Epigenetic control of melanoma cell invasiveness by the stem cell factor SALL4

Melanoma cells rely on developmental programs during tumor initiation and progression. Here we show that the embryonic stem cell (ESC) factor Sall4 is re-expressed in the Tyr::Nras$^{Q61K}$; Cdkn2a$^{−/−}$ melanoma model and that its expression is necessary for primary melanoma formation. Surprisingly, while Sall4 loss prevents tumor formation, it promotes micrometastases to distant organs in this melanoma-prone mouse model. Transcriptional profiling and in vitro assays using human melanoma cells demonstrate that SALL4 loss induces a phenotype switch and the acquisition of an invasive phenotype. We show that SALL4 negatively regulates invasiveness through interaction with the histone deacetylase (HDAC) 2 and direct co-binding to a set of invasiveness genes. Consequently, SALL4 knock down, as well as HDAC inhibition, promote the expression of an invasive signature, while inhibition of histone acetylation partially reverts the invasiveness program induced by SALL4 loss. Thus, SALL4 appears to regulate phenotype switching in melanoma through an HDAC2-mediated mechanism

Yin Yang 1 sustains biosynthetic demands during brain development in a stage-specific manner

The transcription factor Yin Yang 1 (YY1) plays an important role in human disease. It is often overexpressed in cancers and mutations can lead to a congenital haploinsufficiency syndrome characterized by craniofacial dysmorphisms and neurological dysfunctions, consistent with a role in brain development. Here, we show that Yy1 controls murine cerebral cortex development in a stage-dependent manner. By regulating a wide range of metabolic pathways and protein translation, Yy1 maintains proliferation and survival of neural progenitor cells (NPCs) at early stages of brain development. Despite its constitutive expression, however, the dependence on Yy1 declines over the course of corticogenesis. This is associated with decreasing importance of processes controlled by Yy1 during development, as reflected by diminished protein synthesis rates at later developmental stages. Thus, our study unravels a novel role for Yy1 as a stage-dependent regulator of brain development and shows that biosynthetic demands of NPCs dynamically change throughout development

Yin Yang 1 Orchestrates a Metabolic Program Required for Both Neural Crest Development and Melanoma Formation

Increasing evidence suggests that cancer cells highjack developmental programs for disease initiation and progression. Melanoma arises from melanocytes that originate during development from neural crest stem cells (NCSCs). Here, we identified the transcription factor Yin Yang 1 (Yy1) as an NCSCs regulator. Conditional deletion of Yy1 in NCSCs resulted in stage-dependent hypoplasia of all major neural crest derivatives due to decreased proliferation and increased cell death. Moreover, conditional ablation of one Yy1 allele in a melanoma mouse model prevented tumorigenesis, indicating a particular susceptibility of melanoma cells to reduced Yy1 levels. Combined RNA sequencing (RNA-seq), chromatin immunoprecipitation (ChIP)-seq, and untargeted metabolomics demonstrated that YY1 governs multiple metabolic pathways and protein synthesis in both NCSCs and melanoma. In addition to directly regulating a metabolic gene set, YY1 can act upstream of MITF/c-MYC as part of a gene regulatory network controlling metabolism. Thus, both NCSC development and melanoma formation depend on an intricate YY1-controlled metabolic program

Antagonistic cross-regulation between Sox9 and Sox10 controls an anti-tumorigenic program in melanoma

Melanoma is the most fatal skin cancer, but the etiology of this devastating disease is still poorly understood. Recently, the transcription factor Sox10 has been shown to promote both melanoma initiation and progression. Reducing SOX10 expression levels in human melanoma cells and in a genetic melanoma mouse model, efficiently abolishes tumorigenesis by inducing cell cycle exit and apoptosis. Here, we show that this anti-tumorigenic effect functionally involves SOX9, a factor related to SOX10 and upregulated in melanoma cells upon loss of SOX10. Unlike SOX10, SOX9 is not required for normal melanocyte stem cell function, the formation of hyperplastic lesions, and melanoma initiation. To the contrary, SOX9 overexpression results in cell cycle arrest, apoptosis, and a gene expression profile shared by melanoma cells with reduced SOX10 expression. Moreover, SOX9 binds to the SOX10 promoter and induces downregulation of SOX10 expression, revealing a feedback loop reinforcing the SOX10 low/SOX9 high ant,m/ii-tumorigenic program. Finally, SOX9 is required in vitro and in vivo for the anti-tumorigenic effect achieved by reducing SOX10 expression. Thus, SOX10 and SOX9 are functionally antagonistic regulators of melanoma development

Experimental suppression of SOX9 expression rescues the effects of SOX10 deregulation in human melanoma cells.

<p><b>A</b>, SOX9 overexpression in human melanoma cells closely resembles the gene expression signature of SOX10 knockdown as revealed by unsupervised hierarchical clustering of control M010817 melanoma cells, SOX9 overexpressing M010817 cells and SOX10 knock down M010817 cells. Microarray gene expression accession number: GSE37059. <b>B</b>, Western blot analysis showing that SOX10 expression is downregulated upon overexpression of SOX9 in two independent human melanoma cell lines (A375 and M010817). <b>C</b>, Chromatin immunoprecipitation assay demonstrating the binding of SOX9 to the promoter of SOX10 in human melanoma M010817 cells. <b>D, E</b>, Quantitative real-time PCR analysis of SOX10 (<b>E</b>) and SOX9 (<b>F</b>) expression after the knockdown of SOX10 and after the double knockdown of SOX10 and SOX9 in M010817 cell line. <b>F</b>, Quantification of number of Annexin V-positive cells based on the FACS analysis in the melanoma M010817 cells upon SOX9 KD, SOX10 KD or double SOX9/SOX10 KD. OE, overexpression; KD, knock down; ChIP, chromatin immunoprecipitation; prom, promoter.</p

Mouse giant congenital naevi and melanoma reveal no expression of Sox9.

