Search

Summary Although transgene-based reporter gene assays have been used to discover small molecules targeting expression of cancer-driving genes, the success is limited due to the fact that reporter gene expression regulated by incomplete cis -acting elements and foreign epigenetic environments does not faithfully reproduce chemical responses of endogenous genes. Here, we present an internal ribosome entry site-based strategy for bicistronically co-expressing reporter genes with an endogenous gene in the native gene locus, yielding an in situ reporter assay closely mimicking endogenous gene expression without disintegrating its function. This strategy combines the CRISPR-Cas9-mediated genome-editing tool with the recombinase-mediated cassette-exchange technology, and allows for rapid development of orthogonal assays for excluding false hits generated from primary screens. We validated this strategy by developing a screening platform for identifying compounds targeting oncogenic eIF4E, and demonstrated that the novel reporter assays are powerful in searching for transcription-targeted lead compounds with high confidence. [ABSTRACT FROM AUTHOR]

Simple Summary: Metabolic reprogramming has a central role in the initiation and progression of cancer, including high-risk neuroblastoma, a deadly childhood malignant tumor of the sympathetic nervous system. This rewiring of cellular metabolism increases the manufacture of fuel and building blocks for biomass production, which is essential to sustain the growth and proliferation of neuroblastoma cells. However, the rewiring also makes neuroblastoma cells metabolically distinct from normal sympathetic neurons, thereby offering new therapeutic opportunities. In this review, we summarize the recent progress in the study of neuroblastoma metabolic reprogramming and underlying molecular mechanisms. Neuroblastoma is a pediatric cancer responsible for approximately 15% of all childhood cancer deaths. Aberrant MYCN activation, as a result of genomic MYCN amplification, is a major driver of high-risk neuroblastoma, which has an overall survival rate of less than 50%, despite the best treatments currently available. Metabolic reprogramming is an integral part of the growth-promoting program driven by MYCN, which fuels cell growth and proliferation by increasing the uptake and catabolism of nutrients, biosynthesis of macromolecules, and production of energy. This reprogramming process also generates metabolic vulnerabilities that can be exploited for therapy. In this review, we present our current understanding of metabolic reprogramming in neuroblastoma, focusing on transcriptional regulation as a key mechanism in driving the reprogramming process. We also highlight some important areas that need to be explored for the successful development of metabolism-based therapy against high-risk neuroblastoma. [ABSTRACT FROM AUTHOR]

Assesses the effect of the high-mobility group protein 14 (HMG-14) on transcription by RNA polymerase II. Enhancement of transcription on chromatin templates; Stimulation of rate of elongation; Association of HMG-14 with nucleosomes.

The neural crest is a transient embryonic tissue that initially generates neural crest stem cells, which then migrate throughout the body to give rise to a variety of mature tissues. It was proposed that the fate of neural crest cells is gradually determined via environmental cues from the surrounding tissues. In the present study, neural crest cells were isolated and identified from mouse embryos. Bone morphogenetic protein 4 (BMP4) and Neuregulin (NRG) were employed to induce the differentiation of neural crest cells. Treatment with BMP4 revealed neuron-associated differentiation; cells treated with NRG exhibited differentiation into the Schwann cell lineage, a type of glia. Soft agar clonogenic and neurosphere formation assays were conducted to investigate the effects of N-Myc (MYCN) overexpression in neural crest cells; the number of colonies and neurospheres notably increased after 14 days. These findings demonstrated that the direction of cell differentiation may be affected by altering the factors present in the surrounding environment. In addition, MYCN may serve a key role in regulating neural crest cell differentiation. [ABSTRACT FROM AUTHOR]

Simple Summary: Breast cancer is the second most common cancer in women worldwide. Regulation of breast cancer metastasis remains an elusive phenomenon. Elucidating the mechanistic pathway of metastatic signaling may identify targets for regulating cancer metastatic potential. Raf-1 kinase inhibitor protein (RKIP) has been shown to negatively regulate signaling pathways involved in cancer progression and metastasis. RKIP may suppress metastasis of breast cancer cells by downregulating elements of the immune system. Raf-1 kinase inhibitor protein was first identified as a negative regulator of the Raf signaling pathway. Subsequently, it was shown to have a causal role in containing cancer progression and metastasis. Early studies suggested that RKIP blocks cancer progression by inhibiting the Raf-1 pathway. However, it is not clear if the RKIP tumor and metastasis suppression function involve other targets. In addition to the Raf signaling pathway, RKIP has been found to modulate several other signaling pathways, affecting diverse biological functions including immune response. Recent advances in medicine have identified both positive and negative roles of immune response in cancer initiation, progression and metastasis. It is possible that one way that RKIP exerts its effect on cancer is by targeting an immune response mechanism. Here, we provide evidence supporting the causal role of tumor and metastasis suppressor RKIP in downregulating signaling pathways involved with immune response in breast cancer cells and discuss its potential ramification on cancer therapy. [ABSTRACT FROM AUTHOR]

