Differential Inhibition of Sox10 Functions by Notch-Hes Pathway.

In the developing central nervous system, the terminal differentiation of oligodendrocytes (OLs) is regulated by both extrinsic and intrinsic factors. Recent studies have suggested that the Notch-Hes signaling pathway influences the maturation of oligodendrocytes in culture and during development. However, the specific Notch receptors and their downstream effectors Hes genes that are involved in oligodendrocyte maturation have not been investigated systematically. In this study, we showed that Notch1 and Notch3 are expressed in oligodendrocyte precursor cells (OPCs) during gliogenesis, and Hes5 is the major Notch downstream transcription factor that is transiently expressed in OPCs. Overexpression of Notch intracellular domain (NICD) and Hes5 proteins in embryonic chicken spinal cord suppressed both the endogenous and Sox10-induced Mbp gene expression. Unexpectedly, overexpression of NICD/Hes5 did not inhibit Sox10 induction of Olig2 expression and Myrf induced Mbp expression, suggesting the differential inhibitory effects of NICD/Hes5 signaling on Sox10 activation of myelin-related genes and early progenitor genes.

MEK inhibition remodels the active chromatin landscape and induces SOX10 genomic recruitment in BRAF(V600E) mutant melanoma cells.

BACKGROUND: The MAPK/ERK signaling pathway is an essential regulator of numerous cell processes that are crucial for normal development as well as cancer progression. While much is known regarding MAPK/ERK signal conveyance from the cell membrane to the nucleus, the transcriptional and epigenetic mechanisms that govern gene expression downstream of MAPK signaling are not fully elucidated. RESULTS: This study employed an integrated epigenome analysis approach to interrogate the effects of MAPK/ERK pathway inhibition on the global transcriptome, the active chromatin landscape, and protein-DNA interactions in 501mel melanoma cells. Treatment of these cells with the small-molecule MEK inhibitor AZD6244 induces hyperpigmentation, widespread gene expression changes including alteration of genes linked to pigmentation, and extensive epigenomic reprogramming of transcriptionally distinct regulatory regions associated with the active chromatin mark H3K27ac. Regulatory regions with differentially acetylated H3K27ac regions following AZD6244 treatment are enriched in transcription factor binding motifs of ETV/ETS and ATF family members as well as the lineage-determining factors MITF and SOX10. H3K27ac-dense enhancer clusters known as super-enhancers show similar transcription factor motif enrichment, and furthermore, these super-enhancers are associated with genes encoding MITF, SOX10, and ETV/ETS proteins. Along with genome-wide resetting of the active enhancer landscape, MEK inhibition also results in widespread SOX10 recruitment throughout the genome, including increased SOX10 binding density at H3K27ac-marked enhancers. Importantly, these MEK inhibitor-responsive enhancers marked by H3K27ac and occupied by SOX10 are located near melanocyte lineage-specific and pigmentation genes and overlap numerous human SNPs associated with pigmentation and melanoma phenotypes, highlighting the variants located within these regions for prioritization in future studies. CONCLUSIONS: These results reveal the epigenetic reprogramming underlying the re-activation of melanocyte pigmentation and developmental transcriptional programs in 501mel cells in response to MEK inhibition and suggest extensive involvement of a MEK-SOX10 axis in the regulation of these processes. The dynamic chromatin changes identified here provide a rich genomic resource for further analyses of the molecular mechanisms governing the MAPK pathway in pigmentation- and melanocyte-associated diseases.

Neuropilin-1 upregulation elicits adaptive resistance to oncogene-targeted therapies.

Cancer cell dependence on activated oncogenes is therapeutically targeted, but acquired resistance is virtually unavoidable. Here we show that the treatment of addicted melanoma cells with BRAF inhibitors, and of breast cancer cells with HER2-targeted drugs, led to an adaptive rise in neuropilin-1 (NRP1) expression, which is crucial for the onset of acquired resistance to therapy. Moreover, NRP1 levels dictated the efficacy of MET oncogene inhibitors in addicted stomach and lung carcinoma cells. Mechanistically, NRP1 induced a JNK-dependent signaling cascade leading to the upregulation of alternative effector kinases EGFR or IGF1R, which in turn sustained cancer cell growth and mediated acquired resistance to BRAF, HER2, or MET inhibitors. Notably, the combination with NRP1-interfering molecules improved the efficacy of oncogene-targeted drugs and prevented or even reversed the onset of resistance in cancer cells and tumor models. Our study provides the rationale for targeting the NRP1-dependent upregulation of tyrosine kinases, which are responsible for loss of responsiveness to oncogene-targeted therapies.

Long Non-Coding RNA H19 Protects H9c2 Cells against Hypoxia-Induced Injury by Targeting MicroRNA-139.

