Polyphyllin D punctures hypertrophic lysosomes to reverse drug resistance of hepatocellular carcinoma by targeting acid sphingomyelinase.

Hypertrophic lysosomes are critical for tumor progression and drug resistance; however, effective and specific lysosome-targeting compounds for cancer therapy are lacking. Here we conducted a lysosomotropic pharmacophore-based in silico screen in a natural product library (2,212 compounds), and identified polyphyllin D (PD) as a novel lysosome-targeted compound. PD treatment was found to cause lysosomal damage, as evidenced by the blockade of autophagic flux, loss of lysophagy, and the release of lysosomal contents, thus exhibiting anticancer effects on hepatocellular carcinoma (HCC) cell both in vitro and in vivo. Closer mechanistic examination revealed that PD suppressed the activity of acid sphingomyelinase (SMPD1), a lysosomal phosphodieserase that catalyzes the hydrolysis of sphingomyelin to produce ceramide and phosphocholine, by directly occupying its surface groove, with Trp148 in SMPD1 acting as a major binding residue; this suppression of SMPD1 activity irreversibly triggers lysosomal injury and initiates lysosome-dependent cell death. Furthermore, PD-enhanced lysosomal membrane permeabilization to release sorafenib, augmenting the anticancer effect of sorafenib both in vivo and in vitro. Overall, our study suggests that PD can potentially be further developed as a novel autophagy inhibitor, and a combination of PD with classical chemotherapeutic anticancer drugs could represent a novel therapeutic strategy for HCC intervention.

ZSWIM1 Promotes the Proliferation and Metastasis of Lung Adenocarcinoma Cells through the STK38/MEKK2/ERK1/2 Axis.

Investigating the functions of the proteins with no or less functional annotations is an important goal of the HPP (Human Proteome Project) Grand Project. In this study, we investigated the function of such a protein, ZSWIM1 (C20orf162), its gene located on chromosome 20. Its expression is upregulated in lung adenocarcinoma compared with the adjacent normal tissues and negatively correlated with the overall survival. Overexpressing ZSWIM1 markedly promotes the proliferation, migration, invasion as well as epithelial-to-mesenchymal transition in lung adenocarcinoma cells, while knocking down ZSWIM1 functions oppositely. The interactome of ZSWIM1 was identified by immunoprecipitation-mass spectrometry, and we verified the interaction of ZSWIM1 with the potential partner, STK38. ZSWIM1 antagonized the function of STK38. Mechanically, ZSWIM1 promoted the activation of MEKK2/ERK1/2 pathway through interacting with STK38, leading to the release of MEKK2. Taken together, ZSWIM1 can be annotated as an oncogene in lung adenocarcinoma, and the STK38/MEKK2/ERK1/2 axis mediates its promoting role in lung adenocarcinoma.

Exosomal PPARγ derived from macrophages suppresses LPS-induced peritonitis by negative regulation of CD14/TLR4 axis.

BACKGROUND: Intercellular communication between macrophages and peritoneal mesothelial cells (PMCs) has been suggested as a key factor regulating peritonitis development. Here, we explored whether PPARγ (peroxisome proliferator-activated receptor gamma) can be packaged into macrophage exosomes to mediate intercellular communication and regulate peritonitis. METHODS: Macrophage exosomes were isolated by ultracentrifugation and identified by nanoparticle tracking analysis and transmission electron microscopy. Proteomic analysis of macrophage-derived exosomes was performed using mass spectrometry. Co-culture models of supernatants or exosomes with PMCs, as well as a mouse peritonitis model induced by lipopolysaccharide (LPS), were employed. RESULTS:  In this study, using stable Raw264.7 cells overexpressing GFP-FLAG-PPARγ (OE-PPARγ), we found that PPARγ inhibited LPS-induced inflammatory responses in Raw264.7 cells and that PPARγ was incorporated into macrophage exosomes during this process. Overexpression of PPARγ mainly regulated the secretion of differentially expressed exosomal proteins involved in the biological processes of protein transport, lipid metabolic process, cell cycle, apoptotic process, DNA damage stimulus, as well as the KEGG pathway of salmonella infection. Using co-culture models and mouse peritonitis model, we showed that exosomes from Raw264.7 cells overexpressing PPARγ inhibited LPS-induced inflammation in co-cultured human PMCs and in mice through downregulating CD14 and TLR4, two key regulators of the salmonella infection pathway. Pretreatment of the PPARγ inhibitor GW9662 abolished the anti-inflammatory effect of exosomes from Raw264.7 OE-PPARγ cells on human PMCs. CONCLUSIONS: These results suggested that overexpression of PPARγ largely altered the proteomic profile of macrophage exosomes and that exosomal PPARγ from macrophages acted as a regulator of intercellular communication to suppress LPS-induced inflammatory responses in vitro and in vivo via negatively regulating the CD14/TLR4 axis.

