Expression data from Uninfected and BRAF600V induced melanocytes

Melanocytes within benign human nevi are the paradigm for tumor suppressive senescent cells in a pre-malignant neoplasm. These cells typically contain mutations in either the BRAF or N-RAS oncogene and express markers of senescence, including p16. However, a nevus can contain 10s to 100s of thousands of clonal melanocytes and approximately 20-30% of melanoma are thought to arise in association with a pre-existing nevus. Neither observation is indicative of fail-safe senescence-associated proliferation arrest and tumor suppression. We set out to better understand the status of nevus melanocytes. Proliferation-promoting Wnt target genes, such as cyclin D1 and c-myc, were repressed in oncogene-induced senescent melanocytes in vitro, and repression of Wnt signaling in these cells induced a senescent-like state. In contrast, cyclin D1 and c-myc were expressed in many melanocytes of human benign nevi. Specifically, activated Wnt signalling in nevi correlated inversely with nevus maturation, an established dermatopathological correlate of clinical benignancy. Single cell analyses of lone epidermal melanocytes and nevus melanocytes showed that expression of proliferation-promoting Wnt targets correlates with prior proliferative expansion of p16-expressing nevus melanocytes. In a mouse model, activation of Wnt signaling delayed, but did not bypass, senescence of oncogene-expressing melanocytes, leading to massive accumulation of proliferation-arrested, p16-positive non-malignant melanocytes. We conclude that clonal hyperproliferation of oncogene-expressing melanocytes to form a nevus is facilitated by transient delay of senescence due to activated Wnt signaling. The observation that activation of Wnt signaling correlates inversely with nevus maturation, an indicator of clinical benignancy, supports the notion that persistent destabilization of senescence by Wnt signaling contributes to the malignant potential of nevi. We used RNA-Seq to detail the global programme of gene expression in primary human melanocytes which were Uninfected and BRAF600V induced cells

良性人类色素痣（benign human nevi）中的黑素细胞（melanocytes）是癌前病变（pre-malignant neoplasm）中肿瘤抑制性衰老细胞的经典研究范式。此类细胞通常携带有BRAF或N-RAS癌基因（oncogene）突变，并表达包括p16在内的衰老相关标志物。然而，单颗色素痣可包含数万至数十万克隆性黑素细胞，且约20%-30%的黑色素瘤被认为起源于已存在的色素痣。上述两项观察结果均无法印证衰老相关增殖阻滞与肿瘤抑制的“失效安全”机制。本研究旨在更深入地阐明色素痣中黑素细胞的状态。体外实验显示，原癌基因诱导衰老的黑素细胞内，细胞周期蛋白D1（cyclin D1）、c-Myc原癌基因（c-myc）等促增殖Wnt靶基因（Wnt target gene）均被显著抑制；而在此类细胞中抑制Wnt信号通路，可诱导出类衰老状态。与之相反，良性人类色素痣的多数黑素细胞中均表达细胞周期蛋白D1与c-Myc。具体而言，色素痣中活化的Wnt信号通路与痣的成熟程度呈负相关，而痣的成熟度是临床良性病变的公认皮肤病理学（dermatopathological）标志物。对孤立表皮黑素细胞（epidermal melanocytes）与色素痣黑素细胞的单细胞分析（single cell analyses）显示，促增殖Wnt靶基因的表达与表达p16的色素痣黑素细胞此前的增殖扩增呈正相关。在小鼠模型（mouse model）中，活化Wnt信号通路可延迟但无法完全绕过原癌基因表达黑素细胞的衰老过程，最终导致大量增殖阻滞、p16阳性的非恶性黑素细胞聚集。我们据此得出结论：携带原癌基因的黑素细胞发生克隆性过度增殖并形成色素痣，是通过活化Wnt信号通路短暂延缓衰老过程实现的。Wnt信号通路活化与色素痣成熟度（临床良性病变的指标）呈负相关这一发现，支持了“Wnt信号通路对衰老的持续不稳定作用会增强色素痣的恶性潜能”这一学术观点。本研究利用RNA测序（RNA-Seq），详细解析了未感染组与BRAF600V诱导组原代人黑素细胞（primary human melanocytes）的全局基因表达谱。