Domain Family Member Research Articles

A Novel BRD Family PROTAC Inhibitor dBET1 Exerts Great Anti-Cancer Effects by Targeting c-MYC in Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) represents an aggressive hematopoietic malignancy with a prognosis inferior to that of other leukemias. Recent targeted therapies offer new opportunities to achieve better treatment outcomes. However, due to the complex heterogeneity of AML, its prognosis remains dismal. In this study, we first identified the correlation between high expression of BRD4 and overall survival of patients with AML. Targeted degradation of BRD2, BRD3, and BRD4 proteins by dBET1, a proteolysis-targeting chimera (PROTAC) against the bromodomain and extra-terminal domain (BET) family members, showed cytotoxic effects on Kasumi (AML1-ETO), NB4 (PML-RARa), THP-1 (MLL-AF9), and MV4-11 (MLL-AF4) AML cell lines representing different molecular subtypes of AML. Furthermore, we determined that dBET1 treatment arrested cell cycling and enhanced apoptosis and c-MYC was identified as the downstream target. Collectively, our results indicated that dBET1 had broad anti-cancer effects on AML cell lines with different molecular lesions and provided more benefits to patients with AML.

The investigation of hippo signaling pathway in mouse uterus during peri-implantation period.

Events in the uterus during the peri-implantation period include embryo development, acquisition of uterine receptivity, implantation and decidualization. Hippo signaling pathway regulates cell proliferation, apoptosis and differentiation. We aimed to determine localization and expressions of pYAP (Phospho Yes-associated protein), YAP (Yes-associated protein), TEAD1 (TEA domain family member 1) and CTGF (Connective tissue growth factor), members of the Hippo signaling pathway, in the mouse uterus during the peri-implantation period. Pregnant mice were randomly separated into 5 groups: 1st, 4th, 5th, 6th, and 8th days of pregnancy groups. Non-pregnant female mice in estrous phase were included in the estrous group. Uteri and implantation sites were collected. Also, inter-implantation sites were collected from the 5th day of pregnancy group. pYAP, YAP, TEAD-1 and CTGF were detected by immunohistochemistry and Western blotting. We observed that the expressions of YAP, TEAD-1 and CTGF were increased in the luminal and glandular epithelium on the 1st and 4th days of pregnancy when epithelial proliferation occurred. pYAP expression was high, and YAP and CTGF expressions were low in the luminal epithelium of the implantation sites on the 5th day of pregnancy, when epithelial differentiation occurred. pYAP expression was low, YAP and CTGF expressions were high at implantation sites on the 6th and 8th days of pregnancy, where decidua was formed. Our findings suggest that the Hippo signaling pathway might be involved in implantation and decidualization. Our findings will guide further studies and may help to elucidate underlying causes of implantation failure and pregnancy loss.

Abstract 155: An endosomal type Igamma PIP 5-kinase controls endosome maturation, lysosome function, and autophagy by modulating Rab7a

Abstract Endosomes are major protein-sorting stations in cells. Defects in endosomal sorting are implicated in various human pathologies, such as cancer and neurodegenerative disorders. The small GTPase Ras-related protein Rab-7a (Rab7a) serves as a key organizer of the endosomal-lysosomal system. However, molecular mechanisms controlling Rab7a activation levels and subcellular translocation are still poorly defined. Here, we demonstrate that type Igamma phosphatidylinositol phosphate 5-kinase i5 (PIPKIgammai5), an endosome-localized enzyme that produces phosphatidylinositol 4,5-bisphosphate (PI4,5P2), directly interacts with Rab7a and plays critical roles in the control of endosomal-lysosomal system. The loss of PIPKIgammai5 blocks Rab7a recruitment to early endosomes, which prevents the maturation of early to late endosomes. In this way, PIPKIgammai5 is required for Epidermal Growth Factor Receptor (EGFR) sorting from endosomes to lysosomes, which controls the down-regulation of EGFR signaling. Furthermore, PIPKIgammai5 loss disturbs retromer complex connection with Rab7a, which blocks the retrograde sorting of Cation-independent Mannose 6-Phosphate Receptor (CI-MPR) from late endosomes. This leads to the decreased sorting of hydrolases to lysosomes. Thus, loss of PIPKIgammai5 causes lysosome dysfunction and reduces the autophagic degradation. By modulating retromer-Rab7a connection, PIPKIgammai5 is also required for the recruitment of the GTPase-activating protein (GAP) TBC1 domain family member 5 (TBC1D5) to late endosomes, which controls the conversion of Rab7a from the active state to the inactive state. Altogether, PIPKIgammai5 is critical for the modulation of Rab7a activity, localization, and function in the endosomal-lysosomal system. Citation Format: Ming Sun, Gary Luong, Faiz Plastikwala, Yue Sun. An endosomal type Igamma PIP 5-kinase controls endosome maturation, lysosome function, and autophagy by modulating Rab7a [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 155.

