Upregulation of the long noncoding RNA GJA9-MYCBP and PVT1 is a potential diagnostic biomarker for acute lymphoblastic leukemia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common type of blood cancer in children. Aberrant expression of long noncoding RNAs (lncRNAs) may set stages for ALL development. LncRNAs are emerging as a novel diagnostic and prognostic biomarker for ALL. Herein, we aimed to evaluate the expression of lncRNA GJA9-MYCBP and PVT1 in blood samples of ALL and healthy individuals. METHODS: As a case-control study, 40 pairs of ALL and healthy individual samples were used. The expression of MYC and each candidate lncRNA was measured using quantitative real-time PCR. Any possible association between the expression of putative noncoding RNAs and clinicopathological characteristics was also evaluated. RESULTS: LncRNA GJA9-MYCBP and PVT1 were significantly upregulated in ALL samples compared with healthy ones. Similarly, mRNA levels of MYC were increased in ALL samples than control ones. Receiver operating characteristic curve analysis indicated a satisfactory diagnostic efficacy (p-value <.0001), suggesting that lncRNA GJA9-MYCBP and PVT1 may serve as a diagnostic biomarker for ALL. Linear regression analysis unveiled positive correlations between the expression level of MYC and lncRNA GJA9-MYCBP and PVT1 in ALL patients (p-values <.01). CONCLUSIONS: In this study, we provided approval for the clinical diagnostic significance of lncRNA GJA9-MYCBP and PVT1that their upregulations may be a diagnostic biomarker for ALL.

Ginsenoside Rg1 Regulates Immune Microenvironment and Neurological Recovery After Spinal Cord Injury Through MYCBP2 Delivery via Neuronal Cell-Derived Extracellular Vesicles.

Spinal cord injury (SCI) is a severe neurological condition that frequently leads to significant sensory, motor, and autonomic dysfunction. This study sought to delineate the potential mechanistic underpinnings of extracellular vesicles (EVs) derived from ginsenoside Rg1-pretreated neuronal cells (Rg1-EVs) in ameliorating SCI. These results demonstrated that treatment with Rg1-EVs substantially improved motor function in spinal cord-injured mice. Rg1-EVs enhance microglial polarization toward the M2 phenotype and repressed oxidative stress, thereby altering immune responses and decreasing inflammatory cytokine secretion. Moreover, Rg1-EVs substantially diminish reactive oxygen species accumulation and enhanced neural tissue repair by regulating mitochondrial function. Proteomic profiling highlighted a significant enrichment of MYCBP2 in Rg1-EVs, and functional assays confirmed that MYCBP2 knockdown counteracted the beneficial effects of Rg1-EVs in vitro and in vivo. Mechanistically, MYCBP2 is implicated in the ubiquitination and degradation of S100A9, thereby promoting microglial M2-phenotype polarization and reducing oxidative stress. Overall, these findings substantiated the pivotal role of Rg1-EVs in neuronal protection and functional recovery following SCI through MYCBP2-mediated ubiquitination of S100A9. This research offers novel mechanistic insights into therapeutic strategies against SCI and supports the clinical potential of Rg1-EVs.

Circ_0002669 promotes osteosarcoma tumorigenesis through directly binding to MYCBP and sponging miR-889-3p.

Circular RNAs (circRNAs) are a class of highly multifunctional single-stranded RNAs that play crucial roles in cancer progression, including osteosarcoma (OS). Circ_0002669, generated from the dedicator of cytokinesis (DOCK) gene, was highly expressed in OS tissues, and negatively correlated with OS patient survival. Elevated circ_0002669 promoted OS cell growth and invasion in vivo and in vitro. By biotin pulldown and mass spectroscopy, we found that circ_0002669 directly bound to MYCBP, a positive regulator of c-myc, to prevent MYCBP from ubiquitin-mediated proteasome degradation. In addition, circ_0002669 interacted with miR-889-3p and served as a miRNA sponge to increase the expression of MYCBP, as determined by luciferase assays and RNA immunoprecipitation. Functional rescue experiments indicated MYCBP acted as a key factor for circ_0002669- and miR-889-3p-regulated OS cell proliferation and migration. Increased expression of c-myc-associated genes, such as CCND1, c-Jun and CDK4, were found in circ_0002669- and MYCBP-overexpressing OS cells. Our data thus provide evidence that circ_0002669 promotes OS malignancy by protecting MYCBP from protein ubiquitination and degradation and blocking miR-889-3p-mediated inhibition of MYCBP expression.