ANXA1-GSK3ß interaction and its involvement in NSCLC metastasis.

Although initially discovered and extensively studied for its role in inflammation, Annexin A1 (ANXA1) has been reported to be closely related to cancer in recent years, and its role in cancer is specific to tumor types and tissues. In the present study, we identified ANXA1 as an interaction partner of glycogen synthase kinase 3 beta (GSK3ß), a multi-functional serine/threonine kinase tightly associated with cell fate determination and cancer, and assessed the functional significance of GSK3ß-ANXA1 interaction in the metastasis of non-small cell lung cancer (NSCLC). We confirmed the interaction between GSK3ß and ANXA1 in vitro and in H1299 and A549 cells by Glutathione-S-transferase (GST) pull-down assay and co-immunoprecipitation. We found that ANXA1 negatively regulated the phosphorylation of GSK3ß and inhibited the epithelial-mesenchymal transformation (EMT) process and migration and invasion of NSCLC cells. By functional rescue assay, we confirmed that ANXA1 inhibited EMT through the regulation of GSK3ß activity and thereby inhibited the migration and invasion of NSCLC cells. Our study sheds light on the function of ANXA1 and GSK3ß and provides new elements for the understanding of NSCLC pathogenesis.

The emerging roles of hnRNPK.

Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an DNA/RNA-binding protein and regulates a wide range of biological processes and disease pathogenesis. It contains 3 K-homologous (KH) domains, which are conserved in other RNA-binding proteins, mediate nucleic acid binding activity, and function as an enhancer or repressor of gene transcription. Phosphorylation of the protein alters its regulatory function, which also enables the protein to serve as a docking platform for the signal transduction proteins. In terms of the function of hnRNPK, it is central to many cellular events, including long noncoding RNA (lncRNA) regulation, cancer development and bone homoeostasis. Many studies have identified hnRNPK as an oncogene, where it is overexpressed in cancer tissues compared with the nonneoplastic tissues and its expression level is related to the prognosis of different types of host malignancies. However, hnRNPK has also been identified as a tumour suppressor, as it is important for the activation of the p53/p21 pathway. Recently, the protein is also found to be exclusively related to the regulation of paraspeckles and lncRNAs such as Neat1, Lncenc1 and Xist. Interestingly, hnRNPK has been found to associate with the Kabuki-like syndrome and Au-Kline syndrome with prominent skeletal abnormalities. In vitro study revealed that the hnRNPK protein is essential for the formation of osteoclast, in line with its importance in the skeletal system.

Regulation and related mechanism of GSN mRNA level by hnRNPK in lung adenocarcinoma cells.

Gelsolin (GSN) is an actin filament-capping protein that plays a key role in cell migration. Here we show that heterogeneous nuclear ribonucleoprotein K (hnRNPK) regulates GSN expression level by binding to the 3'-untranslated region (3'UTR) of GSN mRNA in non-small cell lung cancers (NSCLC) H1299 cells which are highly metastatic and express high level of GSN. We found that hnRNPK overexpression increased the mRNA and protein level of GSN, whereas hnRNPK knockdown by siRNA decreased the mRNA and protein level of GSN in both H1299 and A549 cells, indicating a positive role of hnRNPK in the regulation of GSN expression. Furthermore, hnRNPK knockdown affected the migration ability of H1299 and A549 cells which could be rescued by ectopic expression of GSN in those cells. Conversely, GSN knockdown in hnRNPK-overexpressing cells could abort the stimulatory effect of hnRNPK on the cell migration. These results suggest that hnRNPK function in the regulation of cell migration is GSN-dependent. Taken together, these data unveiled a new mechanism of regulation of the GSN expression by hnRNPK and provides new clues for the discovery of new anti-metastatic therapy.

YAP mediates the positive regulation of hnRNPK on the lung adenocarcinoma H1299 cell growth.

