Gene expression profiling of enforced HOXA1 expression in melanoma cell line (SkMel30)

We recently reported an oncogenomics-guided screening approach designed to identify genetic drivers of early stage melanoma metastasis, and in this study we functionally validate the top-scoring candidate, homeobox transcription factor A1 (HOXA1), by demonstrating HOXA1 robust effects on melanoma cell invasion, metastasis and tumorigenicity. Transcriptome and pathway profiling analyses of cells expressing HOXA1 reveal up-regulation of factors involved in diverse cytokine pathways that include the TGF-beta signaling axis, which we further demonstrate to be required for HOXA1-mediated cell invasion. Transcriptome profiling also informed HOXA1 ability to potently down-regulate expression of microphthalmia-associated transcription factor (MITF) and other genes required for melanocyte differentiation, suggesting a mechanism by which HOXA1 expression de-differentiates cells into a pro-invasive precursor cell state concomitant with TGF-beta activation. Our analysis of publicly available datasets indicate that the HOXA1-induced gene signature successfully categorizes melanoma specimens based on their metastatic potential and, importantly, is capable of stratifying melanoma patient risk for metastasis based on expression in primary tumors. The HOXA1-induced transcription analysis was conducted using RNAs extracted from SkMel30 cells transduced with either control or HOXA1, followed by hybridization of labeled cDNA onto Affymetrix GeneChips (Human Genome U133Plus2.0).

本团队此前报道了一种以肿瘤基因组学为指导的筛选方法，用于识别早期黑色素瘤转移的遗传驱动因素；本研究则对得分最高的候选靶点——同源框转录因子A1（homeobox transcription factor A1, HOXA1）进行了功能验证，通过实验证实HOXA1对黑色素瘤细胞的侵袭、转移及致瘤性具有显著调控作用。对表达HOXA1的细胞开展转录组及通路谱分析后发现，包括转化生长因子-β（transforming growth factor-beta, TGF-beta）信号轴在内的多种细胞因子通路相关因子均出现上调；本研究进一步证实，该信号轴是HOXA1介导的细胞侵袭所必需的通路。转录组谱分析同时表明，HOXA1可有效下调小眼畸形相关转录因子（microphthalmia-associated transcription factor, MITF）及黑色素细胞分化所需的其他基因的表达，这提示HOXA1的表达可通过伴随TGF-beta激活的过程，将细胞去分化为促侵袭前体细胞状态。对公共可用数据集的分析结果显示，HOXA1诱导的基因特征可根据黑色素瘤样本的转移潜能进行有效分类；更为重要的是，该特征还可通过原发肿瘤的基因表达水平，对黑色素瘤患者的转移风险进行分层。本次HOXA1诱导的转录组分析，以经对照载体或HOXA1载体转导的SkMel30细胞提取的RNA为实验材料，将标记后的cDNA与Affymetrix基因芯片（Human Genome U133Plus2.0）进行杂交完成。