Ubiquitin ligase and signalling hub MYCBP2 is required for efficient EPHB2 tyrosine kinase receptor function.

Eph receptor tyrosine kinases participate in a variety of normal and pathogenic processes during development and throughout adulthood. This versatility is likely facilitated by the ability of Eph receptors to signal through diverse cellular signalling pathways: primarily by controlling cytoskeletal dynamics, but also by regulating cellular growth, proliferation, and survival. Despite many proteins linked to these signalling pathways interacting with Eph receptors, the specific mechanisms behind such links and their coordination remain to be elucidated. In a proteomics screen for novel EPHB2 multi-effector proteins, we identified human MYC binding protein 2 (MYCBP2 or PAM or Phr1). MYCBP2 is a large signalling hub involved in diverse processes such as neuronal connectivity, synaptic growth, cell division, neuronal survival, and protein ubiquitination. Our biochemical experiments demonstrate that the formation of a complex containing EPHB2 and MYCBP2 is facilitated by FBXO45, a protein known to select substrates for MYCBP2 ubiquitin ligase activity. Formation of the MYCBP2-EPHB2 complex does not require EPHB2 tyrosine kinase activity and is destabilised by binding of ephrin-B ligands, suggesting that the MYCBP2-EPHB2 association is a prelude to EPHB2 signalling. Paradoxically, the loss of MYCBP2 results in increased ubiquitination of EPHB2 and a decrease of its protein levels suggesting that MYCBP2 stabilises EPHB2. Commensurate with this effect, our cellular experiments reveal that MYCBP2 is essential for efficient EPHB2 signalling responses in cell lines and primary neurons. Finally, our genetic studies in Caenorhabditis elegans provide in vivo evidence that the ephrin receptor VAB-1 displays genetic interactions with known MYCBP2 binding proteins. Together, our results align with the similarity of neurodevelopmental phenotypes caused by MYCBP2 and EPHB2 loss of function, and couple EPHB2 to a signalling effector that controls diverse cellular functions.

The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA.

Upstream open reading frames (uORFs) are a frequent feature of eukaryotic mRNAs. Upstream ORFs govern main ORF translation in a variety of ways, but, in a nutshell, they either filter out scanning ribosomes or allow downstream translation initiation via leaky scanning or reinitiation. Previous reports concurred that eIF4G2, a long-known but insufficiently studied eIF4G1 homologue, can rescue the downstream translation, but disagreed on whether it is leaky scanning or reinitiation that eIF4G2 promotes. Here, we investigated a unique human mRNA that encodes two highly conserved proteins (POLGARF with unknown function and POLG, the catalytic subunit of the mitochondrial DNA polymerase) in overlapping reading frames downstream of a regulatory uORF. We show that the uORF renders the translation of both POLGARF and POLG mRNAs reliant on eIF4G2. Mechanistically, eIF4G2 enhances both leaky scanning and reinitiation, and it appears that ribosomes can acquire eIF4G2 during the early steps of reinitiation. This emphasizes the role of eIF4G2 as a multifunctional scanning guardian that replaces eIF4G1 to facilitate ribosome movement but not ribosome attachment to an mRNA.

Regulator of G protein signaling 12 drives inflammatory arthritis by activating synovial fibroblasts through MYCBP2/KIF2A signaling.

