumor progression, therapeutic responses and TNBC-related death. However, the underlying mechanisms are not fully understood. In this study, we delineate a previously unrecognized role of aberrant glucose metabolism in regulating the turnover of Snail1, which is a key transcriptional factor of epithelial-mesenchymal transition (EMT) and critically contributes to the acquisition of stemness, metastasis and chemo-resistance. Mechanistically, we demonstrate that AMP-activated protein kinase (AMPK), when activated in response to glucose deprivation, directly phosphorylates Snail1 at Ser11. Such a phosphorylation modification of Snail1 facilitates its recruitment of the E3 ligase FBXO11 and promotes its degradation, thereby suppressing stemness, metastasis and increasing cellular sensitivity to chemotherapies in vitro and in vivo. Clinically, histological analyses reveal a negative correlation between p-AMPKα and Snail1 in TNBC specimens. Taken together, our findings establish a novel mechanism and functional significance of AMPK in linking glucose status to Snail1-dependent malignancies and underscore the potential of AMPK agonists as a promising therapeutic strategy in the management of TNBC.

Moscatilin Reverses EMT Progression and its Resulting Enhanced Invasion and Migration by Affecting the TGF-ß Signaling Pathway in Bladder Cancer.

BACKGROUND: Bladder cancer metastasis is an essential process in the progression of muscle-invasive bladder cancer. EMT plays a crucial role in facilitating the spread of cancer cells. Identifying compounds that can inhibit these abilities of cancer cells is a significant international endeavor. OBJECTIVE: To explore the migration and invasion effect of Moscatilin on the bladder and clarify the mechanism of action Methods: The anti-bladder cancer effect of Moscatilin was observed by a cell proliferation experiment. The migration and invasion of bladder cancer cells inhibited by Moscatilin were detected by Transwell and Wound healing. The effects of Moscatilin on EMT-related proteins E-cadherin, N-cadherin, Snail1, Vimentin, and TGF-ß signaling pathways were detected by Western blot, and nucleic acid levels were verified by qPCR. RESULTS: Our study revealed that Moscatilin reduced the viability of bladder cancer cells in vitro and impeded their migration and invasion in experimental settings. Furthermore, we observed that Moscatilin decreased the activation levels of active proteins, specifically Smad3, Samd2, and MMP2. Additionally, we found that moscatilin significantly reduced the expression level of TGF-ß and was also capable of reversing the overexpression effect of TGF-ß. Treatment with Moscatilin also led to significant inhibition of interstitial cell markers Ncadherin and Snail1, which are associated with EMT. CONCLUSION: These findings indicate that Moscatilin impedes the migration and invasion of bladder cancer cells by influencing cell survival, modulating TGF-ß/Smad signaling, and inhibiting EMT.

CXCR2/Snail-1-Induced Epithelial-Mesenchymal Transition in the Formation and Progression of RCC with Inferior Vena Cava Tumour Thrombus.

BACKGROUND: Renal cell carcinoma (RCC), a common and highly invasive malignant tumour, presents clinical challenges due to its propensity for easy metastasis. Inferior vena cava tumour thrombus is a common RCC complication significantly impacting patient prognosis. This study investigates C-X-C chemokine receptor type 2 (CXCR2)/Snail-1-induced epithelial-mesenchymal transition (EMT) in RCC with inferior vena cava tumour thrombus. METHODS: Tissues from 51 RCC patients were analysed for CXCR2 and Snail-1 Messenger Ribonucleic Acid (mRNA) levels using Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Elevated levels of both were observed in tumour and inferior vena cava tumour thrombus tissues. Using Short Hairpin RNA (shRNA) technology, we inhibited CXCR2 and Snail-1 expression to investigate their impact on EMT, invasiveness, and metastatic potential in RCC cells. RESULTS: Compared with that in the Short Hairpin RNA-Negative Control (ShNC) group, inhibition of CXCR2 and Snail-1 suppressed the degree of EMT, invasiveness, and metastatic ability of RCC cells (p < 0.01). Further mechanistic studies showed that CXCR2/Snail-1 participated in the formation and progression of RCC by regulating the extracellular signal-regulated kinase 1/2 (ERK1/2) signalling pathways. Additionally, compared with that in the ShNC group, knockdown of CXCR2 and Snail-1 significantly inhibited the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9; p < 0.01), thereby regulating the metastasis of RCC. CONCLUSIONS: Our findings suggest that CXCR2/Snail-1-induced EMT plays an important role in the formation and progression of RCC with inferior vena cava tumour thrombus. CXCR2/Snail-1 participates in the invasion and metastasis of RCC by regulating the expression of multiple signalling pathways and related genes. These results provide new insights and directions for the treatment of RCC.