Lung cancer is the leading cause of cancer death worldwide, and non-small cell lung cancer (NSCLC) accounts for 80%-85% of diagnostic cases. The molecular mechanisms of NSCLC pathogenesis are not well understood. Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is a multifunctional protein that regulates gene expression and signal transduction and closely associated with tumorigenesis, but its mechanism of action in the pathogenesis of NSCLC is unclear. In this study, we observed that the expression pattern of hnRNPK in H1299 lung adenocarcinoma cells varied depending on the cell density in culture. Moreover, hnRNPK stimulated the ability of proliferation and colony formation of H1299 cells, which is important for the multilayered cell growth in culture. We further investigated whether there is an association between hnRNPK and the elements involved in the cell contact inhibition pathway. By using quantitative reverse transcriptase-polymerase chain reaction assay and a YAP activity reporter system, we found that hnRNPK upregulated the mRNA and protein levels and transcriptional activity of Yes-associated protein 1 (YAP), a master negative regulator of Hippo contact inhibition pathway. Furthermore, YAP knockdown with siRNA abolished the stimulatory effect of hnRNPK on H1299 cell proliferation. These results suggested that YAP could be one of the effectors of hnRNPK. Our data may provide new clues for further understanding the biological functions of hnRNPK, particularly in the context of lung adenocarcinoma oncogenesis.

PKCα-GSK3ß-NF-κB signaling pathway and the possible involvement of TRIM21 in TRAIL-induced apoptosis.

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is a highly promising therapeutic agent for cancer treatment, owing to its ability to selectively target tumor cells for cell death while having little effect on most normal cells. However, recent research has found that many cancers, including non-small cell lung cancer (NSCLC), display resistance to TRAIL. Therefore, it is important to elucidate the molecular mechanisms governing the resistance of tumor cells to TRAIL treatment. In this study, we show that GSK3ß antagonized TRAIL-induced apoptosis in H1299 NSCLC cells, and determined that the PKCα isozyme is an upstream regulator of GSK3ß that phosphorylates and inactivates GSK3ß, thereby sensitizing cancer cells to TRAIL-induced apoptosis. Furthermore, we demonstrated that the anti-apoptotic effect of GSK3ß is mediated by the NF-κB pathway, whereas the tripartite motif 21 (TRIM21) was able to inhibit the activation of NF-κB by GSK3ß, and leads to the promotion of cell apoptosis. Taken together, our study further delineated the underpinning mechanism of resistance to TRAIL-induced apoptosis in H1299 cells, and provided new clues for sensitizing NSCLC cells to TRAIL therapy.

14-3-3ζ reduces DNA damage by interacting with and stabilizing proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA) is a processivity factor of DNA replication which plays critical roles in the regulation of DNA replication and repair. In this study, we show that PCNA interacts directly in vitro and in cells with 14-3-3ζ, an adaptor protein that regulates cell growth and response to DNA damage in eukaryotes. The interaction is mediated by at least two PCNA-binding sites on 14-3-3ζ, one of which is a novel non-canonical PIP (PCNA interacting protein) box. We find that DNA damages induced by UVC irradiation and MMS (methyl methanesulfonate) can enhance both the interaction of these two proteins and their co-localization with chromatin. Functional analyses suggest that 14-3-3ζ stabilizes PCNA possibly by regulating its ubiquitination, which impacts on DNA damage repair and cell viability.

Ser9-phosphorylated GSK3ß induced by 14-3-3ζ actively antagonizes cell apoptosis in a NF-κB dependent manner.

The activity of glycogen synthase kinase beta (GSK3ß) is mainly regulated by its Ser9 phosphorylation. It has been believed for a long time that Ser9 phosphorylation regulates the functions of GSK3ß through inhibition of its kinase activity. In this study, we have confirmed the interaction of Ser9-phosphorylated GSK3ß with 14-3-3ζ by using GST pull-down assays. We show that 14-3-3ζ enhances Ser9 phosphorylation of GSK3ß by PKC. Surprisingly, using a NF-κB luciferase reporter system, we find that Ser9-phosphorylation of GSK3ß promoted by 14-3-3ζ is critical for the activation of NF-κB pathway, which may thwart the pro-apoptotic activity of GSK3ß. Inhibition of either NF-κB or GSK3ß significantly abolishes the anti-apoptotic effect of 14-3-3ζ and Ser9-phosphorylated GSK3ß, suggesting that Ser9-phosphorylated GSK3ß actively antagonizes cell apoptosis in a NF-κB dependent manner.

