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.

Tbl1 promotes Wnt-ß-catenin signaling-induced degradation of the Tcf7l1 protein in mouse embryonic stem cells.

Activation of the Wnt-ß-catenin signaling pathway by CHIR99021, a specific inhibitor of GSK3ß, induces Tcf7l1 protein degradation, which facilitates the maintenance of an undifferentiated state in mouse embryonic stem cells (mESCs); however, the precise mechanism is still unclear. Here, we showed that the overexpression of transducin-ß-like protein 1 (Tbl1, also known as Tbl1x) or its family member Tblr1 (also known as Tbl1xr1) can decrease Tcf7l1 protein levels, whereas knockdown of each gene increases Tcf7l1 levels without affecting Tcf7l1 transcription. Interestingly, only Tbl1, and not Tblr1, interacts with Tcf7l1. Mechanistically, Tbl1 translocates from the cytoplasm into the nucleus in association with ß-catenin (CTNNB1) after the addition of CHIR99021 and functions as an adaptor to promote ubiquitylation of the Tcf7l1 protein. Functional assays further revealed that enforced expression of Tbl1 is capable of delaying mESC differentiation. In contrast, knockdown of Tbl1 attenuates the effect of CHIR99021 on Tcf7l1 protein stability and mESC self-renewal. Our results provide insight into the regulatory network of the Wnt-ß-catenin signaling pathway involved in promoting the maintenance of naïve pluripotency.

The m6A modification-mediated positive feedback between glycolytic lncRNA SLC2A1-DT and c-Myc promotes tumorigenesis of hepatocellular carcinoma.

Glycolysis exerts a key role in the metabolic reprogramming of cancer. Specific long non-coding RNAs (lncRNAs) have been identified to exhibit oncogenic glycolysis regulation. Nevertheless, the precise mechanisms by which glycolysis-related lncRNAs control hepatocellular carcinoma (HCC) are still unknown. We profiled and analyzed glycolysis-associated lncRNA signatures using HCC specimens from The Cancer Genome Atlas (TCGA) dataset. Considerable upregulation of the glycolysis-related lncRNA SLC2A1-DT was noted in HCC tissues; this upregulation was strongly linked with advanced tumor stage and poor prognosis. Cell culture and animal-related studies indicated that knockdown or overexpression of SLC2A1-DT obviously restrained or promoted glycolysis, propagation, and metastasis in HCC cells. Mechanistically, SLC2A1-DT enhanced the interaction of protein between ß-catenin and YWHAZ, suppressing the binding between ß-catenin and ß-TrCP, an E3 ubiquitin ligase. Thereby, SLC2A1-DT impeded the ß-TrCP-dependent ubiquitination and ß-catenin degradation. The upregulated ß-catenin activated the transcription of c-Myc, which then increased the transcription of glycolytic genes including SLC2A1, LDHA, and HK2. Additionally, we revealed that c-Myc transcriptionally induced the expression of methyltransferase 3 (METTL3), which increased N6-methyladenosine (m6A) modification and stability of SLC2A1-DT in a YTHDF1 dependent manner. Collectively, we show that the lncRNA SLC2A1-DT promotes glycolysis and HCC tumorigenesis by a m6A modification-mediated positive feedback mechanism with glycolytic regulator c-Myc and suggested as an innovative treatment option and indicator for HCC.

Inhibition of glycogen synthase kinase-3 enhances NRF2 protein stability, nuclear localisation and target gene transcription in pancreatic beta cells.