CircSOD2: Disruption of intestinal mucosal barrier function in ulcerative colitis by regulating the miR-378g/Snail1 axis.

BACKGROUND AND AIM: Circular RNA (circRNA) has been found to mediate ulcerative colitis (UC) progression by regulating intestinal mucosal barrier function. However, the role of circSOD2 in UC process and its underlying molecular mechanism still need to be further elucidated. METHODS: Lipopolysaccharide (LPS)-induced Caco2 cells were used to mimic UC cell models. CircSOD2, miR-378g, and Snail1 levels were determined by quantitative real-time PCR. Cell viability was detected using MTT assay, and inflammatory cytokine levels were measured using ELISA. The intestinal mucosal barrier function was evaluated by testing transepithelial electrical resistance and fluorescein isothiocyanate (FITC)-dextran permeability. Snail1 and tight junction-related markers (Zo-1 and Claudin2) protein levels were examined using western blot. The interaction between miR-378g and circSOD2 or Snail1 was confirmed by dual-luciferase reporter assay. Dextran sulfate sodium (DSS) was used to induce UC rat models in vivo. RESULTS: CircSOD2 was overexpressed in UC patients, and its knockdown significantly increased cell viability, transepithelial electrical resistance, and tight junction-related protein expression, while reduced inflammation cytokine levels and the permeability of FITC-dextran in LPS-induced Caco2 cells. In terms of mechanism, circSOD2 sponged miR-378g to positively regulate Snail1 expression. MiR-378g inhibitor reversed the effect of circSOD2 knockdown on intestinal mucosal barrier injury and Snail1 expression in LPS-induced Caco2 cells. In DSS-induced UC rat models, circSOD2 knockdown also could repair the intestinal mucosal barrier injury through regulating miR-378g/Snail1 axis. CONCLUSION: CircSOD2 could destroy intestinal mucosal barrier function in LPS-induced Caco2 cells and DSS-induced UC rats by miR-378g/Snail1 axis.

Snail transcription factors - Characteristics, regulation and molecular targets relevant in vital cellular activities of ovarian cancer cells.

Snail transcription factors play essential roles in embryonic development and participate in many physiological processes. However, these genes have been implicated in the development and progression of various types of cancer. In epithelial ovarian cancer, high expression of these transcription factors is usually associated with the acquisition of a more aggressive phenotype and thus, considered to be a poor prognostic factor. Numerous molecular signals create a complex network of signaling pathways regulating the expression and stability of Snails, which in turn control genes involved in vital cellular functions of ovarian cancer cells, such as invasion, survival, proliferation and chemoresistance.

Ubiquitin-specific Protease 35 Promotes Gastric Cancer Metastasis by Increasing the Stability of Snail1.