BACKGROUND/AIMS: Acute myocardial infarction (AMI) occurs when blood supply to the heart is diminished (ischemia) for long time; ischemia is primarily caused due to hypoxia. The present study evaluated the effects of long non-coding RNA H19 on hypoxic rat H9c2 cells and mouse HL-1 cells. METHODS: Hypoxic injury was confirmed by measuring cell viability, migration and invasion, and apoptosis using MTT, Transwell and flow cytometry assays, respectively. H19 expression after hypoxia was estimated by qRT-PCR. We then measured the effects of non-physiologically expressed H19, knockdown of miR-139 with or without H19 silence, and abnormally expressed Sox8 on hypoxia-induced H9c2 cells. Moreover, the interacted miRNA for H19 and downstream target gene were virtually screened and verified. The involved signaling pathways and the effects of abnormally expressed H19 on contractility of HL-1 cells were explored via Western blot analysis. RESULTS: Hypoxia induced decreases of cell viability, migration and invasion, increase of cell apoptosis and up-regulation of H19. Knockdown of H19 increased hypoxia-induced injury in H9c2 cells. H19 acted as a sponge for miR-139 and H19 knockdown aggravated hypoxia-induced injury by up-regulating miR-139. Sox8 was identified as a target of miR-139, and its expression was negatively regulated by miR-139. The mechanistic studies revealed that overexpression of Sox8 might decrease hypoxia-induced cell injury by activating the PI3K/AKT/mTOR pathway and MAPK. Besides, H19 promoted contractility of HL-1 cells. CONCLUSION: These findings suggest that H19 alleviates hypoxia-induced myocardial cell injury by miR-139-mediated up-regulation of Sox8, along with activation of the PI3K/AKT/mTOR pathway and MAPK.

RNAi silencing of the SoxE gene suppresses cell proliferation in silkworm BmN4 cells.

The transcription factor SoxE is mainly expressed in the gonad and involved in the regulation of gonad development and sex determination in animals. Here, we used the silkworm ovary-derived BmN4-SID1 cell line to survey the roles of the silkworm SoxE protein (BmSoxE) and predict its candidate binding targets. RNAi-mediated silencing of BmSoxE expression suppressed cell proliferation in BmN4-SID1 cells. A further cell cycle analysis revealed that this inhibition of cell proliferation was largely due to cell cycle arrest in G1 phase when BmSoxE expression was blocked in BmN4-SID1 cells. Genome-wide microarray expression analyses demonstrated that the expression levels of a set of genes were significantly altered following BmSoxE RNAi. More than half of these genes contained conserved binding sites for HMG box domain of the Sox proteins and were predicted to be candidate binding targets for BmSoxE. Importantly, some of the candidate targets may be associated with the effect of BmSoxE on cell proliferation. Several candidate target genes showed gonad-specific expression in silkworm larvae. Taken together, these data demonstrate that BmSoxE is required for cell proliferation in silkworm BmN4-SID1 cells and provide valuable information for further investigations of the molecular control exerted by the BmSoxE protein over cell proliferation and gonad development in the silkworm.

SOX10 promotes melanoma cell invasion by regulating melanoma inhibitory activity.

The transcription factor SOX10 (SRY (sex determining region Y)-box 10) has a key role in the embryonic development of melanocytes. Recently, it has been suggested that SOX10 is highly relevant for melanoma development and survival. However, the distinct functions and downstream targets of SOX10 in melanoma remain widely unknown. In this study, we inhibited SOX10 via RNA interference in different human melanoma cell lines and found a significantly reduced invasion capacity in vitro and in the chick embryo model. At later time points, SOX10 inhibition reduced proliferation and induced cell death. We identified melanoma inhibitory activity (MIA) as a direct target gene of SOX10, which is an essential protein for melanoma cell migration and invasion. Expression levels of SOX10 and MIA strictly correlated in melanoma cell lines, and SOX10 inhibition reduced MIA expression and promoter activity. Direct binding of SOX10 to the MIA promoter was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation. Ectopic expression of MIA in SOX10-inhibited melanoma cells restored the invasion capacity, supporting the hypothesis that MIA is responsible for SOX10-mediated melanoma cell invasion. Our data provide evidence for a critical role of SOX10 in melanoma cell invasion through the regulation of MIA and highlight its role as a therapeutic target in melanoma.

Transforming growth factor ß-mediated Sox10 suppression controls mesenchymal progenitor generation in neural crest stem cells.

During vertebrate development, neural crest stem cells (NCSCs) give rise to neural cells of the peripheral nervous system and to a variety of mesenchymal cell types, including smooth muscle, craniofacial chondrocytes, and osteocytes. Consistently, mesenchymal stem cells (MSCs) have recently been shown to derive in part from the neural crest (NC), although the mechanisms underlying MSC generation remains to be identified. Here, we show that transforming growth factor ß (TGFß)-mediated suppression of the NCSC transcription factor Sox10 induces a switch in neural to mesenchymal potential in NCSCs. In vitro and in vivo, TGFß signal inactivation results in persistent Sox10 expression, decreased cell cycle exit, and perturbed generation of mesenchymal derivatives, which eventually leads to defective morphogenesis. In contrast, TGFß-mediated downregulation of Sox10 or its genetic inactivation suppresses neural potential, confers mesenchymal potential to NC cells in vitro, and promotes cell cycle exit and precocious mesenchymal differentiation in vivo. Thus, negative regulation of Sox10 by TGFß signaling promotes the generation of mesenchymal progenitors from NCSCs. Our study might lay the grounds for future applications demanding defined populations of MSCs for regenerative medicine.