Clostridium botulinum neurotoxin type A (BoNT/A) is one of the most potent biotoxins ever known. Its entry into neurons could block vesicle exocytosis to abolish the release of neurotransmitters from nerve terminals, thus leading to muscle paralysis. Although there are so many peptides, antibodies and chemical compounds claimed to have anti-toxin activity, no drug is available in the clinical application except equine antitoxin serum. In the present work, a short peptide inhibitor RRGW of BoNT/A was firstly identified by computer-aided ligand-receptor binding simulation, then an RRGW derived peptide was rational designed based on the fragment of SNAP-25 (141-206 aa). Proteolytic assay showed that the anti-toxin activity of the RRGW derived peptide was much higher than that of RRGW. Digit abduction score assay demonstrated that the derived peptide delayed BoNT/A-induced muscle paralysis at a lower concentration by 20-fold than RRGW. The results supported that RRGW derived peptide can be a potential BoNT/A inhibitor candidate for further treating botulism.

Recent studies have showed that IKBKE is overexpressed in several kinds of cancers and that IKBKE-knockdown inhibits tumor progression. In this article, we first verified that two glioblastoma cell lines, U87-MG and LN-229, were sensitive to CYT387 by measuring the half maximal inhibitory concentration (IC50) with a CCK-8 assay and then demonstrated that CYT387, as a potent IKBKE inhibitor, suppressed glioblastoma cell proliferation, migration and invasion. Additionally, CYT387 induced cell apoptosis and arrested the cell cycle at the G2/M checkpoint in vitro. Furthermore, we showed that CYT387 did not simply inhibit IKBKE activity but also decreased IKBKE expression at the protein level rather than at the mRNA level. We discovered that CYT387 restrained malignant tumor progression by activating the Hippo pathway in vitro. By coimmunoprecipitation (co-IP), we showed that IKBKE interacted with TEAD2 and YAP1, thus accelerating TEAD2 and YAP1 transport into the nucleus. In subsequent in vivo experiments, we found that CYT387 inhibited subcutaneous nude mouse tumor growth but had little impact on intracranial orthotopic xenografts, probably due to a limited ability to penetrate the blood-brain barrier (BBB). These results suggest that CYT387 has potential as a new antiglioblastoma drug, but an approach to allow passage through the blood-brain barrier (BBB) is needed.

Indoleamine 2,3-dioxygenase 1 (IDO1) activity links to immune escape of cancers. Inhibition of IDO1 provides a new approach for cancer treatment. Most clinical IDO1 drugs show marginal efficacy as single agents. On basis of molecular docking and pharmacophore modelling, a novel inhibitor Roxyl-WL was discovered with a half maximal inhibitory concentration (IC50) value of 1 nM against IDO1 and 10-100-fold increased potent activity compared with IDO1 drugs in clinical trials. Roxyl-WL displayed excellent kinase spectrum selectivity with no activity out of the 337 protein kinases. In vitro, Roxyl-WL effectively augmented the proliferation of T cells and reduced the number of regulatory T cell (Tregs).When administered to melanoma (B16F10) tumor-bearing mice orally, Roxyl-WL significantly suppressed tumor growth and induced immune response.

Although immune checkpoint inhibitors (ICIs) have influenced the treatment paradigm for multiple solid tumors, increasing evidence suggests that primary and adaptive resistance may limit the long-term efficacy of ICIs. New therapeutic strategies with other drug combinations are hence warranted to enhance the antitumor efficacy of ICIs. As a novel tumor suppressor, histone deacetylase (HDAC) inhibitor tucidinostat has been successfully confirmed to act against hematological malignancies. However, the underlying mechanisms of action for tucidinostat and whether it can manipulate the tumor microenvironment (TME) in solid tumors remain unclear.

Hepatocellular carcinoma (HCC) is regarded as a leading cause of cancer-related deaths, and its progression is associated with hypoxia and the induction of hypoxia-inducible factor (HIF). Meloxicam, a selective cyclooxygenase-2 (COX-2) inhibitor, induces cell death in various malignancies. However, the underlying mechanism remains to be elucidated in HCC, especially under hypoxic conditions. The alteration of COX-2 and HIF-1α oncogenicity was evaluated in HCC specimens by tissue microarray. Cell viability, angiogenesis assays, and xenografted nude mice were used to evaluate the effects of meloxicam, along with flow cytometry to detect the cell cycle, apoptosis, and mitochondrial membrane potential (ΔΨm) of HCC. qRT-PCR, Western blotting, immunofluorescence, immunohistochemistry, luciferase assay, and RNAi were carried out to determine the HIF-1α signaling affected by meloxicam. In this study, we showed that meloxicam exerts antiproliferative and antiangiogenesis efficacy in vitro and in vivo and causes disruption of mitochondrial membrane potential (ΔΨm), thus leading to caspase-dependent apoptosis under hypoxic environments. Exposure to meloxicam significantly reduced HIF-1α transcriptional activation and expression through sequestering it in the cytoplasm and accelerating degradation via increasing the von Hippel-Lindau tumor suppressor protein (pVHL) in HCC. These data demonstrated that inhibition of HIF-1α by meloxicam could suppress angiogenesis and enhance apoptosis of HCC cells. This discovery highlights that COX-2 specific inhibitors may be a promising therapy in the treatment of HCC.

