Discovery of tetrazolo-pyridazine-based small molecules as inhibitors of MACC1-driven cancer metastasis.

Metastasis is directly linked to poor prognosis of cancer patients and warrants search for effective anti-metastatic drugs. MACC1 is a causal key molecule for metastasis. High MACC1 expression is prognostic for metastasis and poor survival. Here, we developed novel small molecule inhibitors targeting MACC1 expression to impede metastasis formation. We performed a human MACC1 promoter-driven luciferase reporter-based high-throughput screen (HTS; 118.500 compound library) to identify MACC1 transcriptional inhibitors. HTS revealed 1,2,3,4-tetrazolo[1,5-b]pyridazine-based compounds as efficient transcriptional inhibitors of MACC1 expression, able to decrease MACC1-induced cancer cell motility in vitro. Structure-activity relationships identified the essential inhibitory core structure. Best candidates were evaluated for metastasis inhibition in xenografted mouse models demonstrating metastasis restriction. ADMET showed high drug-likeness of these new candidates for cancer therapy. The NFκB pathway was identified as one mode of action targeted by these compounds. Taken together, 1,2,3,4-tetrazolo[1,5-b]pyridazine-based compounds are effective MACC1 inhibitors and pose promising candidates for anti-metastatic therapies particularly for patients with MACC1-overexpressing cancers, that are at high risk to develop metastases. Although further preclinical and clinical development is necessary, these compounds represent important building blocks for an individualized anti-metastatic therapy for solid cancers.

Long non-coding RNA Homeobox D gene cluster antisense growth-associated long noncoding RNA/microRNA-182-5p/Homeobox protein A10 alleviates postmenopausal osteoporosis via accelerating osteoblast differentiation of bone marrow mesenchymal stem cells.

BACKGROUND: Studies have illuminated that long non-coding RNA (lncRNA) influences bone cell differentiation and formation. Nevertheless, whether lncRNA Homeobox D gene cluster antisense growth-associated long noncoding RNA (HAGLR) was implicated in postmenopausal osteoporosis (PMOP) was yet uncertain. PURPOSE: The research was to explore HAGLR's role in the osteogenic differentiation (OD) process of bone marrow mesenchymal stem cells (BMSCs). METHODS: BMSCs were isolated from mouse bone marrow tissues and identified by electron microscope and flow cytometry. HAGLR, microRNA (miR)-182-5p, and homeobox protein A10 (Hoxa10) levels in BMSCs were detected. Mouse BMSC OD process was induced, and calcium deposition and alkaline phosphatase content were analyzed, as well as expressions of runt-related transcription factor 2, osteopontin, and osteocalcin, and cell apoptosis. Bilateral ovaries were resected from mice to construct the ovariectomized model and bone mineral density, maximum bending stress, maximum load, and elastic modulus of the femur were tested, and the femur was histopathologically evaluated. Chondrocyte apoptosis in the articular cartilage of mice was analyzed. Analysis of the interaction of HAGLR, miR-182-5p with Hoxa10 was conducted. RESULTS: HAGLR and Hoxa10 were down-regulated and miR-182-5p was elevated in PMOP patients. During the BMSC OD process, HAGLR and Hoxa10 levels were suppressed, while miR-182-5p was elevated. Promotion of HAGLR or suppression of miR-182-5p accelerated OD of BMSCs. Inhibition of miR-182-5p reversed the inhibitory effect of HAGLR on BMSC OD. In in vivo experiments, up-regulating HAGLR alleviated PMOP, while silencing Hoxa10 reversed the effects of upregulating HAGLR. HAGLR performed as a sponge for miR-182-5p, while miR-182-5p targeted Hoxa10. CONCLUSION: In general, HAGLR boosted the OD process of BMSCs and relieved PMOP via the miR-182-5p/Hoxa10 axis. These data preliminarily reveal the key role of HAGLR in PMOP, and the research results have a certain reference for the treatment of PMOP.

Wnt signalling pathway in bladder cancer.

