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Cisplatin is one of the most used anticancer chemotherapy agents; however, over time, patients develop resistance to the treatment, and survival rates drop dramatically. Investigation of tumor cell resistance mechanisms could increase sensitivity and prevent cancer progression. Here, we investigated the role of the E3 ubiquitin ligase SCF (β‐TrCP) in cisplatin resistance in different tumor cell lines, analyzing its role in the stability of BRCA1 and CtIP, proteins involved in DNA damage repair by homologous recombination. We showed that SCF(β‐TrCP) plays a key role in cisplatin response, as overexpression of wild‐type β‐TrCP increased DNA damage and cisplatin‐induced apoptosis, while overexpression of a dominant‐negative mutant or siRNA‐mediated downregulation of β‐TrCP decreased the damage and conferred treatment resistance. Furthermore, we demonstrated that BRCA1 and CtIP interacted with β‐TrCP in vivo , and their levels changed when β‐TrCP expression was modulated. We also described that β‐TrCP‐mediated BRCA1 degradation involves both lysosomal and proteasomal pathways. Mechanistically, the failure of β‐TrCP to regulate the degradation of BRCA1 enables a more efficient DNA damage repair and thereby the acquisition of cisplatin resistance. Overall, β‐TrCP overexpression sensitizes cisplatin‐induced DNA damage by depleting BRCA1.

Background Circular RNAs (circRNAs) act as gene expression regulators and are involved in cancer progression. However, their functions have not been sufficiently investigated in nasopharyngeal carcinoma (NPC). Methods The expression profiles of circRNAs in NPC cells within different metastatic potential were reanalyzed. Quantitative reverse transcription PCR and in situ hybridization were used to detect the expression level of circPVT1 in NPC cells and tissue samples. The association of expression level of circPVT1 with clinical properties of NPC patients was evaluated. Then, the effects of circPVT1 expression on NPC metastasis were investigated by in vitro and in vivo functional experiments. RNA immunoprecipitation, pull-down assay and western blotting were performed to confirm the interaction between circPVT1 and β-TrCP in NPC cells. Co-immunoprecipitation and western blotting were performed to confirm the interaction between β-TrCP and c-Myc in NPC cells. Results We find that circPVT1 , a circular RNA, is significantly upregulated in NPC cells and tissue specimens. In vitro and in vivo experiments showed that circPVT1 promotes the invasion and metastasis of NPC cells. Mechanistically, circPVT1 inhibits proteasomal degradation of c-Myc by binding to β-TrCP, an E3 ubiquiting ligase. Stablization of c-Myc by circPVT1 alters the cytoskeleton remodeling and cell adhesion in NPC, which ultimately promotes the invasion and metastasis of NPC cells. Furthermore, c-Myc transcriptionally upregulates the expression of SRSF1, an RNA splicing factor, and recruits SRSF1 to enhance the biosynthesis of circPVT1 through coupling transcription with splicing, which forms a positive feedback for circPVT1 production. Conclusions Our results revealed the important role of circPVT1 in the progression of NPC through the β-TrCP/c-Myc/SRSF1 positive feedback loop, and circPVT1 may serve as a prognostic biomarker or therapeutic target in patients with NPC.