Abstract 991: OVO-like genes are master regulators of the epithelial fate in breast cancer

Abstract The OVO-like genes are evolutionary conserved C2H2 zinc finger transcription factors (TFs) with SNAG repressor domains involved in the maintenance of epithelial homeostasis. From the three Ovo-like family genes identified in mammals, Ovol-like1 (Ovol1) and Ovol-like2 (Ovol2) are the most studied members with multiple reports pinpointing relevant roles in development and disease. In cancer, one of the hallmarks of invasive carcinoma involves the loss of E-cadherin expression by the repression of SNAG transcription factors. Interestingly, contrary to the other SNAG domain family members the OVO-like genes regulate epithelial cell fate. We performed data analysis in breast cancer cell lines and patient data obtained from GEO, CCLE, TCGA and METABRIC to assess the potential of the OVO-like genes as relevant regulators of gene expression in breast carcinoma. In addition, we stablished breast cancer models with inducible expression or repression of the OVO-like genes. We also analyzed the cellular systems we generated using naïve high-throughput methods such as RNA-Seq. Likewise, we studied the effect of OVO-like genes on tumor progression and metastasis using inducible expression systems in both xenograft and syngeneic breast cancer models. We observed that Ovol1 and Ovol2 are constitutively expressed in human breast cancer cell lines, and they are upregulated due to chromosomal gains in breast cancer patients. We observed how the introduction of Ovol1 and Ovol2 into mesenchymal-like breast cancer cell lines modified their transcriptome in a way that promoted expression of epithelial marker genes (e.g., Amphiregulin) and downregulated mesenchymal markers (e.g., Vimentin). In addition, introducing Ovo-like genes in mesenchymal-like cells greatly affected their viability and function. Surprisingly, we observed a similar effect by downregulating the Ovo-like genes in epithelial-like cells. We propose a model where a common epigenetic machinery is shared between structurally related transcription factors with opposing effects, such as OVO-like proteins and other SNAG-containing transcription factors, such as Snail and Slug, such that the abundance of the transcription factors and their co-repressors defines the state of the system as either epithelial or mesenchymal. Citation Format: Diana Drago Garcia, Yosef Yarden. OVO-like genes are master regulators of the epithelial fate in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 991.

TEAD4 regulates trophectoderm differentiation upstream of CDX2 in a GATA3-independent manner in the human preimplantation embryo.

TEAD4 regulates trophectoderm differentiation upstream of CDX2 in a GATA3-independent manner in the human preimplantation embryo.

CircCERS6 Suppresses the Development of Epithelial Ovarian Cancer Through Mediating miR-630/RASSF8.