Varied metal-binding properties of lipoprotein PsaA in Streptococcus pneumoniae.

Streptococcus pneumoniae is a Gram-positive pathogen responsible for pneumonia, otitis media, and meningitis. Manganese and zinc ions are essential for this bacterium, playing regulatory, structural, or catalytic roles as the critical cofactors in the bacterial proteins and metabolic enzymes. Lipoprotein PsaA has been found to mediate Mn(2+) and Zn(2+) transportation in Streptococcus pneumoniae. In the present work, we conducted a systemic study on the contributions from key amino acids in the metal-binding site of PsaA using various spectroscopic and biochemical methods. Our experimental data indicate that four metal-binding residues contribute unequally to the Mn(2+) and Zn(2+) binding, and His139 is most important for both the structural stability and metal binding of the protein. PsaA-Mn(2+) has a lower thermal stability than PsaA-Zn(2+), possibly due to the different coordination preferences of the metals. Kinetics analysis revealed that PsaA-Mn(2+) binding is a fast first-order reaction, whereas PsaA-Zn(2+) binding is a slow second-order reaction, implying that PsaA kinetically prefers binding Mn(2+) to Zn(2+). The present results provide complementary information for understanding the mechanisms of metal transport and bacterial virulence via lipoproteins in Streptococcus pneumoniae.

Ferroptosis in cancer (Review).

Ferroptosis is a type of programmed cell death depending on iron and reactive oxygen species. This unique cell death process has attracted a great deal of attention in the field of cancer research over the past decade. Research on the association of ferroptosis signal pathways and cancer development indicated that targeting ferroptosis has great potential for cancer therapy. In the present study, the latest research progress of ferroptosis was reviewed, focusing on the relationship between ferroptosis and the development of cancer, in order to further promote the clinical application of ferroptosis in cancer.

Artesunate induces ferroptosis by regulating MT1G and has an additive effect with doxorubicin in diffuse large B-cell lymphoma cells.

Diffuse Large B-cell lymphoma (DLBCL) is a highly aggressive disease with heterogeneous outcomes and marked variability in the response to chemotherapy. DLBCL comprises two major subtypes: germinal centre B-cell-like (GCB) and activated B-cell-like (ABC). Our study highlights the extensive antitumour activity of artesunate (ART) against both major DLBCL subtypes. Transcriptome analysis suggests the potential involvement of ferroptosis in artesunate-induced cell death. Because of low glutathione (GSH) and glutathione peroxidase 4 (GPX4) levels, along with the accumulation of free iron (Fe2+), artesunate induces the excessive production of reactive oxygen species (ROS), ultimately leading to ferroptosis, a form of cell death driven by phospholipid peroxidation. A putative target of artesunate, metallothionein 1G (MT1G), was selected for further analysis. Subsequent studies revealed that inhibiting MT1G expression in vitro significantly impedes the ferroptosis-promoting activity of artesunate by reducing lipid peroxidation and iron accumulation. We also showed that the combination of artesunate and doxorubicin had a marked additive inhibitory effect on GCB and ABC DLBCL cells. In conclusion, artesunate induces ferroptotic death in GCB and ABC DLBCL cells by attenuating the GPX4/GSH antioxidant defence system and increasing intracellular iron levels, indicating its therapeutic potential for relapsed or refractory DLBCL.

Identification and analysis of glycogen synthase kinase 3 beta1 interactome.

Glycogen synthase kinase-3 beta (GSK3ß) was initially identified as a key protein in glucose metabolism. GSK3ß might be involved in cell growth, motility and apoptosis. Systematic identification of GSK3ß-associated proteins is crucial for the exhaustive understanding of its functions. Using GST pull-down experiment and LCMS/MS analysis coupled to bioinformatics tools, we have identified 114 proteins that interacted with GSK3ß1 in hepatocellular carcinoma HepG2 cells. Most of the identified proteins are implicated in metabolic process, whereas other proteins are important for cell proliferation or migration, and have been associated with cancer development and metastasis. Several representative proteins, such as hnRNPK, PCNA, Ezrin and STAT1, have been confirmed to interact with GSK-3ß1 by co-immunoprecipitation in HepG2 cells. Further studies of these interactions may discover the precise roles and the underlying mechanisms of GSK-3ß1 in tumour growth and metastasis.