Triple-negative breast cancer (TNBC) is particularly sensitive to cyclin-dependent kinase 7 inhibitor, THZ1, compared to hormone receptor (HR)+ breast cancer, but our data found that different TNBC cell lines had a wide range of IC50 values of THZ1, suggesting a possible heterogeneity in sensitivity to THZ1 in TNBC. To seek potential biomarkers of THZ1 sensitivity, we re-analyzed the mRNAs profile in breast cancer cells treated with THZ1 from the previous study and demonstrated that elevated expression of SOX9 was significantly associated with the sensitivity of THZ1 in TNBC. We also verified that SOX9 expression promoted cell proliferation, migration, stemness, and predicted poor prognosis. Moreover, based on the tissue array of 278 patients and over 900 samples from TCGA data, we found that SOX9 expression was significantly higher in TNBC than HR+ breast cancers. Furthermore, ChIP-sequencing indicated that SOX9 binding to enhancer near transcription factor FOXC1, was remarkably inhibited by THZ1. And we also demonstrated that SOX9 and FOXC1 interacted with each other, which might co-operate and co-regulate the MYC signaling pathway in TNBC. Mechanistically, SOX9 may sensitize TNBC cells to THZ1, in a FOXC1-related manner, suggesting that SOX9 could be as a predictive factor of THZ1.

Diffuse large B‑cell lymphoma (DLBCL), the most common type of non‑Hodgkin's lymphoma, is classified into germinal center and activated B cell (ABC) subtypes. The myeloid differentiation primary response gene 88 (MYD88) L265P mutation is the most prevalent oncogenic mutation among patients with ABC DLBCL, the subtype that has the more inferior outcome. MYD88 oligomerization driven by the L265P mutant augments myddosome assembly and triggers the activation of nuclear factor kappa‑light‑chain‑enhancer of activated B cells (NF‑κB) signaling, highlighting MYD88 oligomerization as a potential therapeutic target for this malignancy. The synthetic peptidomimetic compound ST2825, which has previously been used as an anti‑inflammatory agent, has been reported to inhibit MYD88 dimerization. In the present study, the anticancer effects of ST2825 were investigated using L265P‑expressing ABC DLBCL cell lines. Using confocal microscopy and high‑molecular‑weight fraction experiments, it was revealed that L265P‑associated myddosome assembly was disrupted by ST2825. The results also revealed that disrupting myddosome assembly promoted the death of ABC DLBCL cells harboring the L265P mutation, as well as downregulating survival signals, including the inhibition of NF‑κB and the suppression of IL‑10 and interferon‑β production. Further co‑immunoprecipitation studies demonstrated that MYD88 bound to BTK in L265P‑DLBCL cells, and that this binding was abrogated following ST2825 treatment. Furthermore, the combination of myddosome‑assembly disruption and BTK or BCL‑2 signaling inhibition led to synergistic ABC DLBCL cell death, and more robust inhibition of NF‑κB activity or increased apoptosis, respectively. The results of the present study provide evidence that the synthetic peptidomimetic compound ST2825, which targets myddosome assembly, may serve as a pharmacological inhibitor. ST2825 has the potential for clinical use in patients with L265P DLBCL, and other B‑cell neoplasms driven by activated MYD88 signaling.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and human papillomavirus (HPV) has been increasingly recognized as a pathogenic factor for the initiation and development of HNSCC. E6 oncogene, an essential component of the HPV 16 virus, acts as a leading cause of the malignant transformation of cancer cells. Therefore, investigating the biological effect and potential mechanisms of E6 oncogene on HNSCC cells and exploring potential therapeutic methods is of great value.