Synovial fibroblasts are the active and aggressive drivers in the progression of arthritis, but the cellular and molecular mechanisms remain unknown. Here, our results showed that regulator of G protein signaling 12 (RGS12) maintained ciliogenesis in synovial fibroblasts, which is critical for the development of inflammatory arthritis. Deletion of RGS12 led to a significant decrease in ciliogenesis, adhesion, migration, and secretion of synovial fibroblasts. Mechanistically, RGS12 overexpression in synovial fibroblasts increased the length and number of cilia but decreased the protein level of kinesin family member 2A (KIF2A). The results of LC-MS analyses showed that RGS12 interacted with MYC binding protein 2 to enhance its ubiquitination activity, through which the KIF2A protein was degraded in synovial fibroblasts. Moreover, overexpression of KIF2A blocked the increases in cilia length and number. Mice with RGS12 deficiency or treatment of RGS12 shRNA nanoparticles significantly decreased the clinical score, paw swelling, synovitis, and cartilage destruction during inflammatory arthritis by inhibiting the activation of synovial fibroblasts. Therefore, this study provides evidence that RGS12 activates synovial fibroblasts' pathological function via promoting MCYBP2-mediated degradation of KIF2A and ciliogenesis. Our data suggest that RGS12 may be a potential drug target for the treatment of inflammatory arthritis.

Novel MYCBP::EHD2 and RUNX1::ZNF780A Fusion Genes in T-cell Acute Lymphoblastic Leukemia.

BACKGROUND/AIM: T-cell acute lymphoblastic leukemia (T-ALL) is a rare malignancy characterized by proliferation of early T-cell precursors that replace normal hematopoietic cells. T-ALL cells carry non-random chromosome aberrations, fusion genes, and gene mutations, often of prognostic significance. We herein report the genetic findings in cells from a T-ALL patient. MATERIALS AND METHODS: Bone marrow cells from a patient with T-ALL were examined using G-banding, array comparative genomic hybridization (aCGH), RNA sequencing, reverse transcription polymerase chain reaction (RT-PCR), Sanger sequencing, and fluorescence in situ hybridization. RESULTS: G-banding revealed del(1)(p34), add(5)(q14), trisomy 8, and monosomy 21 in the leukemic cells. aCGH detected the gross unbalances inferred from the karyotyping results, except that heterozygous loss of chromosome 21 did not include its distal part; 21q22.12-q22.3 was undeleted. In addition, aCGH detected a submicroscopic interstitial 7.56 Mbp deletion in the q arm of chromosome 19 from 19q13.2 to 19q13.33. RNA sequencing detected and RT-PCR/Sanger sequencing confirmed the presence of two novel chimeras, MYCBP::EHD2 and RUNX1::ZNF780A. They were generated from rearrangements involving subbands 1p34.3 (MYCBP), 19q13.2 (ZNF780A), 19q13.33 (EHD2), and 21q22.12 (RUNX1), i.e., at the breakpoints of chromosomal deletions. CONCLUSION: The leukemic cells showed the heterozygous loss of many genes as well as the generation of MYCBP::EHD2 and RUNX1::ZNF780A chimeras. Because the partner genes in the chimeras were found at the breakpoints of the chromosomal deletions, we believe that both the heterozygous losses and the generation of the two chimeras occurred simultaneously, and that they were pathogenetically important.

Loss-of-function variants in MYCBP2 cause neurobehavioural phenotypes and corpus callosum defects.