Dysregulation of autophagy in diabetic kidney disease (DKD) has been reported, but the underlying mechanism and its pathogenic role remain elusive. We show that autophagy was inhibited in DKD models and in human diabetic kidneys. Ablation of autophagy-related gene 7 (Atg7) from kidney proximal tubules led to autophagy deficiency and worse renal hypertrophy, tubular damage, inflammation, fibrosis, and albuminuria in diabetic mice, indicating a protective role of autophagy in DKD. Autophagy impairment in DKD was associated with the downregulation of unc-51-like autophagy-activating kinase 1 (ULK1), which was mediated by the upregulation of microRNA-214 (miR-214) in diabetic kidney cells and tissues. Ablation of miR-214 from kidney proximal tubules prevented a decrease in ULK1 expression and autophagy impairment in diabetic kidneys, resulting in less renal hypertrophy and albuminuria. Furthermore, blockade of p53 attenuated miR-214 induction in DKD, leading to higher levels of ULK1 and autophagy, accompanied by an amelioration of DKD. Compared with nondiabetic samples, renal biopsies from patients with diabetes showed induction of p53 and miR-214, associated with downregulation of ULK1 and autophagy. We found a positive correlation between p53/miR-214 and renal fibrosis, but a negative correlation between ULK1/LC3 and renal fibrosis in patients with diabetes. Together, these results identify the p53/miR-214/ULK1 axis in autophagy impairment in diabetic kidneys, pinpointing possible therapeutic targets for DKD. [ABSTRACT FROM AUTHOR]

Leflunomide as an immunosuppressive drug is generally used in the treatment of rheumatoid arthritis. It inhibits DHODH (dihydroorotate dehydrogenase ), which is one of the essential enzymes in the de novo pyrimidine biosynthetic pathway. Here we showed that leflunomide significantly reduced cell proliferation and self-renewal activity. Annexin V-FITC/PI staining assay revealed that leflunomide induced S-phase cell cycle arrest, and promoted cell apoptosis. In vivo xenograft study in SCID mice showed that leflunomide inhibited tumor growth and development. We also observed that DHODH was commonly expressed in neuroblastoma. When treated with leflunomide, the neuroblastoma cell lines BE(2)-C, SK-N-DZ, and SK-N-F1 showed dramatic inhibition of DHODH at mRNA and protein levels. Considering the favorable toxicity profile and the successful clinical experience with leflunomide in rheumatoid arthritis, this drug represents a potential new candidate for targeted therapy in neuroblastoma. [ABSTRACT FROM AUTHOR]

Abstract: Adeno-associated virus (AAV) is a nonpathogenic parvovirus that efficiently replicates in the presence of adenovirus (Ad). Exogenous expression of the AAV replication proteins induces caspase-dependent apoptosis, but determining if AAV infection causes apoptosis during viral infection is complicated by Ad-mediated programmed cell death. To eliminate Ad-induced cytolysis, we used an E3 adenoviral death protein (ADP) mutant, pm534. AAV and pm534-coinfected cells exhibited increased cell killing compared to pm534 alone. Relative to cells infected with Ad alone, AAV and wild-type Ad-infected cells displayed decreased ADP expression, increased cytolysis until the third day of the infection, and decreased cytolysis thereafter. Biochemical and morphological characteristics of apoptosis were observed during coinfections with AAV and pm534 or Ad, including a moderate degree of caspase activation that was not present during infections with pm534 or Ad alone. AAV coinfection also increased extracellular pH. These studies suggest that AAV induces caspase-dependent and caspase-independent apoptosis. [Copyright &y& Elsevier]

Nuclear factor-κB (NF-κB) promotes cell survival by upregulating expression of anti-apoptotic genes, a process that is antagonized by inhibitors of κB (IκB) factors. The only NF-κB family member known to be mutated in human cancer is NF-κB2 p100 (ref. 2), a factor with IκB activity. Here, we report the isolation from irradiated mouse tumour cells of a complex that induces caspase-8 activity in cell-free assays and identify p100 as an essential component of this complex. Expression of p100 profoundly sensitizes cells to death-receptor-mediated apoptosis through a pathway that is independent of IκB-Iike activity. The carboxyl terminus of p100 contains a death domain that is absent from all known tumour-derived mutants. This death domain mediates recruitment of p100 into death machinery complexes after ligand stimulation and is essential for p100's pro-apoptotic activity. p100 also sensitizes NIH3T3 cells to apoptosis triggered by oncogenic Ras, resulting in a marked inhibition of transformation that is rescued by suppression of endogenous caspase-8. These observations thus identify an IκBindependent apoptotic activity of NF-κB2 pl00 and help explain its unique tumour suppressor role. [ABSTRACT FROM AUTHOR]

Background: Abnormal NF-κB2 activation has been implicated in the pathogenesis of multiple myeloma, a cancer of plasma cells. However, a causal role for aberrant NF-κB2 signaling in the development of plasma cell tumors has not been established. Also unclear is the molecular mechanism that drives the tumorigenic process. We investigated these questions by using a transgenic mouse model with lymphocyte-targeted expression of p80HT, a lymphoma-associated NF-κB2 mutant, and human multiple myeloma cell lines.Methods: We conducted a detailed histopathological characterization of lymphomas developed in p80HT transgenic mice and microarray gene expression profiling of p80HT B cells with the goal of identifying genes that drive plasma cell tumor development. We further verified the significance of our findings in human multiple myeloma cell lines.Results: Approximately 40% of p80HT mice showed elevated levels of monoclonal immunoglobulin (M-protein) in the serum and developed plasma cell tumors. Some of these mice displayed key features of human multiple myeloma with accumulation of plasma cells in the bone marrow, osteolytic bone lesions and/or diffuse osteoporosis. Gene expression profiling of B cells from M-protein-positive p80HT mice revealed aberrant expression of genes known to be important in the pathogenesis of multiple myeloma, including cyclin D1, cyclin D2, Blimp1, survivin, IL-10 and IL-15. In vitro assays demonstrated a critical role of Stat3, a key downstream component of IL-10 signaling, in the survival of human multiple myeloma cells.Conclusions: These findings provide a mouse model for human multiple myeloma with aberrant NF-κB2 activation and suggest a molecular mechanism for NF-κB2 signaling in the pathogenesis of plasma cell tumors by coordinated regulation of plasma cell generation, proliferation and survival. [ABSTRACT FROM AUTHOR]