Accumulation of reactive oxygen species (i.e., oxidative stress) is a leading cause of beta cell dysfunction and apoptosis in diabetes. NRF2 (NF-E2 p45-related factor-2) regulates the adaptation to oxidative stress, and its activity is negatively regulated by the redox-sensitive CUL3 (cullin-3) ubiquitin ligase substrate adaptor KEAP1 (Kelch-like ECH-associated protein-1). Additionally, NRF2 is repressed by the insulin-regulated Glycogen Synthase Kinase-3 (GSK3). We have demonstrated that phosphorylation of NRF2 by GSK3 enhances ß-TrCP (beta-transducin repeat-containing protein) binding and ubiquitylation by CUL1 (cullin-1), resulting in increased proteasomal degradation of NRF2. Thus, we hypothesise that inhibition of GSK3 activity or ß-TrCP binding upregulates NRF2 and so protects beta cells against oxidative stress. We have found that treating the pancreatic beta cell line INS-1 832/13 with the KEAP1 inhibitor TBE31 significantly enhanced NRF2 protein levels. The presence of the GSK3 inhibitor CT99021 or the ß-TrCP-NRF2 protein-protein interaction inhibitor PHAR, along with TBE31, resulted in prolonged NRF2 stability and enhanced nuclear localisation (P < 0.05). TBE31-mediated induction of NRF2-target genes encoding NAD(P)H quinone oxidoreductase 1 (Nqo1), glutamate-cysteine ligase modifier (Gclm) subunit and heme oxygenase (Hmox1) was significantly enhanced by the presence of CT99021 or PHAR (P < 0.05) in both INS-1 832/13 and in isolated mouse islets. Identical results were obtained using structurally distinct GSK3 inhibitors and inhibition of KEAP1 with sulforaphane. In summary, we demonstrate that GSK3 and ß-TrCP/CUL1 regulate the proteasomal degradation of NRF2, enhancing the impact of KEAP1 regulation, and so contributes to the redox status of pancreatic beta cells. Inhibition of GSK3, or ß-TrCP/CUL1 binding to NRF2 may represent a strategy to protect beta cells from oxidative stress.

CRL4DCAF1 ubiquitin ligase regulates PLK4 protein levels to prevent premature centriole duplication.

Centrioles play important roles in the assembly of centrosomes and cilia. Centriole duplication occurs once per cell cycle and is dependent on polo-like kinase 4 (PLK4). To prevent centriole amplification, which is a hallmark of cancer, PLK4 protein levels need to be tightly regulated. Here, we show that the Cullin4A/B-DDB1-DCAF1, CRL4DCAF1, E3 ligase targets PLK4 for degradation in human cells. DCAF1 binds and ubiquitylates PLK4 in the G2 phase to prevent premature centriole duplication in mitosis. In contrast to the regulation of PLK4 by SCFß-TrCP, the interaction between PLK4 and DCAF1 is independent of PLK4 kinase activity and mediated by polo-boxes 1 and 2 of PLK4, suggesting that DCAF1 promotes PLK4 ubiquitylation independently of ß-TrCP. Thus, the SCFSlimb/ß-TrCP pathway, targeting PLK4 for ubiquitylation based on its phosphorylation state and CRL4DCAF1, which ubiquitylates PLK4 by binding to the conserved PB1-PB2 domain, appear to be complementary ways to control PLK4 abundance to prevent centriole overduplication.

miR-15b-5p promotes HgCl2-induced chicken embryo kidney cells ferroptosis by targeting ß-TrCP-mediated ATF4 ubiquitin degradation.

Mercuric chloride (HgCl2), a widespread environmental pollutant, induces ferroptosis in chicken embryonic kidney (CEK) cells. Whereas activating transcription factor 4 (ATF4), a critical mediator of oxidative homeostasis, plays a dual role in ferroptosis, but its precise mechanisms in HgCl2-induced ferroptosis remain elusive. This study aims to investigate the function and molecular mechanism of ATF4 in HgCl2-induced ferroptosis. Our results revealed that ATF4 was downregulated during HgCl2-induced ferroptosis in CEK cells. Surprisingly, HgCl2 exposure has no significant impact on ATF4 mRNA level. Further investigation indicated that HgCl2 enhanced the expression of the E3 ligase beta-transducin repeat-containing protein (ß-TrCP) and increased ATF4 ubiquitination. Subsequent findings identified that miR-15b-5p as an upstream modulator of ß-TrCP, with miR-15b-5p downregulation observed in HgCl2-exposed CEK cells. Importantly, miR-15b-5p mimics suppressed ß-TrCP expression and reversed HgCl2-induced cellular ferroptosis. Mechanistically, HgCl2 inhibited miR-15b-5p, and promoted ß-TrCP-mediated ubiquitin degradation of ATF4, thereby inhibited the expression of antioxidant-related target genes and promoted ferroptosis. In conclusion, our study highlighted the crucial role of the miR-15b-5p/ß-TrCP/ATF4 axis in HgCl2-induced nephrotoxicity, offering a new therapeutic target for understanding the mechanism of HgCl2 nephrotoxicity.

Modulation of Cullin-RING E3 ubiquitin ligase-dependent ubiquitination by small molecule compounds.

