A novel functional splice variant of AKT3 defined by analysis of alternative splice expression in HPV-positive oropharyngeal cancers

Theresa Guo, Akihiro Sakai, Bahman Afsari, Michael Considine, Liudmila V Danilova, Alexander Favorov, S Yegnasubramanian, Dylan Z. Kelley, Emily Flam, Patrick K. Ha, Zubair Khan, Sarah Wheelan, J. Silvio Gutkind, Elana Fertig, Daria Gaykalova, Joseph Califano

Research output: Contribution to journalArticle

Abstract

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. 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 the development of cancer.

Original languageEnglish (US)
Pages (from-to)5248-5258
Number of pages11
JournalCancer Research
Volume77
Issue number19
DOIs
StatePublished - Oct 1 2017

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Oropharyngeal Neoplasms
Alternative Splicing
Methylation
RNA Sequence Analysis
Squamous Cell Carcinoma
Neoplasms
Carcinogenesis
Genome
Genes
Mutation
Atlases
Head and Neck Neoplasms
Genetic Promoter Regions
Neck
Head
Cell Line
Polymerase Chain Reaction
DNA
Incidence
Growth

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

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A novel functional splice variant of AKT3 defined by analysis of alternative splice expression in HPV-positive oropharyngeal cancers. / Guo, Theresa; Sakai, Akihiro; Afsari, Bahman; Considine, Michael; Danilova, Liudmila V; Favorov, Alexander; Yegnasubramanian, S; Kelley, Dylan Z.; Flam, Emily; Ha, Patrick K.; Khan, Zubair; Wheelan, Sarah; Gutkind, J. Silvio; Fertig, Elana; Gaykalova, Daria; Califano, Joseph.

In: Cancer Research, Vol. 77, No. 19, 01.10.2017, p. 5248-5258.

Research output: Contribution to journalArticle

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abstract = "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. 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 the development of cancer.",
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T1 - A novel functional splice variant of AKT3 defined by analysis of alternative splice expression in HPV-positive oropharyngeal cancers

AU - Guo, Theresa

AU - Sakai, Akihiro

AU - Afsari, Bahman

AU - Considine, Michael

AU - Danilova, Liudmila V

AU - Favorov, Alexander

AU - Yegnasubramanian, S

AU - Kelley, Dylan Z.

AU - Flam, Emily

AU - Ha, Patrick K.

AU - Khan, Zubair

AU - Wheelan, Sarah

AU - Gutkind, J. Silvio

AU - Fertig, Elana

AU - Gaykalova, Daria

AU - Califano, Joseph

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