The corpus callosum is a bundle of axon fibres that connects the two hemispheres of the brain. Neurodevelopmental disorders that feature dysgenesis of the corpus callosum as a core phenotype offer a valuable window into pathology derived from abnormal axon development. Here, we describe a cohort of eight patients with a neurodevelopmental disorder characterized by a range of deficits including corpus callosum abnormalities, developmental delay, intellectual disability, epilepsy and autistic features. Each patient harboured a distinct de novo variant in MYCBP2, a gene encoding an atypical really interesting new gene (RING) ubiquitin ligase and signalling hub with evolutionarily conserved functions in axon development. We used CRISPR/Cas9 gene editing to introduce disease-associated variants into conserved residues in the Caenorhabditis elegans MYCBP2 orthologue, RPM-1, and evaluated functional outcomes in vivo. Consistent with variable phenotypes in patients with MYCBP2 variants, C. elegans carrying the corresponding human mutations in rpm-1 displayed axonal and behavioural abnormalities including altered habituation. Furthermore, abnormal axonal accumulation of the autophagy marker LGG-1/LC3 occurred in variants that affect RPM-1 ubiquitin ligase activity. Functional genetic outcomes from anatomical, cell biological and behavioural readouts indicate that MYCBP2 variants are likely to result in loss of function. Collectively, our results from multiple human patients and CRISPR gene editing with an in vivo animal model support a direct link between MYCBP2 and a human neurodevelopmental spectrum disorder that we term, MYCBP2-related developmental delay with corpus callosum defects (MDCD).

MYCBP2 expression correlated with inflammatory cell infiltration and prognosis immunotherapy in thyroid cancer patients.

Introduction: Immune checkpoint inhibitors (ICIs) have shown promising results for the treatment of multiple cancers. ICIs and related therapies may also be useful for the treatment of thyroid cancer (TC). In TC, Myc binding protein 2 (MYCBP2) is correlated with inflammatory cell infiltration and cancer prognosis. However, the relationship between MYCBP2 expression and ICI efficacy in TC patients is unclear. Methods: We downloaded data from two TC cohorts, including transcriptomic data and clinical prognosis data. The Tumor Immune Dysfunction and Exclusion (TIDE) algorithm was used to predict the efficacy of ICIs in TC patients. MCPcounter, xCell, and quanTIseq were used to calculate immune cell infiltration scores. Gene set enrichment analysis (GSEA) and single sample GSEA (ssGSEA) were used to evaluate signaling pathway scores. Immunohistochemical (IHC) analysis and clinical follow up was used to identify the MYCBP2 protein expression status in patients and associated with clinical outcome. Results: A higher proportion of MYCBP2-high TC patients were predicted ICI responders than MYCBP2-low patients. MYCBP2-high patients also had significantly increased infiltration of CD8+ T cells, cytotoxic lymphocytes (CTLs), B cells, natural killer (NK) cells and dendritic cells (DC)s. Compared with MYCBP2-low patients, MYCBP2-high patients had higher expression of genes associated with B cells, CD8+ T cells, macrophages, plasmacytoid dendritic cells (pDCs), antigen processing and presentation, inflammatory stimulation, and interferon (IFN) responses. GSEA and ssGSEA also showed that MYCBP2-high patients had significantly increased activity of inflammatory factors and signaling pathways associated with immune responses.In addiation, Patients in our local cohort with high MYCBP2 expression always had a better prognosis and greater sensitivity to therapy while compared to patients with low MYCBP2 expression after six months clinic follow up. Conclusions: In this study, we found that MYCBP2 may be a predictive biomarker for ICI efficacy in TC patients. High MYCBP2 expression was associated with significantly enriched immune cell infiltration. MYCBP2 may also be involved in the regulation of signaling pathways associated with anti-tumor immune responses or the production of inflammatory factors.

An atypical ubiquitin ligase at the heart of neural development and programmed axon degeneration.