Cullin (CUL)-RING (Really Interesting New Gene) E3 ubiquitin (Ub) ligases (CRLs) are the largest E3 family. The E3 CRL core ligase is a subcomplex formed by the CUL C-terminal domain bound with the ROC1/RBX1 RING finger protein, which acts as a hub that mediates and organizes multiple interactions with E2, Ub, Nedd8, and the ARIH family protein, thereby resulting in Ub transfer to the E3-bound substrate. This report describes the modulation of CRL-dependent ubiquitination by small molecule compounds including KH-4-43, #33, and suramin, which target the CRL core ligases. We show that both KH-4-43 and #33 inhibit the ubiquitination of CK1α by CRL4CRBN. However, either compound's inhibitory effect on this reaction is significantly reduced when a neddylated form of CRL4CRBN is used. On the other hand, both #33 and KH-4-43 inhibit the ubiquitination of ß-catenin by CRL1ß-TrCP and Nedd8-CRL1ß-TrCP almost equally. Thus, neddylation of CRL1ß-TrCP does not negatively impact the sensitivity to inhibition by #33 and KH-4-43. These findings suggest that the effects of neddylation to alter the sensitivity of CRL inhibition by KH-4-43/#33 is dependent upon the specific CRL type. Suramin, a compound that targets CUL's basic canyon, can effectively inhibit CRL1/4-dependent ubiquitination regardless of neddylation status, in contrast to the results observed with KH-4-43/#33. This observed differential drug sensitivity of KH-4-43/#33 appears to echo CUL-specific Nedd8 effects on CRLs as revealed by recent high-resolution structural biology efforts. The highly diversified CRL core ligase structures may provide opportunities for specific targeting by small molecule modulators.

Monoubiquitination empowers ubiquitin chain elongation.

Ubiquitination often generates lysine 48-linked polyubiquitin chains that signal proteolytic destruction of the protein target. A significant subset of ubiquitination proceeds by a priming/extending mechanism, in which a substrate is first monoubiquitinated with a priming E2-conjugating enzyme or a set of E3 ARIH/E2 enzymes specific for priming. This is then followed by ubiquitin (Ub) chain extension catalyzed by an E2 enzyme capable of elongation. This report provides further insights into the priming/extending mechanism. We employed reconstituted ubiquitination systems of substrates CK1α (casein kinase 1α) and ß-catenin by Cullin-RING E3 Ub ligases (CRLs) CRL4CRBN and CRL1ßTrCP, respectively, in the presence of priming E2 UbcH5c and elongating E2 Cdc34b (cell division cycle 34b). We have established a new "apyrase chase" strategy that uncouples priming from chain elongation, which allows accurate measurement of the decay rates of the ubiquitinated substrate with a defined chain length. Our work has revealed highly robust turnover of monoubiquitinated ß-catenin that empowers efficient polyubiquitination. The results of competition experiments suggest that the interactions between the ubiquitinated ß-catenin and CRL1ßTrCP are highly dynamic. Moreover, ubiquitination of the Ub-modified ß-catenin appeared more resistant to inhibition by competitors than the unmodified substrate, suggesting tighter binding with CRL1ßTrCP. These findings support a role for conjugated Ub in enhancing interactions with E3.

Novel targets of ß-TrCP cooperatively accelerate carbohydrate and fatty acid consumption.

Cellular energy is primarily produced from glucose and fat through glycolysis and fatty acid oxidation (FAO) followed by the tricarboxylic acid cycle in mitochondria; energy homeostasis is carefully maintained via numerous feedback pathways. In this report, we uncovered a new master regulator of carbohydrate and lipid metabolism. When ubiquitin E3 ligase ß-TrCP2 was inducibly knocked out in ß-TrCP1 knockout adult mice, the resulting double knockout mice (DKO) lost fat mass rapidly. Biochemical analyses of the tissues and cells from ß-TrCP2 KO and DKO mice revealed that glycolysis, FAO, and lipolysis were dramatically upregulated. The absence of ß-TrCP2 increased the protein stability of metabolic rate-limiting enzymes including 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3), adipose triglyceride lipase (ATGL), carnitine palmitoyltransferase 1A (CPT1A), and carnitine/acylcarnitine translocase (CACT). Our data suggest that ß-TrCP is a potential regulator for total energy homeostasis by simultaneously controlling glucose and fatty acid metabolism and that targeting ß-TrCP could be an effective strategy to treat obesity and other metabolic disorders.