Background Human umbilical cord-derived mesenchymal stem cell (hUC-MSC) engraftment is a promising therapy for acute ischemic stroke (AIS). However, the harsh ischemic microenvironment limits the therapeutic efficacy of hUC-MSC therapy. Curcumin is an anti-inflammatory agent that could improve inflammatory microenvironment. However, whether it enhances the neuroprotective efficacy of hUC-MSC transplantation is still unknown. In the present study, we investigated the therapeutic efficacy and the possible mechanism of combined curcumin and hUC-MSC treatment in AIS. Methods Middle cerebral artery occlusion (MCAO) mice and oxygen glucose deprivation (OGD) microglia were administrated hUC-MSCs with or without curcumin. Neurological deficits assessment, brain water content and TTC were used to assess the therapeutic effects of combined treatment. To elucidate the mechanism, MCAO mice and OGD microglia were treated with AKT inhibitor MK2206, GSK3β activator sodium nitroprusside (SNP), GSK3β inhibitor TDZD-8 and Nrf2 gene knockout were used. Immunofluorescence, flow cytometric analysis, WB and RT-PCR were used to evaluate the microglia polarization and the expression of typical oxidative mediators, inflammatory cytokines and the AKT/GSK-3β/β-TrCP/Nrf2 pathway protein. Results Compared with the solo hUC-MSC-grafted or curcumin groups, combined curcumin-hUC-MSC therapy significantly improved the functional performance outcomes, diminished the infarct volumes and the cerebral edema. The combined treatment promoted anti-inflammatory microglia polarization via Nrf2 pathway and decreased the expression of ROS, oxidative mediators and pro-inflammatory cytokines, while elevating the expression of the anti-inflammatory cytokines. Nrf2 knockout abolished the antioxidant stress and anti-inflammation effects mediated with combined treatment. Moreover, the combined treatment enhanced the phosphorylation of AKT and GSK3β, inhibited the β-TrCP nucleus translocation, accompanied with Nrf2 activation in the nucleus. AKT inhibitor MK2206 activated GSK3β and β-TrCP and suppressed Nrf2 phosphorylation in nucleus, whereas MK2206 with the GSK3β inhibitor TDZD-8 reversed these phenomena. Furthermore, combined treatment followed by GSK3β inhibition with TDZD-8 restricted β-TrCP nucleus accumulation, which facilitated Nrf2 expression. Conclusions We have demonstrated that combined curcumin-hUC-MSC therapy exerts anti-inflammation and antioxidant stress efficacy mediated by anti-inflammatory microglia polarization via AKT/GSK-3β/β-TrCP/Nrf2 axis and an improved neurological function after AIS.

Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to DNA-damaging therapies and is driven by oncogenic KRAS signaling. We found that inhibition of the KRAS effector Rac1 disrupts ATR/Chk1 DNA damage signaling by promoting the degradation of Claspin, a fork protection protein required for Chk1 activation. In PDAC and normal pancreatic ductal cells, Rac1 inhibition reduced Claspin protein stability without altering its mRNA levels, shortening its protein half-life more than threefold. Claspin loss required proteasomal degradation and βTrCP1/2, and depended on the Ser30/Ser34 phosphodegron, but occurred independently of PLK1 activity. Although Rac1 inhibition decreased Claspin in both normal and cancer cells, PDAC cells were selectively sensitized to γ-radiation, with Claspin depletion triggering apoptosis only in the tumor cells. These findings identify Rac1 as a key regulator of the replication stress response and suggest that targeting Rac1-mediated Claspin stabilization may enhance PDAC sensitivity to radiation therapy.

Huntington's disease (HD) is a fatal neurodegenerative disorder causing selective neuronal death in the brain. Dysfunction of the ubiquitin–proteasome system may contribute to the disease; however, the exact mechanisms are still unknown. We report here a new pathological mechanism by which mutant huntingtin specifically interferes with the degradation of β‐catenin. Huntingtin associates with the β‐catenin destruction complex that ensures its equilibrated degradation. The binding of β‐catenin to the destruction complex is altered in HD, leading to the toxic stabilization of β‐catenin. As a consequence, the β‐transducin repeat‐containing protein (β‐TrCP) rescues polyglutamine (polyQ)‐huntingtin‐induced toxicity in striatal neurons and in a Drosophila model of HD, through the specific degradation of β‐catenin. Finally, the non‐steroidal anti‐inflammatory drug indomethacin that decreases β‐catenin levels has a neuroprotective effect in a neuronal model of HD and in Drosophila and increases the lifespan of HD flies. We thus suggest that restoring β‐catenin homeostasis in HD is of therapeutic interest.