The degeneration of nerve fibres following injury was first described by Augustus Waller over 170 years ago. Initially assumed to be a passive process, it is now evident that axons respond to insult via regulated cellular signaling events resulting in their programmed degeneration. Pro-survival and pro-degenerative factors have been identified and their regulatory mechanisms are beginning to emerge. The ubiquitin system has been implicated in the pro-degenerative process and a key component is the ubiquitin E3 ligase MYCBP2 (also known as PHR1). Ubiquitin E3 ligases are tasked with the transfer of the small protein modifier ubiquitin to substrates and consist of hundreds of members. They can be classified as single subunit systems or as multi-subunit complexes. Their catalytic domains can also be assigned to three general architectures. Hints that MYCBP2 might not conform to these established formats came to light and it is now clear from biochemical and structural studies that MYCBP2 is indeed an outlier in terms of its modus operandi. Furthermore, the unconventional way in which MYCBP2 transfers ubiquitin to substrates has been linked to neurodevelopmental and pro-degenerative function. Herein, we will summarize these research developments relating to the unusual features of MYCBP2 and postulate therapeutic strategies that prevent Wallerian degeneration. These have exciting potential for providing relief from pathological neuropathies and neurodegenerative diseases.

MiR-26b-5p inhibits cell proliferation and EMT by targeting MYCBP in triple-negative breast cancer.

BACKGROUND: The study was designed to elucidate the association and functional roles of miR-26b-5p and c-MYC binding protein (MYCBP) in triple-negative breast cancer (TNBC). METHOD: Luciferase reporter assay was used to confirm the relationship between miR-26b-5p and MYCBP in TNBC cells. The expression levels of miR-26b-5p and MYCBP in tissue specimens and cell lines were determined using reverse transcription-quantitative PCR. Cell proliferation, migration and invasion were assessed using CCK-8 assay, colony formation and transwell assay. RESULTS: We first observed that miR-26b-5p directly targets the 3'-UTR of MYCBP to inhibit MYCBP expression in MDA-MB-468 and BT-549 cells. The expression of miR-26b-5p was inversely correlated with MYCBP expression in TNBC tissues. We further demonstrated that MYCBP knockdown suppressed the proliferation, migration and invasion of TNBC cells. Furthermore, MYCBP overexpression counteracted the suppressive effect of miR-26b-5p on TNBC cell behaviors. Western blot analysis demonstrated that the E-cadherin protein level was increased, while protein levels of N-cadherin and vimentin were decreased in cells transfected with miR-26b-5p, which were all reversed by ectopic expression of MYCBP. CONCLUSIONS: In summary, our findings revealed the tumor suppressive role of miR-26b-5p in regulating TNBC cell proliferation and mobility, possibly by targeting MYCBP.

Targeting autophagy using small-molecule compounds to improve potential therapy of Parkinson's disease.

Parkinson's disease (PD), known as one of the most universal neurodegenerative diseases, is a serious threat to the health of the elderly. The current treatment has been demonstrated to relieve symptoms, and the discovery of new small-molecule compounds has been regarded as a promising strategy. Of note, the homeostasis of the autolysosome pathway (ALP) is closely associated with PD, and impaired autophagy may cause the death of neurons and thereby accelerating the progress of PD. Thus, pharmacological targeting autophagy with small-molecule compounds has been drawn a rising attention so far. In this review, we focus on summarizing several autophagy-associated targets, such as AMPK, mTORC1, ULK1, IMPase, LRRK2, beclin-1, TFEB, GCase, ERRα, C-Abelson, and as well as their relevant small-molecule compounds in PD models, which will shed light on a clue on exploiting more potential targeted small-molecule drugs tracking PD treatment in the near future.

The Long Noncoding RNA LINC00665 Facilitates c-Myc Transcriptional Activity via the miR-195-5p MYCBP Axis to Promote Progression of Lung Adenocarcinoma.