Osteosarcoma is the most common primary malignant bone tumor in childhood and adolescence and has a propensity for local invasion and early lung metastasis. However, the current therapies often result in chemoresistance, and a therapeutic target is not available in the clinic for osteosarcoma. Here, we report that BRD7 forms a complex with the anaphase-promoting complex/cyclosome (APC/C) and is degraded by APC/C(cdh1) and APC/C(cdc20) during the cell cycle. Moreover, BRD7 is a tumor suppressor in osteosarcoma, and the BRD7 mutant resistant to degradation by APC/C is more efficient than the wild-type protein at suppressing proliferation, colony formation, and tumor growth of osteosarcoma in vitro and in vivo. The combination of proTAME, an inhibitor of APC/C, with chemotherapeutic drugs efficiently targets osteosarcoma in vitro. Furthermore, there is a strong inverse correlation of protein levels between BRD7 and Cdh1 or Cdc20, and lower BRD7 expression is an indicator for poor prognosis in patients with osteosarcoma. Collectively, our results indicate that targeting the APC/C-BRD7 pathway may be a novel strategy for treating osteosarcoma.

The transcription factor B cell lymphoma 6 (BCL6) is an oncogenic driver of diffuse large B cell lymphoma (DLBCL) and mediates lymphomagenesis through transcriptional repression of its target genes by recruiting corepressors to its N-terminal broad-complex/tramtrack/bric-a-brac (BTB) domain. Blocking the protein-protein interactions of BCL6 and its corepressors has been proposed as an effective approach for the treatment of DLBCL. However, BCL6 inhibitors with excellent drug-like properties are rare. Hence, the development of BCL6 inhibitors is worth pursuing. We screened our internal chemical library by luciferase reporter assay and Homogenous Time Resolved Fluorescence (HTRF) assay and a small molecule compound named WK500B was identified. WK500B engaged BCL6 inside cells, blocked BCL6 repression complexes, reactivated BCL6 target genes, killed DLBCL cells and caused apoptosis as well as cell cycle arrest. In animal models, WK500B inhibited germinal center (GC) formation and DLBCL tumour growth without toxic and side effects. Moreover, WK500B displayed strong efficacy and favourable pharmacokinetics and presented superior druggability. Therefore, WK500B is a promising candidate that could be developed as an effective orally available therapeutic agent for DLBCL.

TMEM132D is a human gene identified with multiple risk alleles for panic disorders, anxiety and major depressive disorders. Defining a conserved family of transmembrane proteins, TMEM132D and its homologs are still of unknown molecular functions. By generating loss-of-function mutants of the sole TMEM132 ortholog in C. elegans, we identify abnormal morphologic phenotypes in the dopaminergic PDE neurons. Using a yeast two-hybrid screen, we find that NAP1 directly interacts with the cytoplasmic domain of human TMEM132D, and mutations in C. elegans tmem-132 that disrupt interaction with NAP1 cause similar morphologic defects in the PDE neurons. NAP1 is a component of the WAVE regulatory complex (WRC) that controls F-actin cytoskeletal dynamics. Decreasing activity of WRC rescues the PDE defects in tmem-132 mutants, whereas gain-of-function of TMEM132D in mammalian cells inhibits WRC, leading to decreased abundance of select WRC components, impaired actin nucleation and cell motility. We propose that metazoan TMEM132 family proteins play evolutionarily conserved roles in regulating NAP1 protein homologs to restrict inappropriate WRC activity, cytoskeletal and morphologic changes in the cell.

As a common chronic metabolic disease, the development of diabetes mellitus (DM) may also be accompanied by liver damage and inflammatory disorders. Sitagliptin is an inhibitor of dipeptidyl peptidase-4 (DPP4, also known as CD26), which is clinically used for DM treatment. However, the mechanism of sitagliptin's efficiency in liver diseases is largely unknown. In this study, mice suffering from streptozotocin (STZ) exhibit elevated liver DPP4 expression and activity, as well as inflammatory and chronic liver injury phenotype, whereas specifically inhibiting the activity of DPP4 in mouse liver tissues and hepatocytes by sitagliptin contributes to decreased cytokines, oxidative stress, cell apoptosis, and inflammation in STZ-induced diabetic mice. Moreover, sitagliptin reduced TNFα or LPS-induced cellular reactive oxygen species (ROS) level, cell apoptosis, and protein expression in the NFκB signaling pathway in HepG2 cells or primary mouse hepatocytes. Altogether, our study confirms that sitagliptin may protect liver tissue by alleviating ROS production and NFκB signaling activation, providing a putative mechanism for preventing the development of diabetic liver disease.