Deubiquitinase (DUB) dysregulation is closely associated with multiple diseases, including tumors. In this study, we used data from The Cancer Genome Atlas and Gene Expression Omnibus databases to analyze the expression of 51 ubiquitin-specific proteases (USPs) in gastric cancer (GC) tissues and adjacent non-neoplastic tissues. The Kaplan-Meier Plotter database was used to analyze the association of the differentially expressed USPs with the overall survival of patients with GC. The results showed that five USPs (USP5, USP10, USP13, USP21, and USP35) were highly expressed in GC tissues and were associated with poor prognosis in patients with GC. Because the epithelial-mesenchymal transition enables epithelial cells to acquire mesenchymal features and contributes to poor prognosis, we investigated whether these USPs had regulatory effects on the key epithelial-mesenchymal transition transcription factor Snail1. Our results showed that USP35 exhibited the most significant regulation on Snail1. Overexpression of USP35 increased and its knockdown decreased Snail1 protein levels. Mechanistically, USP35 interacted with Snail1 and removed its polyubiquitinated chain, thereby increasing its stability. Furthermore, USP35 promoted the invasion and migration of GC cells depending on its DUB activity. USP35 knockdown exhibited the opposite effect. Snail1 depletion partially abrogated the biological effects of USP35. Experiments using nude mouse tail vein injections indicated that wild-type USP35, but not the catalytically inactive USP35-C450A mutant, dramatically enhanced cell colonization and tumorigenesis in the lungs of mice. In addition, USP35 positively correlated with Snail1 expression in clinical GC tissues. Helicobacter pylori infection increased USP35 and Snail1 expression levels. Altogether, we found that USP35 can deubiquitinate Snail1 and increase its expression, thereby contributing to the malignant progression of GC. Therefore, USP35 may serve as a viable target for GC treatment.

Dual role of Snail1 as transcriptional repressor and activator.

Snail1 transcriptional factor plays a key role in the control of epithelial to mesenchymal transition, a process that remodels tumor cells increasing their invasion and chemo-resistance as well as reprograms their metabolism and provides stemness properties. During this transition, Snail1 acts as a transcriptional repressor and, as growing evidences have demonstrated, also as a direct activator of mesenchymal genes. In this review, I describe the different proteins that interact with Snail1 and are responsible for these two different functions on gene expression; I focus on the transcriptional factors that associate to Snail1 in their target promoters, both activated and repressed. I also present working models for Snail1 action both as repressor and activator and raise some issues that still need to be investigated.

Deubiquitinating enzyme OTUD4 regulates metastasis in triple-negative breast cancer by stabilizing Snail1.

Metastasis is the primary cause of cancer-related deaths and remains poorly understood. Deubiquitinase OTU domain containing 4 (OTUD4) has been reported to regulate antiviral immune responses and resistance to radio- or chemo-therapies in certain cancers. However, the role of OTUD4 in cancer metastasis remain unknown. Here, we demonstrate that the depletion of OTUD4 in triple-negative breast cancer (TNBC) cells markedly suppress cell clonogenic ability, migration, invasion and cancer stem cell population in vitro as well as metastasis in vivo. Mechanistically, the tumor promoting function of OTUD4 is mainly mediated by deuiquitinating and stabilizing Snail1, one key transcriptional factor in the epithelial-mesenchymal transition. The inhibitory effect of targeting OTUD4 could be largely reversed by the reconstitution of Snail1 in OTUD4-deficient cells. Overall, our study establishes the OTUD4-Snail1 axis as an important regulatory mechanism of breast cancer metastasis and provides a rationale for potential therapeutic interventions in the treatment of TNBC.

Porcine circovirus type 2 and Glaesserella parasuis serotype 4 co-infection activates Snail1 to disrupt the intercellular junctions and facilitate bacteria translocation across the tracheal epithelium.