The ATR/CHK1 pathway is a key effector of cellular response to DNA damage and therefore is a critical regulator of genomic stability. While the ATR/CHK1 pathway is often inactivated by mutations, CHK1 itself is rarely mutated in human cancers. Thus, cellular levels of CHK1 likely play a key role in the maintenance of genomic stability and preventing tumorigenesis. Glucose deprivation is observed in many solid tumors due to high glycolytic rates of cancer cells and insufficient vascularization, yet cancer cells have devised mechanisms to survive in conditions of low glucose. Although CHK1 degradation through the ubiquitin–proteasome pathway following glucose deprivation has been previously reported, the detailed molecular mechanisms remain elusive. Here, we show that CHK1 is ubiquitinated and degraded upon glucose deprivation by the Skp1‐Cullin‐F‐box (β‐TrCP) E3 ubiquitin ligase. Specifically, CHK1 contains a β‐TrCP recognizable degron domain, which is phosphorylated by AMPK in response to glucose deprivation, allowing for β‐TrCP to recognize CHK1 for subsequent ubiquitination and degradation. Our results provide a novel mechanism by which glucose metabolism regulates a DNA damage effector, and imply that glucose deprivation, which is often found in solid tumor microenvironments, may enhance mutagenesis, clonal expansion, and tumor progression by triggering CHK1 degradation. Glucose deprivation is observed in many solid tumors, and cancer cells have devised mechanisms to survive in conditions of low glucose. Here, we show that CHK1, a critical regulator of genomic stability, is ubiquitinated and degraded upon glucose deprivation by the β‐TrCP E3 ubiquitin ligase. Our results indicate that glucose deprivation may enhance tumorigenesis through inactivation of the CHK1 pathway.

RIPK4 is a key player in epidermal differentiation and barrier formation. RIPK4 signaling pathways controlling keratinocyte proliferation and differentiation depend on its kinase activity leading to Dvl2, Pkp1 and IRF6 phosphorylation and NF-kappaB activation. However, the mechanism regulating RIPK4 activity levels remains elusive. We show that cultured keratinocytes display constitutive active phosphorylated RIPK4 while PKC signaling can trigger RIPK4 activation in various non-keratinocyte cell lines, in which RIPK4 is present in a non-phosphorylated state. Interestingly, we identified the SCF(beta-TrCP) ubiquitin E3 ligase complex responsible for regulating the active RIPK4 protein level. The SCF(beta-TrCP) complex binds to a conserved phosphodegron motif in the intermediate domain of RIPK4, subsequently leading to K48-linked ubiquitinylation and degradation. The recruitment of beta-TrCP is dependent on RIPK4 activation and trans-autophosphorylation. beta-TrCP knock-down resulted in RIPK4-dependent formation of actin stress fibers, cell scattering and increased cell motility, suggesting that tight control of RIPK4 activity levels is crucial to maintain cell shape and behavior in keratinocytes.

Previous cesarean scar defect (PCSD), also acknowledged as the myometrium of uterus defects, which commonly results in myometrial discontinuity between the uterine and cervical cavity. Current literatures have indicated the efficacy of MSCs and MSC-derived exosomes (MSC-Exos) for diverse refractory disease administration, yet the feasibility of MSC-Exos for PCSD treatment is largely obscure. In this study, we took advantage of the in vivo myofibrotic model for mimicking the typical manifestation of PCSD and the assessment of fertility. Meanwhile, the ex vivo scratch wound healing model is used for exploring the underlying molecular mechanism. On the one hand, we took advantage of the TGF-β-induced in vitro myofibrotic model and the full-thickness uterine injury rat model to verify the efficacy of human umbilical cord MSC-derived exosomes (hUC-MSC-Exos). On the other hand, with the aid of CHK1 overexpression and β-TrCP knockdown, together with multifaceted biological analyses (e.g., histopathological sections, qRT-PCR assay, western-blotting analysis, Co-IP assay, protein degradation and ubiquitination), we further dissected the underlying regulatory mechanism. We identified hUC-MSC-Exos and verified the suppressive effect of hUC-MSC-Exos upon TGF-β-induced in vitro myofibrotic model and full-thickness uterine injury in rats via delivering β-TrCP. Furthermore, we found that β-TrCP in hUC-MSC-Exos facilitated the ubiquitination degradation of CHK1 and inhibited the myofibrosis. Collectively, our data indicated the preferable ameliorative effect of hUC-MSC-Exos upon both the in vitro and in vivo myofibrotic models, together with the β-TrCP and CHK1-mediated regulatory mechanism. Our findings provided new references of hUC-MSC-Exos-based regimens for PCSD management in future.

Skp1–Cul1–F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface. Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.