The role of T2E mediated CBF-1/RBP-Jκ signaling in metastatic thyroid cancer.

OBJECTIVE: Cancer has been shown to be an independent risk factor for 2019-nCoV. Expression of transmembrane serine protease 2 (TMPRSS2) is abnormal in many cancers. Nevertheless, system analysis of TMPRSS2-ERG (T2E) abnormalities in metastatic thyroid cancer remains to be elucidated. METHOD: Using genomic and chromatin data, we demonstrate a unique cis-regulatory landscape between non-T2E and T2E-positive metastatic thyroid cancers, including clusters of regulatory elements (COREs). We attempt to describe the effect of T2E silencing on the cis-regulatory structure in metastatic thyroid cancers and its phase with the obvious phenotype characteristics of T2E-positive metastatic thyroid cancers. RESULTS: These differences were linked by the ERG (erythroblast transformation-specific related gene) co-opts of FoxA1 and HOXB13, which realized T2E specific transcription profile. The study also demonstrated the T2E-specific CORE in an ERG site of structural rearrangement, which is due to the expansion of the T2E locus and contributes to its up-expression. Ultimately, we demonstrate that T2E-specific transcription profile is the basis of vulnerability of CBF-1/RBP-Jκ pathway. In fact, CBF-1/RBP-Jκ pathway inhibits the invasion and growth of T2E-positive thyroid tumors. CONCLUSION: This study indicates that the overexpression of ERG co-option has a unique cis-regulatory structure in T2E positive thyroid tumors, which induces drug dependence on CBF-1/RBP-Jκ signal. Our study solved the genetic and epigenetic variation of T2E in metastatic thyroid cancer for the first time. It is worth noting that further functional and clinical validation is needed as our study is a bioinformatics analysis.

SIRT2 modulates VEGFD-associated lymphangiogenesis by deacetylating EPAS1 in human head and neck cancer.

Sirtuin 2 (SIRT2) is one of seven mammalian homologs of silent information regulator 2 (Sir2) and an NAD+ -dependent deacetylase; however, its critical role in lymphangiogenesis remains to be explored. We investigate SIRT2 mediated regulation of vascular endothelial growth factor D (VEGFD) expression and lymphangiogenesis by deacetylating endothelial PAS domain protein 1 (EPAS1) in head and neck cancer (HNC) in vitro and in vivo. In this study, we report that SIRT2, rather than other members of the Sir2 family, reduces the expression of VEGFD and lymphangiogenesis in hypoxia-induced HNC cells and transplanted HNC mice models by reducing EPAS1 acetylation at Lys674 and decreasing the transcriptional activity of EPAS1 target genes. The expression of SIRT2 was closely related to the expression of VEGFD, lymphangiogenesis in subcutaneously transplanted mice models, and lymphangiogenesis in patients with HNC. Our results suggest that SIRT2 plays a central role in tumor lymphangiogenesis via deacetylating EPAS1 protein. Reagents targeting the NAD+ -dependent deacetylase activity of SIRT2 would be beneficial for inhibiting tumor lymphangiogenesis and treating other hypoxia-related diseases.

Single-cell RNA sequencing reveals the regenerative potential of thyroid follicular epithelial cells in metastatic thyroid carcinoma.

Thyroid stimulating hormone deficiency is the cornerstone of treatment for metastatic thyroid cancer. Due to the loss of follicular epithelial cells in thyroid cancer, the thyroid gland degenerates to 85% of its original size. When thyroid stimulating hormone is restored, follicular epithelial cells in thyroid cancer regenerate, which is postulated to be related to stem-like cells. By single cell RNA seq, we found a group of rare thyroid follicular epithelial cells in mouse metastatic thyroid cancer, which expressed stem-like genes (CD44V6+ and CD133+) and a large number of differentiated cells (CD44V6+ and CD24+). In mouse and in organoids, the two subsets contribute equally to metastatic thyroid cancer regeneration. The analysis of human metastatic thyroid cancer revealed that the differentiated thyroid follicular epithelial cell subpopulation was similar to that of the stem like epithelial cell subpopulation, and the regeneration potential was also enhanced after thyroid stimulating hormone ablation. Accordingly, we propose that the regeneration of metastatic thyroid cancer is driven by almost all persistent thyroid follicular epithelial cells, not only by few stem-like cells.

Celastrol Reverses Palmitic Acid-Induced Insulin Resistance in HepG2 Cells via Restoring the miR-223 and GLUT4 Pathway.