Clinically, Porcine circovirus type 2 (PCV2) often causes disease through coinfection with other bacterial pathogens, including Glaesserella parasuis (G. parasuis), which causes high morbidity and mortality. However, the mechanism of PCV2 and G. parasuis serotype 4 (GPS4) co-infection is still not fully understood. In this study, swine tracheal epithelial cells (STEC) were used as a barrier model, and our results showed that PCV2 infection increased the adhesion of GPS4 to STEC, while decreasing the levels of ZO-1, Occludin and increasing tracheal epithelial permeability, and ultimately facilitated GPS4 translocation. Snail1 is a transcriptional repressor, and has been known to induce epithelial-to-mesenchymal transition (EMT) during development or in cancer metastasis. Importantly, we found that Snail1, as a transcriptional repressor, was crucial in destroying the tracheal epithelial barrier induced by PCV2, GPS4, PCV2 and GPS4 coinfection. For the first time, we found that PCV2, GPS4, PCV2 and GPS4 coinfection cross-activates TGF-ß and p38/MAPK signaling pathways to upregulate the expression of Snail1, down-regulate the levels of ZO-1 and Occludin, and thus disrupt the integrity of tracheal epithelial barrier then promoting GPS4 translocation. Finally, PCV2 and GPS4 co-infection also can activate TGF-ß and p38/MAPK signaling pathways in vivo and upregulate Snail1, ultimately down-regulating the expression of ZO-1 and Occludin. Our study elucidates how PCV2 infection promotes GPS4 to breach the tracheal epithelial barrier and aggravate clinical manifestations.

Formation of an invasion-permissive matrix requires TGFß/SNAIL1-regulated alternative splicing of fibronectin.

BACKGROUND: As in most solid cancers, the emergence of cells with oncogenic mutations in the mammary epithelium alters the tissue homeostasis. Some soluble factors, such as TGFß, potently modify the behavior of healthy stromal cells. A subpopulation of cancer-associated fibroblasts expressing a TGFß target, the SNAIL1 transcription factor, display myofibroblastic abilities that rearrange the stromal architecture. Breast tumors with the presence of SNAIL1 in the stromal compartment, and with aligned extracellular fiber, are associated with poor survival prognoses. METHODS: We used deep RNA sequencing and biochemical techniques to study alternative splicing and human tumor databases to test for associations (correlation t-test) between SNAIL1 and fibronectin isoforms. Three-dimensional extracellular matrices generated from fibroblasts were used to study the mechanical properties and actions of the extracellular matrices on tumor cell and fibroblast behaviors. A metastatic mouse model of breast cancer was used to test the action of fibronectin isoforms on lung metastasis. RESULTS: In silico studies showed that SNAIL1 correlates with the expression of the extra domain A (EDA)-containing (EDA+) fibronectin in advanced human breast cancer and other types of epithelial cancers. In TGFß-activated fibroblasts, alternative splicing of fibronectin as well as of 500 other genes was modified by eliminating SNAIL1. Biochemical analyses demonstrated that SNAIL1 favors the inclusion of the EDA exon by modulating the activity of the SRSF1 splicing factor. Similar to Snai1 knockout fibroblasts, EDA- fibronectin fibroblasts produce an extracellular matrix  that does not sustain TGFß-induced fiber organization, rigidity, fibroblast activation, or tumor cell invasion. The presence of EDA+ fibronectin changes the action of metalloproteinases on fibronectin fibers. Critically, in an mouse orthotopic breast cancer model, the absence of the fibronectin EDA domain completely prevents lung metastasis. CONCLUSIONS: Our results support the requirement of EDA+ fibronectin in the generation of a metastasis permissive stromal architecture in breast cancers and its molecular control by SNAIL1. From a pharmacological point of view, specifically blocking EDA+ fibronectin deposition could be included in studies to reduce the formation of a pro-metastatic environment.

OTUD4 regulates metastasis and chemoresistance in melanoma by stabilizing Snail1.