The novel ß-TrCP protein isoform hidden in circular RNA confers trastuzumab resistance in HER2-positive breast cancer.

Trastuzumab notably improves the outcome of human epidermal growth factor receptor 2 (HER2)-positive breast cancer patients, however, resistance to trastuzumab remains a major hurdle to clinical treatment. In the present study, we identify a circular RNA intimately linked to trastuzumab resistance. circ-ß-TrCP, derived from the back-splicing of ß-TrCP exon 7 and 13, confers trastuzumab resistance by regulating NRF2-mediated antioxidant pathway in a KEAP1-independent manner. Concretely, circ-ß-TrCP encodes a novel truncated 343-amino acid peptide located in the nucleus, referred as ß-TrCP-343aa, which competitively binds to NRF2, blocks SCFß-TrCP-mediated NRF2 proteasomal degradation, and this protective effect of ß-TrCP-343aa on NRF2 protein requires GSK3 activity. Subsequently, the elevated NRF2 transcriptionally upregulates a cohort of antioxidant genes, giving rise to trastuzumab resistance. Moreover, the translation ability of circ-ß-TrCP is inhibited by eIF3j under both basal and oxidative stress conditions, and eIF3j is transcriptionally repressed by NRF2, thus forming a positive feedback circuit between ß-TrCP-343aa and NRF2, expediting trastuzumab resistance. Collectively, our data demonstrate that circ-ß-TrCP-encoded ß-TrCP protein isoform drives HER2-targeted therapy resistance in a NRF2-dependent manner, which provides potential therapeutic targets for overcoming trastuzumab resistance.

Glutamine regulates the cellular proliferation and cell cycle progression by modulating the mTOR mediated protein levels of ß-TrCP.

The amino acid glutamine plays an important role in cell growth and proliferation. Reliance on glutamine has long been considered a hallmark of highly proliferating cancer cells. Development of strategies for cancer therapy that primarily target glutamine metabolism has been an active area of research. Glutamine depletion is associated with growth arrest and apoptosis-induced cell death; however, the molecular mechanisms involved in this process are not clearly understood. Here, we show that glutamine depletion activates the energetic stress AMPK pathway and inhibits mTORC1 activity. Furthermore, inhibition of mTORC1 reduces the protein levels of ß-TrCP, resulting in aberrant cell cycle progression and reduced proliferation. In agreement with the role of ß-TrCP in glutamine metabolism, knockdown of ß-TrCP resulted in proliferation and cell cycle defects similar to those observed for glutamine depletion. In summary, our results provide mechanistic insights into the role of glutamine metabolism in regulation of cell growth and proliferation via ß-TrCP, uncovering a previously undescribed molecular process involved in glutamine metabolism.

Isolation and characterization of ß-transducin repeat-containing protein ligands screened using a high-throughput screening system.

ß-transducin repeat-containing protein (ß-TrCP) is an F-box protein subunit of the E3 Skp1-Cullin-F box (SCF) type ubiquitin-ligase complex, and provides the substrate specificity for the ligase. To find potent ligands of ß-TrCP useful for the proteolysis targeting chimera (PROTAC) system using ß-TrCP in the future, we developed a high-throughput screening system for small molecule ß-TrCP ligands. We screened the chemical library utilizing the system and obtained several hit compounds. The effects of the hit compounds on in vitro ubiquitination activity of SCFß-TrCP1 and on downstream signaling pathways were examined. Hit compounds NPD5943, NPL62020-01, and NPL42040-01 inhibited the TNFα-induced degradation of IκBα and its phosphorylated form. Hence, they inhibited the activation of the transcription activity of NF-κB, indicating the effective inhibition of ß-TrCP by the hit compounds in cells. Next, we performed an in silico analysis of the hit compounds to determine the important moieties of the hit compounds. Carboxyl groups of NPL62020-01 and NPL42040-01 and hydroxyl groups of NPD5943 created hydrogen bonds with ß-TrCP similar to those created by intrinsic target phosphopeptides of ß-TrCP. Our findings enhance our knowledge of useful small molecule ligands of ß-TrCP and the importance of residues that can be ligands of ß-TrCP.

Differential dysregulation of ß-TrCP1 and -2 by HIV-1 Vpu leads to inhibition of canonical and non-canonical NF-κB pathways in infected cells.

