A Novel Functional Splice Variant of AKT3 Defined by Analysis of Alternative Splice Expression in HPV-Positive Oropharyngeal Cancers.
A Novel Functional Splice Variant of AKT3 Defined by Analysis of Alternative Splice Expression in HPV-Positive Oropharyngeal Cancers.
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DOI：
10.1158/0008-5472.can-16-3106
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发表时间：
2017-10-01
期刊：
影响因子：
11.2
通讯作者：
Califano J
中科院分区：
文献类型：
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作者：
Guo T;Sakai A;Afsari B;Considine M;Danilova L;Favorov AV;Yegnasubramanian S;Kelley DZ;Flam E;Ha PK;Khan Z;Wheelan SJ;Gutkind JS;Fertig EJ;Gaykalova DA;Califano J
在过去的20年中，HPV相关的口咽鳞状细胞癌（OPSCC）的发病率增加了200%以上。最近的基因测序工作已经阐明了头颈癌中的相关基因，但HPV相关肿瘤一直显示出很少的DNA突变。在这项研究中，我们试图分析选择性剪接事件（ASE），可以改变基因功能的突变无关。为了鉴定HPV相关肿瘤特有的ASE，对46例HPV阳性OPSCC和25例正常组织样本进行RNA测序。一种新的算法，使用离群值统计的RNA测序连接表达确定了109剪接事件，这是证实了在一个验证集从癌症基因组图谱（TCGA）。由于最常见的剪接事件是一个替代的起始位点（39%），MBD-seq全基因组CpG甲基化数据进行了分析，在启动子区域的甲基化改变。ASE在6个基因的启动子甲基化和表达的替代转录起始位点，包括AKT 3显着负相关。使用qRT-PCR和qMSP方法证实了新的AKT 3转录变体和甲基化变化。新AKT 3变体的体外沉默导致多种头颈部细胞系的显著生长抑制，这是野生型AKT 3敲低未观察到的效果。HPV相关OPSCC中ASE的分析确定了可能参与癌变的多种改变，包括AKT 3的一种新的功能活性转录变体。我们的数据表明，ASES代表了一个重要的肿瘤发生机制，在理解导致癌症发展的复杂遗传变化方面具有尚未开发的潜力。
The incidence of HPV-related oropharyngeal squamous cell carcinoma (OPSCC) has increased more than 200% in the past 20 years. Recent genetic sequencing efforts have elucidated relevant genes in head and neck cancer, but HPV-related tumors have consistently shown few DNA mutations. In this study, we sought to analyze alternative splicing events (ASE) that could alter gene function independent of mutations. To identify ASE unique to HPV-related tumors, RNA sequencing was performed on 46 HPV-positive OPSCC and 25 normal tissue samples. A novel algorithm using outlier statistics on RNA-sequencing junction expression identified 109 splicing events, which were confirmed in a validation set from the Cancer Genome Atlas (TCGA). Because the most common type of splicing event identified was an alternative start site (39%), MBD-seq genome-wide CpG methylation data were analyzed for methylation alterations at promoter regions. ASE in six genes showed significant negative correlation between promoter methylation and expression of an alternative transcriptional start site, including AKT3. The novel AKT3 transcriptional variant and methylation changes were confirmed using qRT-PCR and qMSP methods. In vitro silencing of the novel AKT3 variant resulted in significant growth inhibition of multiple head and neck cell lines, an effect not observed with wild type AKT3 knockdown. Analysis of ASE in HPV-related OPSCC identified multiple alterations likely involved in carcinogenesis, including a novel, functionally active transcriptional variant of AKT3. Our data indicate that ASEs represent a significant mechanism of oncogenesis with untapped potential for understanding complex genetic changes that result in development of cancer.