OBJECTIVES: The natural triterpenoid compound celastrol ameliorates insulin resistance (IR) in animal models, but the underlying molecular mechanism is unclear. In this study, we investigated how celastrol regulates IR. METHODS: The HepG2 cellular IR model was initially established with palmitic acid (PA). The expression and activity of glucose transporter 4 (GLUT4), insulin receptor substrate-1 (IRS1) and 9 microRNAs (miRNAs) (miR-7, -34a, -96, -113, -126, -145, -150, -223 and -370) were detected before and after celastrol treatment using the PA-induced HepG2 IR model. RESULTS: The results showed that 250 µM PA for ≥2 days was optimal for inducing IR in HepG2 cells; 600 nM celastrol significantly attenuated the PA-induced IR in HepG2 cells. The PA-induced GLUT4 and IRS1 downregulation and Ser307 phosphorylation on IRS1 was reversed by subsequent treatment with 600 nM celastrol for 6 h. We next investigated which IR-related miRNAs were possible upstream regulators of celastrol-mediated reversal of PA-induced HepG2 IR. Two miRNAs, miR-150 and -223, were significantly downregulated by PA and were re-raised by subsequent celastrol treatment; and miR-223 was upstream of miR-150. Moreover, knocking down miR-223 abolished celastrol's anti-IR effects in the PA-induced model. CONCLUSIONS: Collectively, our results demonstrated that celastrol reverses PA-induced IR-related alterations, in part via miR-223 in HepG2 cells. Further investigation is warranted for establishing the clinical potential of celastrol in treating IR-related disorders.

Celastrol reverses palmitic acid (PA)-caused TLR4-MD2 activation-dependent insulin resistance via disrupting MD2-related cellular binding to PA.

Elevated plasma statured fatty acids (FFAs) cause TLR4/MD2 activation-dependent inflammation and insulin tolerance, which account for the occurrence and development of obesity. It has been confirmed that statured palmitic acid (PA) (the most abundant FFA) could bind MD2 to cause cellular inflammation. The natural compound celastrol could improve obesity, which is suggested via inhibiting inflammation, yet the detailed mechanism for celastrol is still unclear. As celastrol is reported to directly target MD2, we thought disrupting the binding between FFAs and MD2 might be one of the ways for celastrol to inhibit FFAs-caused inflammation and insulin resistance. In this study, we found evidence to support our hypothesis: celastrol could reverse PA-caused TLR4/MD2 activation-dependent insulin resistance, as determined by glucose-lowering ability, cellular glucose uptake, insulin action-related proteins and TLR4/MD2/NF-κB activation. Bioinformatics and cellular experiments showed that both celastrol and PA could bind MD2, and that celastrol could expel PA from cells. Finally, celastrol could reverse high fat diet caused hyperglycemia and obesity, and liver NF-kB activations. Taking together, we proved that celastrol could reverses PA-caused TLR4-MD2 activation-dependent insulin resistance via disrupting PA binding to MD2.

Mutations Y493G and K546D in human HSP90 disrupt binding of celastrol and reduce interaction with Cdc37.

Celastrol, a natural compound derived from the Chinese herb Tripterygium wilfordii Hook F, has been proven to inhibit heat shock protein 90 (HSP90) activity and has attracted much attention because of its promising effects in cancer treatment and in ameliorating degenerative neuron diseases. However, the HSP90 structure involved in celastrol interaction is not known. Here, we report a novel celastrol-binding pocket in the HSP90 dimer, predicted by molecular docking. Mutation of the two key binding pocket amino acids (Lys546 and Tyr493) disrupted the binding of celastrol to HSP90 dimers, as detected by isothermal titration calorimetry (ITC). Interestingly, such mutations also reduced binding between HSP90 and the cochaperone Cdc37, thus providing a new explanation for reported findings that celastrol shows more obvious effects in disrupting binding between HSP90 and Cdc37 than between HSP90 and other cochaperones. In short, our work discloses a novel binding pocket in HSP90 dimer for celastrol and provides an explanation as to why celastrol has a strong effect on HSP90 and Cdc37 binding.

Celastrol targets IRAKs to block Toll-like receptor 4-mediated nuclear factor-κB activation.

OBJECTIVE: Celastrol has been established as a nuclear factor-κB (NF-κB) activation inhibitor; however, the exact mechanism behind this action is still unknown. Using text-mining technology, the authors predicted that interleukin-1 receptor-associated kinases (IRAKs) are potential celastrol targets, and hypothesized that targeting IRAKs might be one way that celastrol inhibits NF-κB. This is because IRAKs are key molecules for some crucial pathways to activate NF-κB (e.g., the interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) superfamily). METHODS: The human hepatocellular cell line (HepG2) treated with palmitic acid (PA) was used as a model for stimulating TLR4/NF-κB activation, in order to observe the potential effects of celastrol in IRAK regulation and NF-κB inhibition. The transfection of small interfering RNA was used for down-regulating TLR4, IRAK1 and IRAK4, and the Western blot method was used to detect changes in the protein expressions. RESULTS: The results showed that celastrol could effectively inhibit PA-caused TLR4-dependent NF-κB activation in the HepG2 cells; PA also activated IRAKs, which were inhibited by celastrol. Knocking down IRAKs abolished PA-caused NF-κB activation. CONCLUSION: The results for the first time show that targeting IRAKs is one way in which celastrol inhibits NF-κB activation.