<p><b>A-D</b>, Immunostaining for Sox9 (<b>A, C</b>) and Sox10 (<b>B, D</b>) in the skin sections of <i>Tyr::Nras^Q61K</i> and <i>Tyr::Nras^Q61KINK4a^−/−</i> mice. <b>E-H</b>, Experimental strategy used to abrogate the expression of Sox9 (E) and Sox10 (G) in the mouse melanocytic lineage. Pictures of two representative mice 1 year after tamoxifen injections reveal no reduction in the skin hyperpigmentation in <i>Tyr::Nras^Q61KSox9^fl/+Tyr-CreERT2</i> mice as compared to their <i>Tyr::Nras^Q61K</i> littermates (F) in contrast to a pronounced skin whitening observed upon Sox10 loss (H). BF, bright field; HF, hair follicle; mo, months; P0, postnatal day 0. Scale bars, 25 μm.</p

SOX10 knockdown results in elevated SOX9 expression in mouse and human melanocytes.

<p><b>A</b>, Experimental design used to investigate the level of SOX9 and SOX10 expression <i>in vitro</i>. Cultured human keratinocytes, melanocytes, cells derived from biopsies of patients with giant congential naevi and melanoma cells (M010817 cell line) were subjected to RNA isolation and subsequent Q-RT-PCR analysis. Keratinocytes were used as a control. <b>B, C</b>, Quantitative real-time PCR analysis showing the decline of SOX9 expression (<b>C</b>) and increase of SOX10 expression (<b>B</b>) that correlate with the acquisition of malignant state by human NRAS^Q61K-mutated cells. Data are presented as the mean fold change and are normalized over levels found in melanocytes. <b>D, E</b>, SOX10 and SOX9 expression in a large set of proliferative and invasive cell lines analysed by gene expression using microarrays (<b>D</b>) and Western blot (<b>E</b>) techniques. <b>F</b>, Experimental design used to deregulate SOX10 expression in human cells derived from the biopsy of a patient with NRAS^Q61K-mutated giant congenital naevus. <b>G, H</b>, Quantitative real-time PCR analysis of SOX10 (<b>G</b>) and SOX9 (<b>H</b>) expression after the knockdown of SOX10. <b>I</b>, Experimental design used to analyze the expression of Sox9 in the melanocytic lineage from <i>Tyr::Nras^Q61K</i> and <i>Tyr::Nras^Q61K</i> <i>Sox10^LacZ/+</i> mice. <b>K, L</b>, Cells were isolated from the trunk skin of <i>Tyr::Nras^Q61K</i> and <i>Tyr::Nras^Q61K</i> <i>Sox10^LacZ/+</i> mice and stained for Melan-a and c-Kit antibodies. FACS-sorted cells were subsequently used for the RNA isolation and quantitative real-time PCR with primers specific for the coding regions of <i>Sox9</i> gene. Data are presented as the mean fold change and are normalized to the control. Kerat, keratinocytes; M, melanocytes; Nev, naevus cells; Mel, melanoma cells; KD, knock down.</p

Differential expression of SOX10 and SOX9 in human melanocytes, human giant congenital naevi and human melanoma samples.

<p><b>A</b>, Scheme showing the localization of epidermal melanocytes (in red) in the human skin. <b>B, C</b>, Immunostaining for MITF (green, left panel) and SOX9 (red, right panel) in the human skin demonstrating the lack of SOX9 expression in the epidermal melanocytes. Inserts show higher magnification images of MITF and SOX9 immunostainings. Scale bars, 25 μm. <b>D</b>, Scheme showing the localization of melanocytes (in red) within the hair follicle. <b>E</b>, Immunostaining for MITF (green) and SOX9 (red) in the human skin reveals the expression of SOX9 in the cells of outer root sheath but not in the MITF-positive melanoblasts/melanocytes. Scale bar 100 μm. <b>F, G</b>, High magnification images of immunostaining for MITF and SOX9 in the upper part of human hair follicle (<b>F</b>) and the follicular bulb (<b>G</b>). <b>H</b>, Analysis of SOX9 (red, left panel) and SOX10 (red, right panel) expression in the patients with human giant congenital naevi demonstrates the lack of SOX9 expression in the SOX10-positive giant congenital naevi cells. Inserts show higher magnification. <b>I</b>, Representative examples of immunostaining for SOX9 (green) and SOX10 (red) in a tissue microarray of primary melanoma samples are shown. <b>J-K</b>, Distribution of SOX10 vs. SOX9 expression in human melanoma (based on TCGA database). 334 melanoma patients were divided in two groups, namely SOX10 High/ SOX9 Low and SOX10 Low / SOX9 high based on SOX10 and SOX9 expression levels. DP, dermal papilla; HF, hair follicle; M, melanocytes; ORS, outer root sheath. Scale bars, 25 μm.</p