Accumulating evidence have demonstrated that circular RNAs (circRNAs) exert important roles in tumor initiation and progression. Nevertheless, the role and mechanism of circRNA ceramide synthase 6 (circCERS6) in epithelial ovarian cancer (EOC) remain to be clarified. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assay, we measured RNA and protein expression. We analyzed the effects of circCERS6/microRNA-630 (miR-630)/Ras-association domain family member 8 (RASSF8) axis in cell proliferation, migration, and invasion by 5-Ethynyl-2'-deoxyuridine (EdU) assay, colony formation assay, scratch test, and transwell assay. Using RNA-pull down assay and dual-luciferase reporter assay, the interaction between miR-630 and circCERS6 or RASSF8 was verified. The in vivo role of circCERS6 in tumor growth was analyzed using xenograft mice model. CircCERS6 expression was markedly reduced in EOC tissues and cell lines. CircCERS6 overexpression hampered the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of EOC cells. CircCERS6 directly interacted with miR-630. miR-630 expression was up-regulated in EOC tissues and cell lines. CircCERS6 overexpression-induced anti-tumor effects in EOC were largely reversed by the overexpression of miR-630. RASSF8 was a direct target of miR-630. RASSF8 level was decreased in EOC tissues and cell lines. CircCERS6 up-regulated RASSF8 expression by acting as a molecular sponge for miR-630 in EOC cells. CircCERS6 overexpression-mediated suppressive effects in EOC cells were largely overturned by the silence of RASSF8. CircCERS6 overexpression blocked tumor growth in vivo. CircCERS6 overexpression hampered the proliferation, migration, invasion, and EMT of EOC cells by up-regulating RASSF8 via sponging miR-630.

TBC1D15-Drp1 interaction-mediated mitochondrial homeostasis confers cardioprotection against myocardial ischemia/reperfusion injury

ObjectiveMitochondria are essential for myocardial ischemia/reperfusion (I/R) injury. TBC domain family member 15 (TBC1D15) participates in the regulation of mitochondrial homeostasis although its role remains elusive in I/R injury. Methods and materialsThis study examined the role of TBC1D15 in mitochondrial homeostasis under myocardial I/R injury using inducible cardiac-specific TBC1D15 knockin (TBC1D15CKI) and knockout (TBC1D15CKO) mice. ResultsTBC1D15 mRNA/protein levels were downregulated in human ischemic cardiomyopathy samples, mouse I/R hearts and neonatal mouse cardiomyocytes with H/R injury, consistent with scRNA sequencing finding from patients with coronary heart disease. Cardiac-specific knockin of TBC1D15 attenuated whereas cardiac-specific knockout of TBC1D15 overtly aggravated I/R-induced cardiomyocyte apoptosis and cardiac dysfunction. TBC1D15CKI mice exhibited reduced mitochondrial damage and mitochondrial fragmentation following myocardial I/R injury, while TBC1D15CKO mice displayed opposite results. TBC1D15 preserved mitochondrial function evidenced by safeguarding MMP and oxygen consumption capacity, antagonizing ROS accumulation and cytochrome C release, which were nullified by TBC1D15 knockdown. Time-lapse confocal microscopy revealed that TBC1D15 activated asymmetrical mitochondrial fission through promoting mitochondria-lysosome contacts untethering in NMCMs under H/R injury, whereas overexpression of TBC1D15 mutants (R400K and ∆231–240) failed to regulate asymmetrical fission and knockdown of TBC1D15 slowed down asymmetrical fission. Moreover, TBC1D15-offered benefits were mitigated by knockdown of Fis1 and Drp1. Mechanistically, TBC1D15 recruited Drp1 to mitochondria-lysosome contact sites via direct interaction with Drp1 through its C terminus (574–624) domain. Interfering with interaction between TBC1D15 and Drp1 abrogated asymmetrical mitochondrial fission and mitochondrial function. Cardiac phenotypes of TBC1D15CKO mice upon I/R injury were rescued by adenovirus-mediated overexpression of wild-type but not mutants (R400K, ∆231–240 and ∆574–624) TBC1D15. ConclusionsTBC1D15 ameliorated I/R injury through a novel modality to preserve mitochondrial homeostasis where mitochondria-lysosome contacts (through the TBC1D15/Fis1/RAB7 cascade) regulate asymmetrical mitochondrial fission (TBC1D15/Drp1 interaction), suggesting promises of targeting TBC1D15 in the management of myocardial I/R injury.

RNA bisulfite sequencing reveals NSUN2-mediated suppression of epithelial differentiation in pancreatic cancer.