The HIV-1 Vpu protein is expressed late in the virus lifecycle to promote infectious virus production and avoid innate and adaptive immunity. This includes the inhibition of the NF-κB pathway which, when activated, leads to the induction of inflammatory responses and the promotion of antiviral immunity. Here we demonstrate that Vpu can inhibit both canonical and non-canonical NF-κB pathways, through the direct inhibition of the F-box protein ß-TrCP, the substrate recognition portion of the Skp1-Cul1-F-box (SCF)ß-TrCP ubiquitin ligase complex. There are two paralogues of ß-TrCP (ß-TrCP1/BTRC and ß-TrCP2/FBXW11), encoded on different chromosomes, which appear to be functionally redundant. Vpu, however, is one of the few ß-TrCP substrates to differentiate between the two paralogues. We have found that patient-derived alleles of Vpu, unlike those from lab-adapted viruses, trigger the degradation of ß-TrCP1 while co-opting its paralogue ß-TrCP2 for the degradation of cellular targets of Vpu, such as CD4. The potency of this dual inhibition correlates with stabilization of the classical IκBα and the phosphorylated precursors of the mature DNA-binding subunits of canonical and non-canonical NF-κB pathways, p105/NFκB1 and p100/NFκB2, in HIV-1 infected CD4+ T cells. Both precursors act as alternative IκBs in their own right, thus reinforcing NF-κB inhibition at steady state and upon activation with either selective canonical or non-canonical NF-κB stimuli. These data reveal the complex regulation of NF-κB late in the viral replication cycle, with consequences for both the pathogenesis of HIV/AIDS and the use of NF-κB-modulating drugs in HIV cure strategies. IMPORTANCE The NF-κB pathway regulates host responses to infection and is a common target of viral antagonism. The HIV-1 Vpu protein inhibits NF-κB signaling late in the virus lifecycle, by binding and inhibiting ß-TrCP, the substrate recognition portion of the ubiquitin ligase responsible for inducing IκB degradation. Here we demonstrate that Vpu simultaneously inhibits and exploits the two different paralogues of ß-TrCP by triggering the degradation of ß-TrCP1 and co-opting ß-TrCP2 for the destruction of its cellular targets. In so doing, it has a potent inhibitory effect on both the canonical and non-canonical NF-κB pathways. This effect has been underestimated in previous mechanistic studies due to the use of Vpu proteins from lab-adapted viruses. Our findings reveal previously unappreciated differences in the ß-TrCP paralogues, revealing functional insights into the regulation of these proteins. This study also raises important implications for the role of NF-κB inhibition in the immunopathogenesis of HIV/AIDS and the way that this may impact on HIV latency reversal strategies based on the activation of the non-canonical NF-κB pathway.

Human Neuralized is a novel tumour suppressor targeting Wnt/ß-catenin signalling in colon cancer.

While there is growing evidence that many epigenetically silenced genes in cancer are tumour suppressor candidates, their significance in cancer biology remains unclear. Here, we identify human Neuralized (NEURL), which acts as a novel tumour suppressor targeting oncogenic Wnt/ß-catenin signalling in human cancers. The expression of NEURL is epigenetically regulated and markedly suppressed in human colorectal cancer. We, therefore, considered NEURL to be a bona fide tumour suppressor in colorectal cancer and demonstrate that this tumour suppressive function depends on NEURL-mediated oncogenic ß-catenin degradation. We find that NEURL acts as an E3 ubiquitin ligase, interacting directly with oncogenic ß-catenin, and reducing its cytoplasmic levels in a GSK3ß- and ß-TrCP-independent manner, indicating that NEURL-ß-catenin interactions can lead to a disruption of the canonical Wnt/ß-catenin pathway. This study suggests that NEURL is a therapeutic target against human cancers and that it acts by regulating oncogenic Wnt/ß-catenin signalling.

Small molecule and peptide inhibitors of ßTrCP and the ßTrCP-NRF2 protein-protein interaction.