Celastrol Ameliorates EAE Induction by Suppressing Pathogenic T Cell Responses in the Peripheral and Central Nervous Systems.

Multiple sclerosis (MS) is the prototypical inflammatory demyelinating disease of the central nervous system (CNS), and MS results in physical and cognitive impairments, such as fatigue, pain, depression and bladder dysfunction. Though many therapies for MS have been developed, the safety profile and effectiveness of these therapies still need to be defined. Thus, new therapies for MS must be explored. Celastrol, a quinonemethide triterpene, is a pharmacologically active compound present in Thunder God Vine root extracts used to treat inflammatory and autoimmune diseases. Molecular studies have identified several molecular targets, which are mostly centered on the inhibition of IKK-NF-κB signaling. The animal model of experimental autoimmune encephalomyelitis (EAE) has been widely used in MS studies; thus, we tried to explore the role of celastrol in EAE development in this study. We demonstrated that the intraperitoneal injection of celastrol significantly attenuated EAE disease. Th17 cell responses in the peripheral lymph nodes in EAE mice were also inhibited by celastrol. We determined that celastroldownregulated cytokine production in bone-marrow derived dendritic cells (BMDCs). Accordingly, T cells that were co-cultured with either BMDCs pre-treated with celastrolor splenic DCs and then collected on day 7 after EAE immunizationshowed that Th17 cell polarization is suppressed in the above two situations. Moreover, celastrol was required for tissue-infiltrating DCs to sustain Th17 responses in the central nervous system (CNS). Taken together, the results of our study demonstrate that celastrol ameliorates EAE development by suppressing pathogenic Th17 responses; this finding offers a better understanding of the role of celastrol in EAE development as well as new proposals for clinical interventions.

Celastrol inhibits lung infiltration in differential syndrome animal models by reducing TNF-α and ICAM-1 levels while preserving differentiation in ATRA-induced acute promyelocytic leukemia cells.

All-trans retinoic acid (ATRA) is a revolutionary agent for acute promyelocytic leukemia (APL) treatment via differentiation induction. However, ATRA treatment also increases cytokine, chemokine, and adhesive molecule (mainly ICAM-1) expression, which can cause clinical complications, including a severe situation known as differentiation syndrome (DS) which can cause death. Therefore, it is of clinical significance to find a strategy to specifically blunt inflammatory effects while preserving differentiation. Here we report that the natural compound, celastrol, could effectively block lung infiltrations in DS animal models created by loading ATRA-induced APL cell line NB4. In ATRA-treated NB4 cells, celastrol could potently inhibit ICAM-1 elevation and partially reduce TNF-α and IL-1ß secretion, though treatment showed no effects on IL-8 and MCP-1 levels. Celastrol's effect on ICAM-1 in ATRA-treated NB4 was related to reducing MEK1/ERK1 activation. Strikingly and encouragingly, celastrol showed no obvious effects on ATRA-induced NB4 differentiation, as determined by morphology, enzymes, and surface markers. Our results show that celastrol is a promising and unique agent for managing the side effects of ATRA application on APL, and suggest that hyper-inflammatory ability is accompanied by, but not necessary for, APL differentiation. Thus we offered an encouraging novel strategy to further improve differentiation therapy.

IP-FCM platform detects the existence and regulator-caused dissociation of components in naturally assembled HSP90 complex.

Flow cytometry, in conjunction with immunoprecipitation (IP-FCM), is suggested to have some advantages to conventional IP-western blot technology in analyzing protein complexes. In this paper, to further examine its practicability, we test the use of IP-FCM in detecting the HSP90 complex, which has gained importance in drug research and development and involves more than a dozen components. We found that IP-FCM could effectively detect HSP70, p23, Cdc37, and Cdk6 components in the HSP90 complex naturally formed in U937 cells when this complex was captured by anti-HSP90 antibody-coated polystyrene microspheres. IP-FCM could also detect alteration in components caused by treating cells with HSP90 inhibitors. In a cell-free environment, IP-FCM could detect the direct effects of ATP and/or HSP90 inhibitors (17-N-allylamino-17-demethoxygeldanamycin or celastrol) in causing component dissociation and the time- and dose-effects of inhibitor-caused dissociation. IP-FCM is a practical and powerful platform for analyzing HSP90 complex components, and is thus a useful tool in studying HSP90 complex function and screening inhibitors.