Long noncoding RNAs (lncRNAs) have recently received growing substantial attention in cancer research due to their important roles in various cancer types. However, the underlying mechanisms and functions of lncRNAs, especially in lung adenocarcinoma (LUAD), remain elusive. Based on pan-cancer screening analyses, we identified that the noncoding RNA LINC00665 was up-regulated in lung adenocarcinoma, which was subsequently confirmed in clinical samples and cell lines. Higher expression of LINC00665 was positively associated with poor prognosis and advanced T stage. Next, using gain- and loss- of function approaches, we revealed that LINC00665 promotes cell proliferation, cell migration, invasion, and suppresses cell apoptosis in LUAD through in vitro and in vivo experiments. Additionally, our findings showed that LINC00665 was predominately localized in the cytoplasm so as to interact with Ago2 protein, which could function as miRNA sponges. The results of bioinformatics prediction and RNA pull-down assay indicated that LINC00665 directly interacted with miR-195-5p. This was also confirmed by fluorescence colocalization. Furthermore, luciferase reporter assay demonstrated that Myc binding protein (MYCBP, also called AMY-1), which enhanced c-Myc transcriptional activity, was the target gene of LINC00665 dependent on miR-195-5p. Finally, rescue functional assay results uncovered that the oncogenic capability of LINC00665 was dependent on miR-195-5p and c-Myc transcriptional activity. In summary, this work elucidates that LINC00665 accelerates LUAD progression via the miR-195-5p/MYCBP axis by acting as a competing endogenous RNA (ceRNA), suggesting that LINC00665 may represent a potential therapeutic target for clinical intervention of LUAD.

Chlamydomonas FAP70 is a component of the previously uncharacterized ciliary central apparatus projection C2a.

Cilia are essential organelles required for cell signaling and motility. Nearly all motile cilia have a '9+2' axoneme composed of nine outer doublet microtubules plus two central microtubules; the central microtubules together with their projections are termed the central apparatus (CA). In Chlamydomonas reinhardtii, a model organism for studying cilia, 30 proteins are known CA components, and ∼36 more are predicted to be CA proteins. Among the candidate CA proteins is the highly conserved FAP70 (CFAP70 in humans), which also has been reported to be associated with the doublet microtubules. Here, we determined by super-resolution structured illumination microscopy that FAP70 is located exclusively in the CA, and show by cryo-electron microscopy that its N-terminus is located at the base of the C2a projection of the CA. We also found that fap70-1 mutant axonemes lack most of the C2a projection. Mass spectrometry revealed that fap70-1 axonemes lack not only FAP70 but two other conserved candidate CA proteins, FAP65 (CFAP65 in humans) and FAP147 (MYCBPAP in humans). Finally, FAP65 and FAP147 co-immunoprecipitated with HA-tagged FAP70. Taken together, these data identify FAP70, FAP65 and FAP147 as the first defining components of the C2a projection.

lncRNA LUNAR1 accelerates colorectal cancer progression by targeting the miR­495­3p/MYCBP axis.

Colorectal cancer (CRC) is a tumor type characterized by high patient morbidity and mortality. It has been reported that long non­coding (lncRNA) LUNAR1 (LUNAR1) participates in the regulation of tumor progression, such as diffuse large B­cell lymphoma. However, its role and underlying mechanisms in CRC progression have not been elucidated. The present study was designed to investigate the underlying mechanisms by which LUNAR1 regulates CRC progression. RT­qPCR and Pearson's correlation analysis revealed that LUNAR1 was highly expressed and was negatively associated with the overall survival of CRC patients. Moreover, CCK­8, clone formation, wound­healing migration, Transwell chamber and FACs assay analyses showed that LUNAR1 knockdown inhibited CRC cell proliferation, migration and invasion, while accelerating cell apoptosis. Additionally, LUNAR1 was found to function as a sponge of miR­495­3p, which was predicted by TargetScan and confirmed by luciferase reporter assay. Furthermore, functional studies indicated that miR­495­3p overexpression inhibited CRC cell proliferation, migration and invasion, while accelerating cell apoptosis. In addition, bioinformatics and luciferase reporter assays showed that miR­495­3p was found to negatively target Myc binding protein (MYCBP), and functional research showed that LUNAR1 accelerated CRC progression via the miR­495­3p/MYCBP axis. In conclusion, LUNAR1 accelerates CRC progression via the miR­495­3p/MYCBP axis, indicating that LUNAR1 may serve as a prognostic biomarker for CRC patients.