Posttranscriptional modifications in RNA have been considered to contribute to disease pathogenesis and tumor progression. NOL1/NOP2/Sun domain family member 2 (NSUN2) is an RNA methyltransferase that promotes tumor progression in several cancers. Pancreatic cancer relapse inevitably occurs even in cases where primary tumors have been successfully treated. Associations of cancer progression due to reprogramming of the cancer methyl-metabolome and the cancer genome have been noted, but the effect of base modifications, namely 5-methylcytosine (m5C), in the transcriptome remains unclear. Aberrant regulation of 5-methylcytosine turnover in cancer may affect posttranscriptional modifications in coding and noncoding RNAs in disease pathogenesis. Mutations in NSUN2 have been reported as drivers of neurodevelopmental disorders in mice, and upregulated expression of NSUN2 in tumors of the breast, bladder, and pancreas has been reported. In this study, we conducted mRNA whole transcriptomic bisulfite sequencing to categorize NSUN2 target sites in the mRNA of human pancreatic cancer cells. We identified a total of 2829 frequent m5C sites in mRNA from pancreatic cancer cells. A total of 90.9% (2572/2829) of these m5C sites were mapped to annotated genes in autosomes and sex chromosomes X and Y. Immunohistochemistry staining confirmed that the NSUN2 expression was significantly upregulated in cancer lesions in the LSL-KrasG12D/+;Trp53fl/fl;Pdx1-Cre (KPC) spontaneous pancreatic cancer mouse model induced by Pdx1-driven Cre/lox system expressing mutant KrasG12D and p53 deletion. The in vitro phenotypic analysis of NSUN2 knockdown showed mild effects on pancreatic cancer cell 2D/3D growth, morphology and gemcitabine sensitivity in the early phase of tumorigenesis, but cumulative changes after multiple cell doubling passages over time were required for these mutations to accumulate. Syngeneic transplantation of NSUN2-knockdown KPC cells via subcutaneous injection showed decreased stromal fibrosis and restored differentiation of ductal epithelium in vivo. SIGNIFICANCE: Transcriptome-wide mRNA bisulfite sequencing identified candidate m5C sites of mRNAs in human pancreatic cancer cells. NSUN2-mediated m5C mRNA metabolism was observed in a mouse model of pancreatic cancer. NSUN2 regulates cancer progression and epithelial differentiation via mRNA methylation.

FTO Alleviates CdCl2-Induced Apoptosis and Oxidative Stress via the AKT/Nrf2 Pathway in Bovine Granulosa Cells.

Cadmium (Cd) is a common environmental heavy metal contaminant of reproduction toxicity. Cd accumulation in animals leads to the damage of granulosa cells. However, its mechanism needs to be elucidated. This research found that treating granulosa cells with Cd resulted in reduced cell viability. The flow cytometry results showed that Cd increased the degree of apoptosis and level of superoxide anion (O2−) in granulosa cells. Further analysis showed that Cd treatment resulted in reduced expression levels of nuclear factor erythroid 2-related factor-2 (Nrf2), superoxide dismutase (SOD), catalase (CAT) and NAD(P)H: quinone oxidoreductase 1 (NQO1), and an increased expression level of malondialdehyde (MDA); the expression levels of Bcl-2 associated X (Bax) and caspase-3 increased, whereas that of B-cell lymphoma 2 (Bcl-2) decreased. Changes in m6A methylation-related enzymes were noted with Cd-induced damage to granulosa cells. The results of transcriptome and MeRIP sequencing revealed that the AKT pathway participated in Cd-induced damage in granulosa cells, and the MAX network transcriptional repressor (MNT) may be a potential target gene of fat mass and obesity-associated protein (FTO). FTO and YTH domain family member 2 (YTHDF2) regulated MNT expression through m6A modification. FTO overexpression alleviated Cd-induced apoptosis and oxidative stress through the activation of the AKT/Nrf2 pathway; this process could be reversed using siMNT. Overall, these findings associated m6A with Cd-induced damage to granulosa cells and provided insights into Cd-induced granulosa cell cytotoxicity from a new perspective centered on m6A modification.