Melanoma is the most aggressive form of skin cancer with rapidly increased incidence worldwide especially in the Caucasian population. Surgical excision represents the curative treatment choice in patients with early-stage disease. However, the therapeutic outcomes in patients with metastatic melanoma remains unsatisfactory. Thus, understanding molecular mechanisms contributing to metastasis and chemoresistance is critical for new improved therapies of melanoma. Snail1, an important epithelial-mesenchymal transition transcription factors (EMT-TFs), is critical to induce the EMT process, thereby contributing to cancer metastasis. However, the involvement of Snail1 in melanoma metastasis remains elusive and the underlying mechanism to regulate Snail1 in melanoma needs to be further investigated. Here, we identified OTUD4 as a novel deubiquitinase of Snail1 in melanoma. Moreover, the depletion of OTUD4 in melanoma cells markedly inhibited Snail1 stability and Snail1-driven malignant phenotypes both in vitro and in vivo. Overall, our study establishes OTUD4 as a novel therapeutic target in metastasis and chemoresistance of melanoma by stabilizing Snail1 and provides a rationale for potential therapeutic strategies of melanoma.

MicroRNA-143 acts as a tumor suppressor through Musashi-2/DLL1/Notch1 and Musashi-2/Snail1/MMPs axes in acute myeloid leukemia.

BACKGROUND: The previous studies have revealed that abnormal RNA-binding protein Musashi-2 (MSI2) expression is associated with cancer progression through post-transcriptional mechanisms, however mechanistic details of this regulation in acute myeloid leukemia (AML) still remain unclear. Our study aimed to explore the relationship between microRNA-143 (miR-143) and MSI2 and to clarify their clinical significance, biological function and mechanism. METHODS: Abnormal expression of miR-143 and MSI2 were evaluated in bone marrow samples from AML patients by quantitative real time-PCR. Effects of miR-143 on regulating MSI2 expression were investigated using luciferase reporter assay. Functional roles of MSI2 and miR-143 on AML cell proliferation and migration were determined by CCK-8 assay, colony formation, and transwell assays in vitro and in mouse subcutaneous xenograft and orthotopic transplantation models in vivo. RNA immunoprecipitation, RNA stability measurement and Western blotting were performed to assess the effects of MSI2 on AML. RESULTS: We found that MSI2 was significantly overexpressed in AML and exerted its role of promoting AML cell growth by targeting DLL1 and thereby activating Notch signaling pathway. Moreover, we found that MSI2 bound to Snail1 transcript and inhibited its degradation, which in turn upregulated the expression of matrix metalloproteinases. We also found that MSI2 targeting miR-143 is downregulated in AML. In the AML xenograft mouse model, overexpression of MSI2 recapitulated its leukemia-promoting effects, and overexpression of miR-143 partially attenuated tumor growth and prevented metastasis. Notably, low expression of miR-143, and high expression of MSI2 were associated with poor prognosis in AML patients. CONCLUSIONS: Our data demonstrate that MSI2 exerts its malignant properties via DLL1/Notch1 cascade and the Snail1/MMPs axes in AML, and upregulation of miR-143 may be a potential therapeutic approach for AML.

Analyzing the role of cancer-associated fibroblast activation on macrophage polarization.

Snail1 is a transcriptional factor required for cancer-associated fibroblast (CAF) activation, and mainly detected in CAFs in human tumors. In the mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT) model of murine mammary gland tumors, Snai1 gene deletion, besides increasing tumor-free lifespan, altered macrophage differentiation, with fewer expressing low levels of MHC class II. Snail1 was not expressed in macrophages, and in vitro polarization with interleukin-4 (IL4) or interferon-γ (IFNγ) was not altered by Snai1 gene depletion. We verified that CAF activation modified polarization of naïve bone-marrow-derived macrophages (BMDMΦs). When BMDMΦs were incubated with Snail1-expressing (active) CAFs or with conditioned medium derived from these cells, they exhibited a lower cytotoxic capability than when incubated with Snail1-deleted (inactive) CAFs. Gene expression analysis of BMDMΦs polarized by conditioned medium from wild-type or Snai1-deleted CAFs revealed that active CAFs differentially stimulated a complex combination of genes comprising genes that are normally induced by IL4, downregulated by IFNγ, or not altered during the two canonical differentiations. Levels of RNAs relating to this CAF-induced alternative polarization were sensitive to inhibitors of factors specifically released by active CAFs, such as prostaglandin E2 and TGFß. Finally, CAF-polarized macrophages promoted the activation of the immunosuppressive regulatory T cells (T-regs). Our results imply that an active CAF-rich tumor microenvironment induces the polarization of macrophages to an immunosuppressive phenotype, preventing the macrophage cytotoxic activity on tumor cells and enhancing the activation of T-reg cells.