Good or not good: Role of miR-18a in cancer biology.

miR-18a is a member of primary transcript called miR-17-92a (C13orf25 or MIR17HG) which also contains five other miRNAs: miR-17, miR-19a, miR-20a, miR-19b and miR-92a. This cluster as a whole shows specific characteristics, where miR-18a seems to be unique. In contrast to the other members, the expression of miR-18a is additionally controlled and probably functions as its own internal controller of the cluster. miR-18a regulates many genes involved in proliferation, cell cycle, apoptosis, response to different kinds of stress, autophagy and differentiation. The disturbances of miR-18a expression are observed in cancer as well as in different diseases or pathological states. The miR-17-92a cluster is commonly described as oncogenic and it is known as 'oncomiR-1', but this statement is a simplification because miR-18a can act both as an oncogene and a suppressor. In this review we summarize the current knowledge about miR-18a focusing on its regulation, role in cancer biology and utility as a potential biomarker.

Mesenchymal stem cell-secreted extracellular vesicles carrying TGF-ß1 up-regulate miR-132 and promote mouse M2 macrophage polarization.

The effects of mesenchymal stem cells (MSCs) on different types of diseases are controversial, and the inner mechanisms remain unknown, which retards the utilization of MSCs in disease therapy. In this study, we aimed to elucidate the mechanisms of MSCs-extracellular vesicles (EVs) carrying transforming growth factor-beta 1 (TGF-ß1) in M2 polarization in mouse macrophages via the microRNA-132 (miR-132)/E3 ubiquitin ligase myc binding protein 2 (Mycbp2)/tuberous sclerosis complex 2 (TSC2) axis. Mouse MSCs were isolated for adipogenic and osteogenic induction, followed by co-culture with mouse macrophages RAW264.7. Besides, mouse macrophages RAW264.7 were co-cultured with MSCs-EVs in vitro, where the proportion of macrophages and inflammation were detected by flow cytometry and ELISA. The experimental data revealed that MSCs-EVs promoted M2 polarization of macrophages, and elevated interleukin (IL)-10 expression and inhibited levels of IL-1ß, tumour necrosis factor (TNF)-α and IL-6. MSC-EV-treated macrophages RAW264.7 increased TGF-ß1 expression, thus elevating miR-132 expression. MiR-132 directly bound to Mycbp2, as confirmed by luciferase activity assay. Meanwhile, E3 ubiquitin ligase Mycbp2 could ubiquitinate TSC2 protein. Furthermore, silencing TGF-ß1 inhibited M2 polarization of MSC-EV-treated macrophages. Taken conjointly, this study provides evidence reporting that MSC-secreted EVs carry TGF-ß1 to promote M2 polarization of macrophages via modulation of the miR-132/Mycbp2/TSC2 axis.

EYA4 inhibits hepatocellular carcinoma by repressing MYCBP by dephosphorylating ß-catenin at Ser552.

Hepatocellular carcinoma (HCC) is one of the most common malignancies and the fourth leading cause of cancer-related death worldwide. Our previous study showed that EYA4 functioned by suppressing growth of HCC tumor cells, but its molecular mechanism is still not elucidated. Based on the results of gene microassay, EYA4 was inversely correlated with MYCBP and was verified in human HCC tissues by immunohistochemistry and western blot. Overexpressed and KO EYA4 in human HCC cell lines confirmed the negative correlation between EYA4 and MYCBP by qRT-PCR and western blot. Transfected siRNA of MYCBP in EYA4 overexpressed cells and overexpressed MYCBP in EYA4 KO cells could efficiently rescue the proliferation and G2/M arrest effects of EYA4 on HCC cells. Mechanistically, armed with serine/threonine-specific protein phosphatase activity, EYA4 reduced nuclear translocation of ß-catenin by dephosphorylating ß-catenin at Ser552, thereby suppressing the transcription of MYCBP which was induced by ß-catenin/LEF1 binding to the promoter of MYCBP. Clinically, HCC patients with highly expressed EYA4 and poorly expressed MYCBP had significantly longer disease-free survival and overall survival than HCC patients with poorly expressed EYA4 and highly expressed MYCBP. In conclusion, EYA4 suppressed HCC tumor cell growth by repressing MYCBP by dephosphorylating ß-catenin S552. EYA4 combined with MYCBP could be potential prognostic biomarkers in HCC.