The E3 ligase beta-transducin repeat-containing protein (ßTrCP) is an essential component of the ubiquitin-proteasome system that is responsible for the maintenance of cellular protein levels in human cells. Key target substrates for degradation include inhibitor of nuclear factor kappa B, programmed cell death protein 4 and forkhead box protein O3, alongside the transcription factor nuclear factor erythroid-2-related factor 2 (NRF2) that is responsible for cellular protection against oxidative damage. The tumour suppressive nature of many of its substrates and the overexpression of ßTrCP observed in various cancers support a potential therapeutic role for inhibitors in the treatment of cancer. A small molecule substituted pyrazolone, GS143, and the natural product erioflorin have been identified as inhibitors of ßTrCP and protect its targets from proteasomal degradation. Modified peptides based on the sequences of native substrates have also been reported with KD values in the nanomolar range. This review describes the current status of inhibitors of this E3 ligase. The scope for further inhibitor design and the development of PROTAC and molecular glue-type structures is explored in the context of ßTrCP as an example of WD40 domain-containing proteins that are gaining attention as drug targets.

Expression of FBXW11 in normal and disease-associated osteogenic cells.

The ubiquitin-proteasome system (UPS) plays an important role in maintaining cellular homeostasis by degrading a multitude of key regulatory proteins. FBXW11, also known as b-TrCP2, belongs to the F-box family, which targets the proteins to be degraded by UPS. Transcription factors or proteins associated with cell cycle can be modulated by FBXW11, which may stimulate or inhibit cellular proliferation. Although FBXW11 has been investigated in embryogenesis and cancer, its expression has not been evaluated in osteogenic cells. With the aim to explore FBXW11gene expression modulation in the osteogenic lineage we performed molecular investigations in mesenchymal stem cells (MSCs) and osteogenic cells in normal and pathological conditions. In vitro experiments as well as ex vivo investigations have been performed. In particular, we explored the FBXW11 expression in normal osteogenic cells as well as in cells of cleidocranial dysplasia (CCD) patients or osteosarcoma cells. Our data showed that FBXW11 expression is modulated during osteogenesis and overexpressed in circulating MSCs and in osteogenically stimulated cells of CCD patients. In addition, FBXW11 is post-transcriptionally regulated in osteosarcoma cells leading to increased levels of beta-catenin. In conclusion, our findings show the modulation of FBXW11 in osteogenic lineage and its dysregulation in impaired osteogenic cells.

Enhanced binding of ß-catenin and ß-TrCP mediates LMPt's anti-CSCs activity in colorectal cancer.

Cancer stem cells (CSCs), a subpopulation of tumor cells with the features of self-renewal, tumor initiation, and insensitivity to common physical and chemical agents, are the key to cancer relapses, metastasis, and resistance. Accessible CSCs inhibitory strategies are primarily based on small molecule drugs, yet toxicity limits their application. Here, we report a liposome loaded with low toxicity and high effectiveness of miriplatin, lipo-miriplatin (LMPt) with high miriplatin loading, and robust stability, exhibiting a superior inhibitory effect on CSCs and non-CSCs. LMPt predominantly inhibits the survival of oxaliplatin-resistant (OXA-resistant) cells composed of CSCs. Furthermore, LMPt directly blocks stemness features of self-renewal, tumor initiation, unlimited proliferation, metastasis, and insensitivity. In mechanistic exploration, RNA sequencing (RNA-seq) revealed that LMPt downregulates the levels of pro-stemness proteins and that the ß-catenin-mediated stemness pathway is enriched. Further research shows that either in adherent cells or 3D-spheres, the ß-catenin-OCT4/NANOG axis, the vital pathway to maintain stemness, is depressed by LMPt. The consecutive activation of the ß-catenin pathway induced by mutant ß-catenin (S33Y) and OCT4/NANOG overexpression restores LMPt's anti-CSCs effect, elucidating the key role of the ß-catenin-OCT4/NANOG axis. Further studies revealed that the strengthened binding of ß-catenin and ß-TrCP initiates ubiquitination and degradation of ß-catenin induced by LMPt. In addition, the ApcMin/+ transgenic mouse model, in which colon tumors are spontaneously formed, demonstrates LMPt's potent anti-non-CSCs activity in vivo.

All-Trans Retinoic Acid Promotes a Tumor Suppressive OTUD6B-ß-TrCP-SNAIL Axis in Esophageal Squamous Cell Carcinoma and Enhances Immunotherapy.