TBC1D24-related familial infantile multifocal myoclonus: Description of a new Chinese pedigree with a 20 year follow up

IntroductionDisorders associated with mutations in the Tre2/Bub2/Cdc16 (TBC)1 domain family member 24 gene (TBC1D24) present a wide range of phenotypes, ranging from mild to fatal seizure diseases, non-syndromic deafness, and complex syndromes such as deafness, onychodystrophy, osteodystrophy, and mental retardation(DOOR syndrome). In this study, we introduce three siblings of a previously unreported Chinese family with familial infantile myoclonic epilepsy caused by a homozygous TBC1D24 mutation. MethodsGenomic DNA was extracted from whole blood of the proband, his parents, and sisters. TBC1D24 exomes were sequenced by whole exome sequencing then analyzed by genetic analysis with Sanger sequencing validation. The patients were followed up for more than 20 years to summarize their clinical features. ResultsGenetic analysis identified a homozygous TBC1D24 mutation (c.241_252del12) in the proband and his sisters. Prediction models suggest that the mutation leads to an alteration in the properties and structure of the TBC1D24 protein, especially in the folding direction of the loop region, which is likely to decrease protein activity. The patients manifested with early-onset myoclonic epilepsy, were prone to status epilepticus, and seizures only occurred during wakefulness. Imaging characteristics included cerebellar atrophy and abnormal cerebellar signals. ConclusionWe report a pedigree case of infantile myoclonic epilepsy caused by a homozygous TBC1D24 mutation. Our long-term clinical follow-up not only enriches the clinical phenotype of the disease, but also provides a clinical experience for the early diagnosis and clinical treatment of the disease.

Association of Variants in COMT, RASSF1 and GPM6A with the Risk of Paranoid Schizophrenia Patients in Prof HB Saanin Psychiatric Hospital, West Sumatra, Indonesia.

BackgroundSchizophrenia is a multifactorial disease in which genetic factors play a greater role than other factors. The genes of importance in schizophrenia patients are the genes that encode for neurotransmitters associated with low minor allele frequency (MAF) scores. This study was aimed to determine the association of genetic variations in catechol-O-methyl transferase (COMT), Ras association domain family member 1 (RASSF1) and glycoprotein M6A (GPM6A) with the risk of paranoid schizophrenia (PS) in patients admitted to Prof HB Saanin Psychiatric Hospital, West Sumatra, Indonesia.MethodsGenotyping analysis through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and PCR-amplification refractory mutation system (ARMS) was performed in 100 PS patients and 100 healthy controls. Chi-square and Fisher’s exact tests were used to compare the frequencies of genotype and allotype between the PS and control groups. Odds ratio (OR) with 95% confidence interval (95% CI) were calculated to determine the relative risk of PS with respect to genetic variations.ResultsPolymorphism rs13142920 in GPM6A was associated with significantly elevated risk of PS (P = 0.020; OR = 1.60 [95% CI: 1.08, 2.39]). However, COMT rs4680 and RASSF1 rs2073499 polymorphisms were not significantly associated with PS.ConclusionThe GPM6A rs13142920 polymorphism holds great potential as a genetic marker in PS patients.

Fasting potentiates insulin-mediated glucose uptake in rested and prior-contracted rat skeletal muscle.

A single bout of exercise can potentiate the effect of insulin on skeletal muscle glucose uptake via activation of the AMPK-TBC1 domain family member 4 (TBC1D4) pathway, which suggests a positive correlation between AMPK activation and insulin sensitization. In addition, prolonged fasting in rodents is known to upregulate and thereby synergistically enhance the effect of exercise on muscle AMPK activation. Therefore, fasting may potentiate the insulin-sensitizing effect of exercise. In the present study, we mimicked exercise by in situ muscle contraction and evaluated the effect of a 36-h fast on muscle contraction-induced insulin sensitization. Male Wistar rats weighing 150-170 g were allocated to either a 36-h fasting or feeding group. The extensor digitorum longus (EDL) muscles were electrically contracted via the common peroneal nerve for 10 min followed by a 3-h recovery period. EDL muscles were dissected and incubated in the presence or absence of submaximal insulin. Our results demonstrated that acute muscle contraction and 36 h of fasting additively upregulated AMPK pathway activation. Insulin-stimulated muscle glucose uptake and site-specific TBC1D4 phosphorylation were enhanced by prior muscle contraction in 36-h-fasted rats, but not in fed rats. Moreover, enhanced insulin-induced muscle glucose uptake and Akt phosphorylation due to 36 h of fasting were associated with a decrease in tribbles homolog 3 (TRB3), a negative regulator of Akt activation. In conclusion, fasting and prior muscle contraction synergistically enhance insulin-stimulated TBC1D4 phosphorylation and glucose uptake, which is associated with augmented AMPK pathway activation in rodents.NEW & NOTEWORTHY In this study, we revealed that 36 h of fasting additively upregulated acute muscle contraction-induced AMPK pathway activation in rats. Besides, fasting and muscle contraction synergistically enhanced insulin-stimulated site-specific TBC1D4 phosphorylation and glucose uptake, which was associated with augmented AMPK pathway activation. These results contribute to understanding the regulation of muscle insulin sensitivity.