GSK3ß/ITCH/c-FLIP Axis Counteracts TRAIL-induced Apoptosis in Human Lung Adenocarcinoma Cells.

AIMS: Further investigation on the mechanism of action of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in NSCLC would shed light on the understanding of TRAIL resistance and provide new clues for the counter-strategy. BACKGROUND: Cellular FLICE-inhibitory protein (c-FLIP) is a critical inhibitor of TRAIL-induced apoptosis. Our previous study suggested that glycogen synthase kinase 3ß (GSK3ß) positively regulated c-FLIP expression in human lung adenocarcinoma cells. Meanwhile, other studies reported that c-FLIP was degraded by HECT-type E3 ligase ITCH (Itchy E3 Ubiquitin Protein Ligase) via the proteasome pathway. OBJECTIVE: We will explore whether ITCH is involved in the expression regulation of c-FLIP positively controlled by GSK3ß during the treatment of TRAIL. METHODS: Human lung adenocarcinoma cells were used to stably overexpress and knockdown GSK3ß. Quantitative real-time PCR (qRT-PCR) assay was used to test the expressional level of mRNA of genes. Western blot analysis was employed to detect the expression of proteins at the protein level. siRNA of ITCH was used to knock down its expression. TRAIL treatment was used to cause apoptosis. RESULTS: In the present study, we have confirmed the degradation of c-FLIP by ITCH protein and the downregulation of ITCH expression by GSK3ß in lung adenocarcinoma cells. Moreover, ITCH silencing reversed the downregulation of c-FLIP protein caused by GSK3ß-knockdown in the cells. Accordingly, TRAIL-induced apoptosis facilitated by GSK3ß knockdown was blocked by the combined interference of ITCH. CONCLUSION: These results suggested that GSK3ß/ITCH axis regulated the stability of c-FLIP and influenced TRAIL-induced apoptosis. Taken together, our study revealed a GSK3ß/ITCH/c-FLIP axis, which counteracts TRAIL-induced apoptosis in human lung adenocarcinoma cells.

DDX17 promotes the growth and metastasis of lung adenocarcinoma.

DEAD box RNA helicase 17 (DDX17) has been shown to be an RNA binding protein involved in RNA metabolism and associated with cancer progression. However, the biological role of DDX17 in the pathogenesis of lung adenocarcinoma (LUAD) has not been well characterized. Here, we demonstrated that DDX17 promoted the proliferation, migration and invasion of H1299 and A549 lung adenocarcinoma cells. Analyses of public datasets showed that DDX17 is upregulated in LUAD specimens. Our tumor xenograft models confirmed the in vivo promoting role of DDX17 in the growth and metastasis of LUAD. Mechanistic analyses further revealed that DDX17 protein interacts with the mRNA of MYL9 and MAGEA6 and upregulates their levels. MYL9 could mediate the function of DDX17 to regulate the actin cytoskeleton rearrangement and cell adhesion, particularly by modulating the stress fiber and focal adhesion formation, whereas DDX17 might inhibit the autophagy process through MAGEA6/AMPKα1 axis in LUAD cells. Collectively, our study revealed the oncogenic role and pathways of DDX17 in LUAD.

Global identification of miR-373-regulated genes in breast cancer by quantitative proteomics.

Although microRNAs (miRNAs) have been reported to play an important role in carcinogenesis, their molecular mechanism remains largely unknown because of our limited understanding of miRNA target genes. miR-373 was found to be capable of promoting breast cancer invasion and metastasis, but only a target gene was experimentally identified on the basis of mRNA expression analysis. In this study, we used SILAC-based quantitative proteomics to globally identify the genes regulated by miR-373. Totally, 3666 proteins were identified, and 335 proteins were found to be regulated by miR-373. Among the 192 proteins that were downregulated by miR-373, 27 (14.1%) were predicted to have at least one potential match site at their 3'-UTR for miR-373 seed sequence. However, miR-373 did not affect the mRNA level of the five selected candidate targets, TXNIP, TRPS1, RABEP1, GRHL2 and HIP1, suggesting that the protein expressions were regulated by miR-373 via translational inhibition instead of mRNA degradation. Luciferase and mutation assays validated that TXNIP and RABEP1 were the direct target genes of miR-373. More than 30 proteins reported to be involved in cancer invasion and metastasis were found to be regulated by miR-373 in breast cancer for the first time.