Epithelial-Mesenchymal Transition of Breast Cancer Cells Induced by Activation of the Transcription Factor Snail1.

Cancer cells use the program of epithelial-mesenchymal transition (EMT) for initiation of the invasion-metastasis cascade. Using confocal and video-microscopy, reorganization of the cytoskeleton was studied in the MCF-7 breast cancer cells undergoing Snail1-induced EMT. We used the line of MCF-7 cells stably expressing tetOff SNAI1 construct (MCF-7-SNAI1 cells). After tetracycline washout and Snail1 activation MCF-7-SNAI1 cells underwent EMT and acquired a migratory phenotype while retaining expression of E-cadherin. We identified five variants of the mesenchymal phenotype, differing in cell morphology and migration velocity. Migrating cells had high degree of plasticity, which allowed them to quickly change both the phenotype and migration velocity. The changes of the phenotype of MCF-7-SNAI1 cells are based on the Arp2/3-mediated branched actin network polymerization in lamellipodia, myosin-based contractility in the zone behind the nucleus, redistribution of adhesive proteins from cell-cell contacts to the leading edge, and reorganization of intermediate keratin filaments.

Implications of Axis Inhibition Protein 2 in Breast Cancer Progression.

BACKGROUND/AIM: Although axis inhibition protein 2 (Axin2) has been reported to act as a tumour suppressor, recent findings suggest that it exhibits oncogenic effects by mediating Snail1-induced epithelial-mesenchymal transition (EMT) in breast cancer cells. EMT is a crucial biological process involved in the initiation of metastasis in cancer progression. This study elucidated the biological significance and mechanism of Axin2 in breast cancer using transcriptomic and molecular techniques. MATERIALS AND METHODS: The expression of Axin2 and Snail1 in MDA-MB-231 breast cancer cells was determined by western blotting analysis, and the role of Axin2 in breast cancer tumorigenesis was investigated in xenograft mouse models constructed using pLKO-Tet-shAxin2-transfected triple negative (TN) breast cancer cells. Additionally, the expression levels of EMT markers were determined using qRT-PCR, and clinical data were analysed using Kaplan-Meier (KM) plotter and The Cancer Genome Atlas (TCGA). RESULTS: Axin2 knockdown significantly decreased (p<0.001) the proliferation of MDA-MB-231 cells in vitro and attenuated (p<0.05) the tumorigenic potential of the cells in vivo. Moreover, Axin2 knockdown significantly increased the relative mRNA levels of epithelial markers but decreased the expression of mesenchymal markers in MDA-MB-231 cells. CONCLUSION: Axin2 may be involved in the progression of breast cancer, particularly triple-negative breast cancer, through the regulation of Snail1-induced EMT, making it a potential therapeutic target.

FBXO11 regulates bone development.

FBXO11 is the substrate-recognition component of a ubiquitin ligase complex called SKP1-cullin-F-boxes. The role of FBXO11 in bone development is unexplored. In this study, we reported a novel mechanism of how bone development is regulated by FBXO11. FBXO11 gene knockdown by lentiviral transduction in mouse pre-osteoblast MC3T3-E1 cells leads to reduced osteogenic differentiation, while overexpressing FBXO11 accelerates their osteogenic differentiation in vitro. Furthermore, we generated two osteoblastic-specific FBXO11 conditional knockout mouse models, Col1a1-ERT2-FBXO11KO and Bglap2-FBXO11KO mice. In both conditional FBXO11KO mouse models, we found FBXO11 deficiency inhibits normal bone growth, in which the osteogenic activity in FBXO11cKO mice is reduced, while osteoclastic activity is not significantly changed. Mechanistically, we found FBXO11 deficiency leads to Snail1 protein accumulation in osteoblasts, leading to suppression of osteogenic activity and inhibition of bone matrix mineralization. FBXO11 knockdown in MC3T3-E1 cells reduced Snail1 protein ubiquitination and increased Snail1 protein accumulation in the cells, which eventually inhibited osteogenic differentiation. In conclusion, FBXO11 deficiency in osteoblasts inhibits bone formation through Snail1 accumulation, inhibiting osteogenic activity and bone mineralization.

