Inhibitor of DNA Binding 4 (ID4) Is Highly Expressed in Human Melanoma Tissues and May Function to Restrict Normal Differentiation of Melanoma Cells

Melanoma tissues and cell lines are heterogeneous, and include cells with invasive, proliferative, stem cell-like, and differentiated properties. Such heterogeneity likely contributes to the aggressiveness of the disease and resistance to therapy. One model suggests that heterogeneity arises from rare cancer stem cells (CSCs) that produce distinct cancer cell lineages. Another model suggests that heterogeneity arises through reversible cellular plasticity, or phenotype-switching. Recent work indicates that phenotype-switching may include the ability of cancer cells to dedifferentiate to a stem cell-like state. We set out to investigate the phenotype-switching capabilities of melanoma cells, and used unbiased methods to identify genes that may control such switching. We developed a system to reversibly synchronize melanoma cells between 2D-monolayer and 3D-stem cell-like growth states. Melanoma cells maintained in the stem cell-like state showed a striking upregulation of a gene set related to development and neural stem cell biology, which included SRY-box 2 (SOX2) and Inhibitor of DNA Binding 4 (ID4). A gene set related to cancer cell motility and invasiveness was concomitantly downregulated. Intense and pervasive ID4 protein expression was detected in human melanoma tissue samples, suggesting disease relevance for this protein. SiRNA knockdown of ID4 inhibited switching from monolayer to 3D-stem cell-like growth, and instead promoted switching to a highly differentiated, neuronal-like morphology. We suggest that ID4 is upregulated in melanoma as part of a stem cell-like program that facilitates further adaptive plasticity. ID4 may contribute to disease by preventing stem cell-like melanoma cells from progressing to a normal differentiated state. This interpretation is guided by the known role of ID4 as a differentiation inhibitor during normal development. The melanoma stem cell-like state may be protected by factors such as ID4, thereby potentially identifying a new therapeutic vulnerability to drive differentiation to the normal cell phenotype.

黑色素瘤组织与细胞系具有异质性，包含具有侵袭性、增殖性、干细胞样及分化特性的细胞群。这种异质性可能是导致疾病恶性进展及治疗耐受的关键诱因。目前存在两种异质性产生的模型假说：其一认为异质性源于罕见的癌症干细胞（CSCs），这类细胞可分化为不同的癌细胞谱系；其二则认为异质性通过可逆的细胞可塑性即表型转换产生。近期研究表明，表型转换还涵盖癌细胞去分化至干细胞样状态的能力。本研究旨在探究黑色素瘤细胞的表型转换能力，并通过无偏倚方法筛选可能调控该转换的基因。我们构建了一套可逆向同步黑色素瘤细胞于二维单层培养与三维类干细胞生长状态之间的实验体系。维持于干细胞样状态的黑色素瘤细胞，其与发育及神经干细胞生物学相关的基因集出现显著上调，其中包括SRY框转录因子2（SOX2）与DNA结合抑制因子4（ID4）；与此同时，与癌细胞运动及侵袭性相关的基因集则出现下调。在人类黑色素瘤组织样本中检测到ID4蛋白的广泛强表达，提示该蛋白与疾病发生具有相关性。小干扰RNA（siRNA）介导的ID4敲低可抑制细胞从单层培养向三维类干细胞生长状态的转换，反而促进细胞向高度分化的神经元样形态转换。我们认为，ID4在黑色素瘤中上调是类干细胞程序的一部分，该程序可促进进一步的适应性可塑性。ID4可能通过阻止干细胞样黑色素瘤细胞向正常分化状态进展，从而参与疾病进程——这一解读基于ID4在正常发育过程中作为分化抑制因子的已知功能。黑色素瘤干细胞样状态可被ID4等因子维持，这或许为靶向治疗提供了新的脆弱位点，可诱导肿瘤细胞分化为正常细胞表型。