Human single-stranded DNA binding protein 1 (hSSB1) plays a critical role in responding to DNA damage and maintaining genome stability. However, the regulation of hSSB1 remains poorly studied. Here, we determined that hSSB1 acetylation at K94 mediated by the acetyltransferase p300 and the deacetylases SIRT4 and HDAC10 impaired its ubiquitin-mediated degradation by proteasomes. Moreover, we demonstrated that the hSSB1-K94R mutant had reduced cell survival in response to DNA damage by radiation or chemotherapy drugs. Furthermore, the p300/CBP inhibitor C646 significantly enhanced the sensitivity of cancer cells to chemotherapy drugs, and a positive correlation between hSSB1 and p300 level was observed in clinical colorectal cancer samples. Acetylation, a novel regulatory modification of hSSB1, is crucial for its function under both physiological and pathological conditions. This finding supports the notion that the combination of chemotherapy drugs with acetylation inhibitors may benefit cancer patients.

Merkel cell polyomavirus (MCV or MCPyV) is the first human polyomavirus to be definitively linked to cancer. The mechanisms of MCV-induced oncogenesis and much of MCV biology are largely unexplored. In this study, we demonstrate that bromodomain protein 4 (Brd4) interacts with MCV large T antigen (LT) and plays a critical role in viral DNA replication. Brd4 knockdown inhibits MCV replication, which can be rescued by recombinant Brd4. Brd4 colocalizes with the MCV LT/replication origin complex in the nucleus and recruits replication factor C (RFC) to the viral replication sites. A dominant negative inhibitor of the Brd4-MCV LT interaction can dissociate Brd4 and RFC from the viral replication complex and abrogate MCV replication. Furthermore, obstructing the physiologic interaction between Brd4 and host chromatin with the chemical compound JQ1(+) leads to enhanced MCV DNA replication, demonstrating that the role of Brd4 in MCV replication is distinct from its role in chromatin-associated transcriptional regulation. Our findings demonstrate mechanistic details of the MCV replication machinery; providing novel insight to elucidate the life cycle of this newly discovered oncogenic DNA virus.

Rab22a-NeoF fusion protein has recently been reported as a promising target for osteosarcoma lung metastasis. However, how this fusion protein is regulated in cells remains unknown. Here, using multiple screenings, it is reported that Rab22a-NeoF1 fusion protein is degraded by an E3 ligase STUB1 via the autophagy receptor NDP52-mediated lysosome pathway, which is facilitated by PINK1 kinase. Mechanistically, STUB1 catalyzes the K63-linked ubiquitin chains on lysine112 of Rab22a-NeoF1, which is responsible for the binding of Rab22a-NeoF1 to NDP52, resulting in lysosomal degradation of Rab22a-NeoF1. PINK1 is able to phosphorylate Rab22a-NeoF1 at serine120, which promotes ubiquitination and degradation of Rab22a-NeoF1. Consistently, by upregulating PINK1, Sorafenib and Regorafenib can inhibit osteosarcoma lung metastasis induced by Rab22a-NeoF1. These findings reveal that the lysosomal degradation of Rab22a-NeoF1 fusion protein is targetable for osteosarcoma lung metastasis, proposing that Sorafenib and Regorafenib may benefit cancer patients who are positive for the RAB22A-NeoF1 fusion gene.

Transforming growth factor-β membrane associated protein (TIMAP) is an endothelial cell (EC)-predominant PP1 regulatory subunit and a member of the myosin phosphatase target (MYPT) protein family. The MYPTs preferentially bind the catalytic protein phosphatase 1 subunit PP1cβ, forming myosin phosphatase holoenzymes. We investigated whether TIMAP/PP1cβ could also function as a myosin phosphatase. Endogenous PP1cβ, myosin light chain 2 (MLC2), and myosin IIA heavy chain coimmunoprecipitated from EC lysates with endogenous TIMAP, and endogenous MLC2 colocalized with TIMAP in EC projections. Purified recombinant GST-TIMAP interacted directly with purified recombinant His-MLC2. However, TIMAP overexpression in EC enhanced MLC2 phosphorylation, an effect not observed with a TIMAP mutant that does not bind PP1cβ. Conversely, MLC2 phosphorylation was reduced in lung lysates from TIMAP-deficient mice and upon silencing of endogenous TIMAP expression in ECs. Ectopically expressed TIMAP slowed the rate of MLC2 dephosphorylation, an effect requiring TIMAP-PP1cβ interaction. The association of MYPT1 with PP1cβ was profoundly reduced in the presence of excess TIMAP, leading to proteasomal MYPT1 degradation. In the absence of TIMAP, MYPT1-associated PP1cβ readily bound immobilized microcystin-LR, an active-site inhibitor of PP1c. By contrast, TIMAP-associated PP1cβ did not interact with microcystin-LR, indicating that the active site of PP1cβ is blocked when it is bound to TIMAP. Thus, TIMAP inhibits myosin phosphatase activity in ECs by competing with MYPT1 for PP1cβ and blocking the PP1cβ active site.