YAP mediates compensatory cardiac hypertrophy through aerobic glycolysis in response to pressure overload.

The heart utilizes multiple adaptive mechanisms to maintain pump function. Compensatory cardiac hypertrophy reduces wall stress and oxygen consumption, thereby protecting the heart against acute blood pressure elevation. The nuclear effector of the Hippo pathway, Yes-associated protein 1 (YAP), is activated and mediates compensatory cardiac hypertrophy in response to acute pressure overload (PO). In this study, YAP promoted glycolysis by upregulating glucose transporter 1 (GLUT1), which in turn caused accumulation of intermediates and metabolites of the glycolytic, auxiliary, and anaplerotic pathways during acute PO. Cardiac hypertrophy was inhibited and heart failure was exacerbated in mice with YAP haploinsufficiency in the presence of acute PO. However, normalization of GLUT1 rescued the detrimental phenotype. PO induced the accumulation of glycolytic metabolites, including l-serine, l-aspartate, and malate, in a YAP-dependent manner, thereby promoting cardiac hypertrophy. YAP upregulated the GLUT1 gene through interaction with TEA domain family member 1 (TEAD1) and HIF-1α in cardiomyocytes. Thus, YAP induces compensatory cardiac hypertrophy through activation of the Warburg effect.

Multifaceted roles of YEATS domain-containing proteins and novel links to neurological diseases.

The so-called Yaf9, ENL, AF9, Taf14, and Sas5 (YEATS) domain-containing proteins, hereafter referred to as YD proteins, take control over the transcription by multiple steps of regulation either involving epigenetic remodelling of chromatin or guiding the processivity of RNA polymerase II to facilitate elongation-coupled mRNA 3' processing. Interestingly, an increasing amount of evidence suggest a wider repertoire of YD protein's functions spanning from non-coding RNA regulation, RNA-binding proteins networking, post-translational regulation of a few signalling transduction proteins and the spindle pole formation. However, such a large set of non-canonical roles is still poorly characterized. Notably, four paralogous of human YEATS domain family members, namely eleven-nineteen-leukaemia (ENL), ALL1-fused gene from chromosome 9 protein (AF9), YEATS2 and glioma amplified sequence 41 (GAS41), have a strong link to cancer yet new findings also highlight a potential novel role in neurological diseases. Here, in an attempt to more comprehensively understand the complexity of four YD proteins and to gain more insight into the novel functions they may accomplish in the neurons, we summarized the YD protein's networks, systematically searched and reviewed the YD genetic variants associated with neurodevelopmental disorders and finally interrogated the model organism Drosophila melanogaster.

Adaptor protein 3BP2 regulates dectin-1-mediated cellular signalling to induce cytokine expression and NF-κB activation.