SPAG5 upregulation contributes to enhanced c-MYC transcriptional activity via interaction with c-MYC binding protein in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that lacks effective therapeutic targets. Sperm-associated antigen 5 (SPAG5) is a mitotic spindle-associated protein that is involved in various biological processes in cervical cancer and bladder urothelial carcinoma. However, the role of SPAG5 in TNBC remains undefined. METHODS: The expression of SPAG5 was examined in TNBC patients via quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry (IHC). The biological functions of SPAG5 in TNBC and the underlying mechanisms were investigated in vitro and in vivo. RESULTS: SPAG5 expression was significantly upregulated in TNBC tissues compared with that in paired adjacent noncancerous tissues (ANTs). High SPAG5 expression was associated with increased lymph node metastasis and high risk of local recurrence. SPAG5 protein expression was significantly associated with poor disease-free survival in TNBC. Gene set enrichment analysis of TNBC data from The Cancer Genome Atlas (TCGA) indicated that high SPAG5 expression was significantly associated with cell cycle and the ATR-BRCA pathway. Functional assays demonstrated that SPAG5 expression promoted tumor growth in vitro and in vivo. In addition, SPAG5-silenced cells were more sensitive to the PARP inhibitor (PARPi) olaparib. Mechanistically, SPAG5 interacted with c-MYC binding protein (MYCBP), thereby increasing MYCBP protein levels and leading to increased c-MYC transcriptional activity, which promoted the expression of the c-MYC target genes: CDC20, CDC25C, BRCA1, BRCA2, and RAD51.Knockdown of MYCBP or c-MYC abolished the SPAG5-induced cell-cycle progression and cell proliferation of TNBC. CONCLUSIONS: Collectively, our results indict that SPAG5 is an efficient prognostic factor in TNBC, and that SPAG5 knockdown increases the sensitivity of TNBC to the PARPi olaparib. SPAG5 promotes tumor growth and DNA repair by increasing c-MYC transcriptional activity via interaction with MYCBP. The SPAG5/MYCBP/c-MYC axis may represent a potential therapeutic target for TNBC treatment.

PAM forms an atypical SCF ubiquitin ligase complex that ubiquitinates and degrades NMNAT2.

PHR (PAM/Highwire/RPM-1) proteins are conserved RING E3 ubiquitin ligases that function in developmental processes, such as axon termination and synapse formation, as well as axon degeneration. At present, our understanding of how PHR proteins form ubiquitin ligase complexes remains incomplete. Although genetic studies indicate NMNAT2 is an important mediator of PHR protein function in axon degeneration, it remains unknown how PHR proteins inhibit NMNAT2. Here, we decipher the biochemical basis for how the human PHR protein PAM, also called MYCBP2, forms a noncanonical Skp/Cullin/F-box (SCF) complex that contains the F-box protein FBXO45 and SKP1 but lacks CUL1. We show FBXO45 does not simply function in substrate recognition but is important for assembly of the PAM/FBXO45/SKP1 complex. Interestingly, we demonstrate a novel role for SKP1 as an auxiliary component of the target recognition module that enhances binding of FBXO45 to NMNAT2. Finally, we provide biochemical evidence that PAM polyubiquitinates NMNAT2 and regulates NMNAT2 protein stability and degradation by the proteasome.