USP22 aggravated diabetic renal tubulointerstitial fibrosis progression through deubiquitinating and stabilizing Snail1.

Renal tubulointerstitial fibrosis (TIF) is one of the main pathological changes induced by diabetic kidney disease (DKD), and epithelial-to-mesenchymal transition (EMT) induced by high glucose (HG) can promote TIF. Our previous study has shown that ubiquitin-specific protease 22 (USP22) could affect the process of DKD by deubiquitinating and stabilizing Sirt1 in glomerular mesangial cells. However, whether USP22 could regulate EMT occurrence in renal tubular epithelial cells and further aggravate the pathological process of TIF in DKD remains to be elucidated. In this study, we found that USP22 expression was upregulated in kidney tissues of db/db mice and HG-treated NRK-52E cells. In vitro, USP22 overexpression promoted the EMT process of NRK-52E cells stimulated by HG and further increased the levels of extracellular matrix (ECM) components such as fibronectin, Collagen I, and Collagen Ⅳ. Meanwhile, USP22 deficiency exhibited the opposite effects. Mechanism studies showed that USP22, depending on its deubiquitinase activity, deubiquitinated and stabilized the EMT transcriptional factor Snail1. In vivo experiment showed that interfering with USP22 could improve the renal pathological damages and renal function of the db/db spontaneous diabetic mice by decreasing Snail1 expression, which could inhibit EMT occurrence, and reduce the production of ECM components. These results suggested that USP22 could accelerate renal EMT and promote the pathological progression of diabetic TIF by deubiquitinating Snail1, providing an experimental basis for using USP22 as a potential target for DKD.

TGFß1-Induced EMT in the MCF10A Mammary Epithelial Cell Line Model Is Executed Independently of SNAIL1 and ZEB1 but Relies on JUNB-Coordinated Transcriptional Regulation.

Epithelial-mesenchymal transition (EMT) fosters cancer cell invasion and metastasis, the main cause of cancer-related mortality. Growing evidence that SNAIL and ZEB transcription factors, typically portrayed as master regulators of EMT, may be dispensable for this process, led us to re-investigate its mechanistic underpinnings. For this, we used an unbiased computational approach that integrated time-resolved analyses of chromatin structure and differential gene expression, to predict transcriptional regulators of TGFß1-inducible EMT in the MCF10A mammary epithelial cell line model. Bioinformatic analyses indicated comparatively minor contributions of SNAIL proteins and ZEB1 to TGFß1-induced EMT, whereas the AP-1 subunit JUNB was anticipated to have a much larger impact. CRISPR/Cas9-mediated loss-of-function studies confirmed that TGFß1-induced EMT proceeded independently of SNAIL proteins and ZEB1. In contrast, JUNB was necessary and sufficient for EMT in MCF10A cells, but not in A549 lung cancer cells, indicating cell-type-specificity of JUNB EMT-regulatory capacity. Nonetheless, the JUNB-dependence of EMT-associated transcriptional reprogramming in MCF10A cells allowed to define a gene expression signature which was regulated by TGFß1 in diverse cellular backgrounds, showed positively correlated expression with TGFß signaling in multiple cancer transcriptomes, and was predictive of patient survival in several cancer types. Altogether, our findings provide novel mechanistic insights into the context-dependent control of TGFß1-driven EMT and thereby may lead to improved diagnostic and therapeutic options.