Bladder cancer (BC) is one of the most common tumours of the urinary system and is also known as a highly malignant tumour. In addition to conventional diagnosis and treatment methods, recent research has focused on studying the molecular mechanisms related to BC, in the hope that new, less toxic and effective targeted anticancer drugs and new diagnostic markers can be discovered. It is known that the Wingless (Wnt) signalling pathway and its related genes, proteins and other substances are involved in multiple biological processes of various tumours. Clarifying the contribution of the Wnt signalling pathway in bladder tumours will help establish early diagnosis indicators, develop new therapeutic drugs and evaluate the prognosis for BC. This review aims to summarise previous studies related to BC and the Wnt signalling pathway, with a focus on exploring the participating substances and their mechanisms in the regulation of the Wnt signalling pathway to better determine how to promote new chemotherapeutic drugs, potential therapeutic targets and diagnostic biomarkers.

Identification of unique long non-coding RNAs as putative biomarkers for chromophobe renal cell carcinoma.

Aim: To investigate whether long non-coding RNAs (lncRNAs) can be utilized as molecular biomarkers in predicting the occurrence and progression of chromophobe renal cell carcinoma. Methods & results: Genetic and related clinical traits of chromophobe renal cell carcinoma were downloaded from the Cancer Genome Atlas and used to construct modules using weighted gene coexpression network analysis. In total, 44,889 genes were allocated into 21 coexpression modules depending on intergenic correlation. Among them, the green module was the most significant key module identified by module-trait correlation calculations (R2 = 0.43 and p = 4e-04). Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses demonstrated that genes in the green module were enriched in many pathways. Coexpression, protein-protein interaction networks, screening for differentially expressed genes, and survival analysis were used to select hub lncRNAs. Five hub lncRNAs (TTK, CENPE, KIF2C, BUB1, and RAD51AP1) were selected out. Conclusion: Our findings suggest that the five lncRNAs may act as potential biomarkers for chromophobe renal cell carcinoma progression and prognosis.

A novel murine model of differentiation-mediated cytomegalovirus reactivation from latently infected bone marrow haematopoietic cells.

CD34+ myeloid lineage progenitor cells are an important reservoir of latent human cytomegalovirus (HCMV), and differentiation to macrophages or dendritic cells (DCs) is known to cause reactivation of latent virus. Due to its species-specificity, murine models have been used to study mouse CMV (MCMV) latency and reactivation in vivo. While previous studies have shown that MCMV genomic DNA can be detected in the bone marrow (BM) of latently infected mice, the identity of these cells has not been defined. Therefore, we sought to identify and enrich for cellular sites of MCMV latency in the BM haematopoietic system, and to explore the potential for establishing an in vitro model for reactivation of latent MCMV. We studied the kinetics and cellular characteristics of acute infection and establishment of latency in the BM of mice. We found that while MCMV can infect a broad range of haematopoietic BM cells (BMCs), latent virus is only detectable in haematopoietic stem cells (HSCs), myeloid progenitor cells, monocytes and DC-enriched cell subsets. Using three separate approaches, MCMV reactivation was detected in association with differentiation into DC-enriched BMCs cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) followed by lipopolysaccharide (LPS) treatment. In summary, we have defined the kinetics and cellular profile of MCMV infection followed by the natural establishment of latency in vivo in the mouse BM haematopoietic system, including the haematopoietic phenotypes of cells that are permissive to acute infection, establish and harbour detectable latent virus, and can be stimulated to reactivate following DC enrichment and differentiation, followed by treatment with LPS.

Transplant-induced reactivation of murine cytomegalovirus immediate early gene expression is associated with recruitment of NF-κB and AP-1 to the major immediate early promoter.

Reactivation of latent human cytomegalovirus is a significant infectious complication of organ transplantation and current therapies target viral replication once reactivation of latent virus has already occurred. The specific molecular pathways that activate viral gene expression in response to transplantation are not well understood. Our studies aim to identify these factors, with the goal of developing novel therapies that prevent transcriptional reactivation in transplant recipients. Murine cytomegalovirus (MCMV) is a valuable model for studying latency and reactivation of CMV in vivo. We previously demonstrated that transplantation of MCMV-latently infected kidneys into allogeneic recipients induces reactivation of immediate early (IE) gene expression and epigenetic reprogramming of the major immediate early promoter (MIEP) within 48 h. We hypothesize that these events are mediated by activation of signalling pathways that lead to binding of transcription factors to the MIEP, including AP-1 and NF-κB. Here we show that transplantation induces rapid activation of several members of the AP-1 and NF-κB transcription factor family and we demonstrate that canonical NF-κB (p65/p50), the junD component of AP-1, and nucleosome remodelling complexes are recruited to the MIEP following transplantation. Proteomic analysis of recipient plasma and transcriptome analysis of kidney RNA identified five extracellular ligands, including TNF, IL-1ß, IL-18, CD40L and IL-6, and three intracellular signalling pathways associated with reactivation of IE gene expression. Identification of the factors that mediate activation of these signalling pathways may eventually lead to new therapies to prevent reactivation of CMV and its sequelae.

