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DrugMap: A quantitative pan-cancer analysis of cysteine ligandability (RNA-Seq)

ABSTRACT: SOX10 is a lineage-restricted transcription factor important for melanoma proliferation and survival. Our data demonstrate that SOX10 genetic depletion by CRISPR/Cas9 significantly impaired melanoma proliferation. We chose 7 different melanoma, all of which exhibited growth dependency on SOX10 with different levels. To define the SOX10 transcriptional signature shared with different SOX10-dependent melanoma, we performed bulk RNA-seq on 7 melanoma transfected with siSOX10 or siControl. Each melanoma cell line was plated and transfected with each siRNA to deplete SOX10 protein expression. After 72 hrs, cells were lysed with Buffer TCL(QIAGEN) supplemented with 2-ME and stored at -80C until use. Libraries from each cell line were made with the modified Smart-seq2 protocol. Each replicate was uniquely barcoded and all the samples were pooled for sequencing. (2) We developed a chemical probe (SH-0029) that covalently engages SOX10. To examine the effect of SH-0029 on SOX10 target genes, SOX10-dependent SKMEL5 melanoma were plated and treated with SH-0029, SH-0105 (a control probe), or DMSO(vehicle) for 48h. Total RNA was extracted with NucleoSpin RNA extraction kit (MACHEREY-NAGEL) and mRNA was subsequently purified with poly dT-attached magnetic beads. We found that downregulated genes induced by SH-0029 treatment strongly overlapped with those by SOX10 genetic depletion, suggesting that SOX10 covalent ligand controls SOX10 transcriptional network.

ORGANISM(S): Homo sapiens

PROVIDER: GSE256466 | GEO | 2024/04/24

REPOSITORIES: GEO

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Project description:It is believed that the inherent differentiation program of melanocytes during embryogenesis predisposes melanoma cells to high frequency of metastasis. Sox10, a transcription factor expressed in neural crest stem cells and a subset of progeny lineages, plays a key role in the development of melanocytes. We show that B16F10 melanoma cells transfected with siRNA specific for Sox10 display reduced migratory activity which in turn indicated that a subset of transcriptional regulatory target genes of Sox10 are likely to be involved in migration and metastasis of melanoma cells. We carried out microarray-based gene expression profiling using Sox10-specific siRNA to identify regulatory targets and found that multiple genes including melanocortin-1 receptor (Mc1R) partake in the regulation of migration. We provide evidences that a significant portion of the effect of Sox10 on migration is mediated by Mitf, a transcription factor downstream to Sox10. The involvement of Mc1R in migration was studied in detail in vivo using a murine metastasis model. Specifically, B16F10 melanoma cells treated with a specific siRNA showed reduced tendency in metastasizing to and colonizing the lung after being injected in the tail vein. These data reveal a cadre of novel regulators and mediators involved in migration and metastasis of melanoma cells that represent potential targets of therapeutic intervention. Chemically synthesized siRNA duplex (WT1-Sox10) was used to knock-down the transcription factor Sox10 in murine melanoma cell line B16F10. For the control, siRNA containing 5 nucleotide alterations (MT1-Sox10) were used. Total RNA was subsequently prepared to synthesize probes for microarray screening. A total of three pairs of replicate samples were generated each from separate transfection followed by RNA preparation. Expression values were determined and compared within each pair of WT1-Sox10 and MT1-Sox10 transfections. Genes showing over 2 fold changes in all three replicate pairs were subjected to subsequent analyses.

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DrugMap: A quantitative pan-cancer analysis of cysteine ligandability (RNA-Seq)

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