The adaptor protein c-Abl Src homology 3 domain-binding protein-2 (3BP2) is phosphorylated by spleen tyrosine kinase (Syk), and the phosphorylation of Tyr183 is important in the regulation of immune responses. Recently, we reported that 3BP2 plays important roles in phagocytosis and chemokine expression mediated by the Fc receptor for IgG. Although it is well established that various phagocytic cells express Syk-coupled C-type lectin receptors (CLRs) to induce innate immune responses, the functions of 3BP2 and the physiological relevance of the phosphorylation of Tyr183 remain elusive. In this study, we generated genome-edited mice and observed that 3BP2 influenced the development of bone marrow-derived dendritic cells (BMDCs) induced by granulocyte-macrophage colony-stimulating factor. In addition, we found that 3BP2 was critical for cytokine expression induced by Syk-coupled CLRs - dectin-1 and macrophage-inducible C-type lectin. Immunoblotting analyses revealed that 3BP2 was required for the dectin-1-induced activation of NF-κB p65. The impaired expression of cytokines and activation of NF-κB in 3BP2-mutant cells were restored by wild-type 3BP2, suggesting that 3BP2 was involved in the dectin-1-mediated signalling that led to NF-κB activation. Furthermore, we found that the phosphorylation of Tyr183 is not essential for cytokine expression and that 3BP2 in combination with caspase recruitment domain family member 9 activates NF-κB in HEK-293T cells. Collectively, these results indicate that in addition to the development of BMDCs, 3BP2 plays an important role in the dectin-1-induced activation of NF-κB and cytokine expression.

ROR2 Downregulation Activates the MSX2/NSUN2/p21 Regulatory Axis and Promotes Dental Pulp Stem Cell Senescence.

Cellular senescence severely limits the research and the application of dental pulp stem cells (DPSCs). A previous study conducted by our research group revealed a close implication of ROR2 in DPSC senescence, although the mechanism underlying the regulation of ROR2 in DPSCs remains poorly understood so far. In the present study, it was revealed that the expression of the ROR2-interacting transcription factor MSX2 was increased in aging DPSCs. It was demonstrated that the depletion of MSX2 inhibits the senescence of DPSCs and restores their self-renewal capacity, and the simultaneous overexpression of ROR2 enhanced this effect. Moreover, MSX2 knockdown suppressed the transcription of NOP2/Sun domain family member 2 (NSUN2), which regulates the expression of p21 by binding to and causing the 5-methylcytidine methylation of the 3'- untranslated region of p21 mRNA. Interestingly, ROR2 downregulation elevated the levels of MSX2 protein, and not the MSX2 mRNA expression, by reducing the phosphorylation level of MSX2 and inhibiting the RNF34-mediated MSX2 ubiquitination degradation. The results of the present study demonstrated the vital role of the ROR2/MSX2/NSUN2 axis in the regulation of DPSC senescence, thereby revealing a potential target for antagonizing DPSC aging.

Functions and roles of IFIX, a member of the human HIN-200 family, in human diseases.

Pyrin and hematopoietic expression, interferon-inducible nature, and nuclear localization (HIN) domain family member 1 (PYHIN1), also known as IFIX, belongs to the family of pyrin proteins. This family includes structurally and functionally related mouse (e.g., p202, p203, and p204 proteins) and human (e.g., the interferon-inducible protein 16, absent in melanoma 2 protein, myeloid cell nuclear differentiation antigen, and pyrin and HIN domain family 1 or IFIX) proteins. The IFIX protein belongs to the HIN-200 family of interferon-inducible proteins that have a 200-amino acid signature motif at their C-termini. The increased expression of pyrin proteins in most cell types inhibits cell cycle control and modulates cell survival. Consistent with this role for pyrin proteins, IFIX is a potential antiviral DNA sensor that is essential for immune responses, the detection of viral DNA in the nucleus and cytoplasm, and the binding of foreign DNA via its HIN domain in a sequence non-specific manner. By promoting the ubiquitination and subsequent degradation of MDM2, IFIX acts as a tumor suppressor, thereby leading to p53/TP53 stabilization, HDAC1 regulation via the ubiquitin-proteasome pathway, and tumor-cell-specific silencing of the maspin gene. These data demonstrate that the potential molecular mechanism(s) underlying the action of the IFIX protein might be associated with the development of human diseases, such as viral infections, malignant tumors, and autoimmune diseases. This review summarizes the current insights into IFIX functions and how its regulation affects the outcomes of various human diseases.