Subcellular proteomics revealed the epithelial-mesenchymal transition phenotype in lung cancer.

Subcellular proteomics was used to compare the protein profiles between human lung adenocarcinoma A549 cells and human bronchial epithelial (HBE) cells. In total, 106 differential proteins were identified and the altered expression levels of partial identified proteins were confirmed by Western blot analysis. Importantly, pathway analysis and biological validation revealed epithelial-mesenchymal transition (EMT) phenotype shift in A549 cells as compared with HBE cells. The EMT phenotype of A549 cells can be increased by self-producing TGF-ß1 and significantly decreased by silencing heterogeneous nuclear ribonucleoprotein (hnRNPK) expression. As EMT has been considered as an important event during malignant tumor progression and metastasis, investigating EMT and deciphering the related pathways may lead to more efficient strategies to fight lung cancer progression. By integrating the subcellular proteomic data with EMT-related functional studies, we revealed new insights into the EMT progress of lung carcinogenesis, providing clues for further investigations on the discovery of potential therapeutic targets.

3'untranslated regions (3'UTR) of Gelsolin mRNA displays anticancer effects in non-small cell lung cancer (NSCLC) cells.

RNA-based therapeutics has attracted substantial interest from both academics and pharmaceutical companies. In this study, we investigated the function and the underlying mechanism of Gelsolin (GSN) 3'UTR in NSCLC H1299 and A549 cells. We found that transfected Flag-GSN plasmids significantly increased the proliferation, migration and invasion of NSCLC cells, whereas GSN 3'UTR could suppress the promotional effect of GSN protein on the development of NSCLC in vitro. Interestingly, we observed that these in vitro anticancer effects of GSN 3'UTR was independent of the co-expression with GSN coding sequence. Moreover, transfected GSN 3'UTR affected the actin-cytoskeleton remodeling and epithelial-mesenchymal transition (EMT) processes in H1299 and A549 cells, and targeted the co-expressed proteins to the plasma membrane. Subsequently, RNA pull-down assays have been performed to identify Tra2ß protein as a GSN 3'UTR binder. We then showed that Tra2ß was important for the localized protein expression mediated by GSN 3'UTR. Taken together, our results suggested that GSN 3'UTR may exert anticancer functions in NSCLC cells through regulating the subcellular localized expression of GSN protein mediated by the interaction between GSN 3'UTR-Tra2ß.

TRIM21-regulated Annexin A2 plasma membrane trafficking facilitates osteosarcoma cell differentiation through the TFEB-mediated autophagy.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents, which is characterized by dysfunctional autophagy and poor differentiation. Our recent studies have suggested that the tripartite motif containing-21 (TRIM21) plays a crucial role in regulating OS cell senescence and proliferation via interactions with several proteins. Yet, its implication in autophagy and differentiation in OS is largely unknown. In the present study, we first showed that TRIM21 could promote OS cell autophagy, as determined by the accumulation of LC3-II, and the degradation of cargo receptor p62. Further, we were able to identify that Annexin A2 (ANXA2), as a novel interacting partner of TRIM21, was critical for TIRM21-induced OS cell autophagy. Although TRIM21 had a negligible effect on the mRNA and protein expressions of ANXA2, we did find that TRIM21 facilitated the translocation of ANXA2 toward plasma membrane (PM) in OS cells through a manner relying on TRIM21-mediated cell autophagy. This functional link has been confirmed by observing a nice co-expression of TRIM21 and ANXA2 (at the PM) in the OS tissues. Mechanistically, we demonstrated that TRIM21, via facilitating the ANXA2 trafficking at the PM, enabled to release the transcription factor EB (TFEB, a master regulator of autophagy) from the ANXA2-TFEB complex, which in turn entered into the nucleus for the regulation of OS cell autophagy. In accord with previous findings that autophagy plays a critical role in the control of differentiation, we also demonstrated that autophagy inhibited OS cell differentiation, and that the TRIM21/ANXA2/TFEB axis is implicated in OS cell differentiation through the coordination with autophagy. Taken together, our results suggest that the TRIM21/ANXA2/TFEB axis is involved in OS cell autophagy and subsequent differentiation, indicating that targeting this signaling axis might lead to a new clue for OS treatment.