Nur77 Was Essential for Neurite Outgrowth and Involved in Schwann Cell Differentiation After Sciatic Nerve Injury.

Nur77, together with Nurr1 and NOR-1, constitutes the NR4A subgroup of orphan nuclear receptors and plays critical roles in cell proliferation, differentiation, migration, and apoptosis. Among them, Nur77 is universally well known to contribute to neurite outgrowth. However, information regarding its regulation and possible function in the peripheral nervous system is still limited. In this study, we performed a sciatic nerve injury model in adult rats and detected an increased expression of Nur77 in the sciatic nerve, which was similar to the expression of Oct-6. Immunofluorescence indicated that Nur77 was located in both axons and Schwann cells. In vitro, we observed enhanced expression of Nur77 during the process of both basic fibroblast growth factor (bFGF)-induced Schwann cells differentiation and nerve growth factor (NGF)-induced PC12 cell neurite outgrowth. In vitro and in vivo experiments indicated that inhibiting the function of Nur77 by specific short hairpin RNA could depress Schwann cells myelinization and axons regeneration. Collectively, all these results suggested that upregulation of Nur77 might be involved in Schwann cells differentiation and neurite elongation following sciatic nerve crush.

Up-regulation of NF45 correlates with Schwann cell proliferation after sciatic nerve crush.

Nuclear factor (NF)45 (also known as interleukin enhancer-binding factor (ILF)2), is a transcription factor that interacts with NF90 to regulate gene expression. It has long been implicated in the regulation of cell proliferation. However, the role of NF45 in the process of peripheral nervous system regeneration after injury remains poorly understood. Herein, we investigated the spatiotemporal expression of NF45 in a rat sciatic nerve crush model. We detected the up-regulated expression of NF45 in Schwann cell after sciatic nerve crush. What's more, the expression of the cell proliferation marker proliferating cell nuclear antigen (PCNA) exhibited a similar tendency with that of NF45. In cell cultures, we observed increased expression of NF45 during the process of TNF-α-induced Schwann cell proliferation, whereas the protein level of p21 was down-regulated. Interference of NF45 led to enhanced expression of p21 and also impaired proliferation of Schwan cells. Taken together, our data implicated that NF45 was up-regulated in the sciatic nerve after crush, which was associated with proliferation of Schwann cell.

TNF-alpha signaling is not required for in vivo transcriptional reactivation of latent murine cytomegalovirus.

BACKGROUND: Reactivation of cytomegalovirus (CMV) is frequently observed in recipients of solid organs and bone marrow transplants and is associated with increased risk of acute and chronic allograft rejection, opportunistic infection, graft failure, and patient mortality. The molecular mechanisms by which reactivation occurs are not well understood. Previous studies have suggested that tumor necrosis factor (TNF)-alpha, which is induced by allogeneic transplantation, may have a role in reactivation of CMV through activation of nuclear factor kappa-light-chain-enhancer of activated B cells and subsequent transcriptional reactivation of immediate early (ie) gene expression. METHODS AND RESULTS: We have tested the role of TNF-alpha in the reactivation of CMV directly by testing whether TNF-alpha is required to initiate transcription of ie gene expression in a murine model of allogeneic transplantation of kidneys latently infected with mouse CMV. CONCLUSIONS: Our studies show that although TNF-alpha seems to be sufficient, it is not required for initiating transcription of ie gene expression in this model, suggesting that both TNF-alpha-dependent and -independent pathways play an important role in the reactivation of latent CMV infection.