TBC1D14 inhibits autophagy to suppress lymph node metastasis in head and neck squamous cell carcinoma by downregulating macrophage erythroblast attacher.

Aims: This study aimed to identify the correlation and molecular mechanism between TBC1 domain family member 14 (TBC1D14) and lymph node metastasis (LNM) in head and neck squamous cell carcinoma (HNSCC).Methods: Whole transcriptome sequencing of HNSCC tissues with or without LNM was performed. TBC1D14 expression was quantified in HNSCC tissues. The role of TBC1D14 in HNSCC migration, invasion, autophagy, and LNM was investigated by wound healing, Transwell, western blotting, immunofluorescence, and transmission electron microscopy assays in vitro and in a mouse model in vivo. The correlation between autophagy and LNM was detected by wound healing and Transwell assays in vitro and western blotting in vivo. Mass spectrometry was used to identify the downstream target proteins. The correlation between TBC1D14 expression and macrophage erythroblast attacher (MAEA) expression was identified by qRT-PCR and western blotting assays in vitro and immunohistochemistry in vivo. The gain-of-function strategy was applied to further reveal the role of MAEA in the TBC1D14-induced autophagy of HNSCC cells.Results: TBC1D14 was a co-differentially expressed gene in the sequencing results, The Cancer Genome Atlas Data Portal, and Gene Expression Omnibus databases. TBC1D14 had a lower RNA and protein expression in HNSCC with LNM samples and was a favorable prognostic indicator. TBC1D14 inhibited the migration and invasion of HNSCC in vivo. Mechanistically, TBC1D14-induced autophagy suppression inhibited the migration and invasion of HNSCC. TBC1D14 expression negatively correlated with MAEA expression both in vitro and in vivo. Furthermore, MAEA overexpression could reverse TBC1D14-induced autophagy suppression.Conclusion: TBC1D14 is a novel LNM inhibitor in HNSCC and a favorable prognostic marker. TBC1D14 suppresses autophagy to inhibit LNM in HNSCC by downregulating MAEA expression. The results clarify the molecular mechanism of TBC1D14 in HNSCC.

ONECUT2 as a key mediator of androgen receptor-independent cell growth and neuroendocrine differentiation in castration-resistant prostate cancer.

Despite androgen dependence in a majority of castration-resistant prostate cancers, some cancer cells are independent of androgen receptor (AR) function, a feature of heterogeneity in prostate cancer. One of the aggressive variants of prostate cancer that are AR independent is neuroendocrine prostate cancer (NEPC). This manuscript will focus on the new finding of human one cut domain family member 2 (ONECUT2) transcription factor and its role in castration resistance, especially in NEPC.

TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression.

Intestinal inflammation is a vital precipitating factor of colorectal cancer (CRC), but the underlying mechanisms are still elusive. TANK-binding kinase 1 (TBK1) is a core enzyme downstream of several inflammatory signals. Recent studies brought the impacts of TBK1 in malignant disease to the forefront, we found aberrant TBK1 expression in CRC is correlated with CRC progression. TBK1 inhibition impaired CRC cell proliferation, migration, drug resistance and tumor growth. Bioinformatic analysis and experiments in vitro showed overexpressed TBK1 inhibited mTORC1 signaling activation in CRC along with elevated GLUT1 expression without inducing GLUT1 translation. TBK1 mediated mTORC1 inhibition induces intracellular autophagy, which in turn decreasing GLUT1 degradation. As a rescue, blocking of autophagosome and retromer respectively via autophagy-related gene 7 (ATG7) or TBC1 Domain Family Member 5 (TBC1D5) silence diminished the regulation of TBK1 to GLUT1. GLUT1 staining presented that TBK1 facilitated GLUT1 membrane translocation which subsequently enhanced glucose consumption. Inhibitor of TBK1 also decreased GLUT1 expression which potentiated drug-sensitivity of CRC cell. Collectively, TBK1 facilitates glucose consumption for supporting CRC progression via initiating mTORC1 inhibition induced autophagy which decreases GLUT1 degradation and increases GLUT1 membrane location. The adaptive signaling cascade between TBK1 and GLUT1 proposes a new strategy for CRC therapy.