ß-TrCP is an E3 ubiquitin ligase that plays important roles in multiple human cancers including esophageal squamous cell carcinoma (ESCC). Analysis of ESCC patient samples reveal that only protein level but not transcript level of ß-TrCP associated with patient prognosis, suggesting regulators of ß-TrCP protein stability play an essential role in ESCC progression and may be novel targets to develop ESCC therapies. Although ß-TrCP stability is known to be mediated by the ubiquitin-proteasome system, it is unclear which enzymes play a major role to determine ß-TrCP stability in the context of ESCC. In this study, OTUD6B is identified as a potent deubiquitinase of ß-TrCP that suppress ESCC progression through the OTUD6B-ß-TrCP-SNAIL axis. Low OTUD6B expression is associated with a poor prognosis of ESCC patients. Importantly, all-trans retinoic acid (ATRA) is found to promote OTUD6B translation and thus suppress ESCC tumor growth and enhance the response of ESCC tumors to anti-PD-1 immunotherapies. These findings demonstrate that OTUD6B is a crucial deubiquitinase of ß-TrCP in ESCC and suggest combination of ATRA and anti-PD-1 immune checkpoint inhibitor may benefit a cohort of ESCC patients.

Ubiquitin E3 ligase ß-TrCP negatively regulates surface protein of hepatitis B virus.

Chronic hepatitis B (CHB) still cannot be cured currently, while the pursuit of a functional cure seems to be an accessible goal, in which the condition mainly depends on the serum hepatitis B surface antigen (HBsAg) levels. HBsAg may be downregulated by protein ubiquitination, which may facilitate finding a new potential intervention target for functional cure of CHB. We confirmed that ß-transducin repeat-containing protein (ß-TrCP) was the E3 ubiquitin ligase of HBsAg. ß-TrCP specifically downregulated the expression of Myc-HBsAg. The degradation of Myc-HBsAg occurred via the proteasome pathway. Knockdown of ß-TrCP increased Myc-HBsAg levels in HepG2 cells. The study further indicated that ß-TrCP could affect the K48-linked polyubiquitin chain by acting on Myc-HBsAg. The GS137 G motif of HBsAg protein is required for ß-TrCP-mediated degradation. Furthermore, we found that ß-TrCP could significantly inhibit both intracellular and extracellular HBsAg levels produced by pHBV-1.3. Our study demonstrated that the E3 ubiquitin ligase ß-TrCP induces K48-linked polyubiquitination of HBsAg, promotes the ubiquitination degradation of HBsAg, and downregulates intra- and extracellular HBsAg levels. Therefore, using the ubiquitination degradation pathway of HBsAg, it is possible to reduce HBsAg levels in CHB patients, which may be helpful in obtaining the goal of functional cure in the treatment of CHB patients.

SDCBP promotes pancreatic cancer progression by preventing YAP1 from ß-TrCP-mediated proteasomal degradation.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal tumour with limited treatment options. Here, we identified syndecan binding protein (SDCBP), also known as syntenin1, as a novel targetable factor in promoting PDAC tumour progression. We also explored a therapeutic strategy for suppressing SDCBP expression. DESIGN: We used samples from patients with PDAC, human organoid models, LSL-KrasG12D/+mice, LSL-Trp53R172H/+ and Pdx1-Cre (KPC) mouse models, and PDX mouse models. Immunostaining, colony formation assay, ethynyl-2-deoxyuridine incorporation assay, real-time cell analysis, cell apoptosis assay, automated cell tracking, invadopodia detection and gelatin degradation assays, coimmunoprecipitation, and pull-down assays were performed in this study. RESULTS: The median overall survival and recurrence-free survival rates in the high-SDCBP group were significantly shorter than those in the low-SDCBP group. In vitro and in vivo studies have demonstrated that SDCBP promotes PDAC proliferation and metastasis. Mechanically, SDCBP inhibits CK1δ/ε-mediated YAP-S384/S387 phosphorylation, which further suppresses ß-TrCP-mediated YAP1 ubiquitination and proteasome degradation by directly interacting with YAP1. SDCBP interacts with the TAD domain of YAP1, mainly through its PDZ1 domain. Preclinical KPC mouse cohorts demonstrated that zinc pyrithione (ZnPT) suppresses PDAC tumour progression by suppressing SDCBP. CONCLUSIONS: SDCBP promotes the proliferation and metastasis of PDAC by preventing YAP1 from ß-TrCP-mediated proteasomal degradation. Therefore, ZnPT could be a promising therapeutic strategy to inhibit PDAC progression by suppressing SDCBP.