Apoptosis, autophagy and senescence protect BRAFV600E nevi from malignant transformation

The oncogenic mutation BRAFV600E is a common event in nevi and melanomas, which are aggressive skin tumors characterized by MAPK signaling pathway activation. It has been observed that mutated benign melanocytic lesions can remain unchanged for decades, but also proliferate or undergo oncogene-induced senescence. The purpose of this study was to investigate the presence of a gene expression signature in BRAFV600E mutated nevi compared to wild-type ones, all derived from sun exposed sites. Microdissected tissues from excisional biopsies of acquired nevi were analyzed to detect the presence of BRAF and NRAS mutations and to profile whole genome expression by means of oligonucleotide microarrays. BRAFV600E mutation was evidenced in 64% of nevi, while no NRAS mutations were detected. Functional analysis of genes differentially expressed between wild-type and mutated lesions pointed out the role of oxidative stress in causing the oncogenic BRAF mutation and that the direct consequence of constitutive activation of BRAF-MEK-ERK pathway, in a benign contest, is the activation of apoptosis, autophagy and senescence. Our data also indicate that in mutated lesions, p53 plays a crucial role in the maintenance of the benign status. In our study, in order to understand the role of BRAF mutation in acquired nevi, we performed gene expression profiling analysis on fourteen lesions derived from intermittently exposed sites, using whole genome oligonucleotide microarrays. Expression data were coupled with sequencing results on BRAF and NRAS genes and class comparison algorithms were applied to detect sequences differentially expressed between BRAFV600E and BRAFwt-NRASwt lesions. Some of the obtained results were further validated by Real-Time RT-PCR on an independent cohort of samples. Mutation analysis evidenced BRAF mutations in 64% of the common acquired nevi. All the mutations consisted in the substitution V600E (T1799A). No alteration was found on exon 11 of the BRAF gene nor on exons 1 and 2 of the NRAS gene, as expected since no lesion came from chronically exposed sites. Transcriptome analysis was carried out using oligo-microarrays and differential expression was tested applying the Linear Models package LIMMA available at www.bioconductor.org that uses Bayesian statistics to compute the probability of a gene being differentially expressed. Only 25 transcripts had B value greater then 2. We then performed a one-way ANOVA, dividing samples into three classes according to their BRAF mutational status (wt, ++, +++) as assessed by pyrosequencing. We found 2882 known transcripts by selecting those with ANOVA p-value<0.01 and increasing or decreasing expression trends along the three classes. Functional analysis was performed using the MetaCoreTM software package to identify overrepresented functional processes. BRAF signature, acquired nevi, gene expression profiling "++ and +++" refer to the percentage of alleles carrying the mutation in the *BRAF*V600E lesions. This has been assessed by pyrosequencing analysis (Venesio et al, 2008. PMID= 18408659).

致癌突变BRAFV600E（BRAFV600E）在黑素细胞痣与黑色素瘤中均较为常见，其中黑色素瘤是一类以丝裂原活化蛋白激酶（MAPK, Mitogen-Activated Protein Kinase）信号通路激活为特征的侵袭性皮肤肿瘤。已有研究表明，携带突变的良性黑素细胞病变可数十年保持稳定，亦可发生增殖或出现癌基因诱导的衰老（oncogene-induced senescence）。
本研究旨在探究源自日光暴露部位的BRAFV600E突变型黑素细胞痣与野生型痣之间的基因表达特征差异。研究人员对获得性黑素细胞痣切除活检样本的显微切割组织进行分析，以检测BRAF与NRAS基因突变情况，并通过寡核苷酸微阵列（oligonucleotide microarray）完成全基因组表达谱分析。
64%的黑素细胞痣样本检出BRAFV600E突变，未检测到NRAS基因突变。对野生型与突变型病变间差异表达基因的功能富集分析显示，氧化应激可诱发致癌性BRAF突变；而在良性病变环境中，BRAF-MEK-ERK通路持续激活的直接效应为激活细胞凋亡、自噬与衰老过程。本研究数据同时表明，在突变型病变中，p53蛋白对维持良性表型发挥关键作用。
为明确BRAF突变在获得性黑素细胞痣中的作用，本研究采用全基因组寡核苷酸微阵列，对14例源自间歇性日光暴露部位的病变样本开展基因表达谱分析。研究将基因表达数据与BRAF、NRAS基因的测序结果相结合，并通过类别比较算法，检测BRAFV600E突变型与BRAF野生型-NRAS野生型（BRAFwt-NRASwt）病变之间的差异表达序列。部分研究结果通过实时荧光定量反转录聚合酶链反应（Real-Time RT-PCR）在独立样本队列中得到验证。
突变分析显示，64%的常见获得性黑素细胞痣携带BRAF突变，所有突变均为V600E位点替换（T1799A）。与预期一致，未在BRAF基因第11外显子或NRAS基因第1、2外显子中检测到突变，因所有样本均非源自慢性日光暴露部位。
本研究采用寡核苷酸微阵列完成转录组分析，并通过Bioconductor数据库（www.bioconductor.org）提供的线性模型软件包LIMMA进行差异表达检验，该工具采用贝叶斯统计方法计算基因差异表达的概率。仅25条转录本的B值大于2。随后研究人员开展单因素方差分析（one-way ANOVA），根据焦磷酸测序（pyrosequencing）检测得到的BRAF突变状态将样本分为三类：野生型（wt）、突变等位基因占比++、突变等位基因占比+++。通过筛选ANOVA检验P值<0.01且表达量随三类样本呈递增/递减趋势的转录本，最终得到2882条已知转录本。研究采用MetaCoreTM软件包开展功能富集分析，以鉴定富集的功能生物学过程。
关键词：BRAF特征基因、获得性黑素细胞痣、基因表达谱分析；"++"与"+++"指BRAFV600E突变型病变中携带突变的等位基因占比，该指标通过焦磷酸测序检测得到（Venesio等，2008，PMID=18408659）。