The La-related protein 7 (LARP7) forms a complex with the nuclear 7SK RNA to regulate RNA polymerase II transcription. It has been implicated in cancer and the Alazami syndrome, a severe developmental disorder. Here, we report a so far unknown role of this protein in RNA modification. We show that LARP7 physically connects the spliceosomal U6 small nuclear RNA (snRNA) with a distinct subset of box C/D small nucleolar RNAs (snoRNAs) guiding U6 2'-O-methylation. Consistently, these modifications are severely compromised in the absence of LARP7. Although general splicing remains largely unaffected, transcriptome-wide analysis revealed perturbations in alternative splicing in LARP7-depleted cells. Importantly, we identified defects in 2'-O-methylation of the U6 snRNA in Alazami syndrome siblings carrying a LARP7 mutation. Our data identify LARP7 as a bridging factor for snoRNA-guided modification of the U6 snRNA and suggest that alterations in splicing fidelity contribute to the etiology of the Alazami syndrome.

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV) infection, leads to significant economic losses in the swine industry worldwide. In our studies, we found that glycyrrhizin, the major component of licorice root extracts, could moderately inhibit PEDV infection in Vero cells, when analyzed by western blot, qRT-PCR and a plaque formation assay. We also revealed that glycyrrhizin inhibited the entry and replication of PEDV. In addition, we demonstrated that glycyrrhizin decreased the mRNA levels of proinflammatory cytokines. Since glycyrrhizin is a competitive inhibitor of high mobility group box-1 (HMGB1), we confirmed that TLR4 and RAGE (£ associated with PEDV pathogenesis during the infection in Vero cells. In summary, our studies provide a molecular basis for developing novel therapeutic methods to control PEDV infection, based on glycyrrhizin and its derivatives.

Ovarian cancer is the most lethal gynecological malignancy worldwide with high metastasis and poor prognosis rates. Cancer-associated fibroblasts (CAFs), a heterogeneous population of cells that constitutes a major component of the tumor microenvironment, secrete extracellular vesicles (EVs) loading with proteins, lipids, and RNAs to promote tumorigenesis. However, the specific roles of CAF-derived proteins contained in EVs in ovarian cancer remain poorly understood at present. Using the gene expression microarray analysis, we identified a list of dysregulated genes between the α-SMA+ CAF and FAP+ CAF subpopulations, from which secretory leukocyte protease inhibitor (SLPI) was chosen for further validation. Quantitative PCR, western blot, immunohistochemistry, and enzyme-linked immunosorbent assays were used to assess SLPI expression in ovarian cancer cells, tissues, CAFs, and EVs. Additionally, we evaluated the effects of exogenous SLPI on proliferation, migration, invasion, and adhesion of ovarian cancer cells in vitro. Our results showed SLPI protein was upregulated in CAFs, particularly in the FAPhigh α-SMAlow CAF subpopulation, and associated with increased tumor grade and decreased overall survival (OS). Importantly, CAF-derived SLPI protein could be encapsulated in EVs for delivery to ovarian cancer cells, thus facilitating cell proliferation, migration, invasion, and adhesion via activating the PI3K/AKT and downstream signaling pathways. Moreover, high plasma expression of SLPI encapsulated in EVs was closely correlated with tumor stage in ovarian cancer patients. Our collective results highlight an oncogenic role of plasma EV-encapsulated SLPI secreted by CAFs in tumor progression for the first time, supporting its potential utility as a prognostic biomarker of ovarian cancer.

Zn2+ is required for the activity of many mitochondrial proteins, which regulate mitochondrial dynamics, apoptosis and mitophagy. However, it is not understood how the proper mitochondrial Zn2+ level is achieved to maintain mitochondrial homeostasis. Using Caenorhabditis elegans, we reveal here that a pair of mitochondrion-localized transporters controls the mitochondrial level of Zn2+. We demonstrate that SLC-30A9/ZnT9 is a mitochondrial Zn2+ exporter. Loss of SLC-30A9 leads to mitochondrial Zn2+ accumulation, which damages mitochondria, impairs animal development and shortens the life span. We further identify SLC-25A25/SCaMC-2 as an important regulator of mitochondrial Zn2+ import. Loss of SLC-25A25 suppresses the abnormal mitochondrial Zn2+ accumulation and defective mitochondrial structure and functions caused by loss of SLC-30A9. Moreover, we reveal that the endoplasmic reticulum contains the Zn2+ pool from which mitochondrial Zn2+ is imported. These findings establish the molecular basis for controlling the correct mitochondrial Zn2+ levels for normal mitochondrial structure and functions.