Abstract 5061: Differentially expressed microRNA signatures based on human papillomavirus status in head and neck squamous cell carcinoma
Authors: Howard Jason D. , Ratner Elena , Yarbrough Wendell G. , Slebos Robbert J. , Weidhaas Joanne , Chung Christine H. 
Published: June 2012

Introduction: Recent studies have demonstrated that human papillomavirus (HPV) status is a favorable prognostic factor in patients with head and neck squamous cell carcinoma (HNSCC). Additionally, microRNAs (miRNAs) have emerged as an important regulator of global gene expression, and their signaling is often altered in cancer. Thus, we assessed the differences in global miRNA expression in normal mucosal epithelia (NME), HPV-positive (+), and HPV-negative (−) tumors to gain insight into patient prognosis given the current standard of care. An understanding of these mechanistic differences could lead to potential therapeutic targets and improvement in clinical outcomes for patients with HPV (−) tumors.

Methods: miRNAs were isolated from 6 NME and 20 frozen tumors using PureLink RNA Isolation Kit, and miRNA using PureLink microRNA Isolation Kit (Invitrogen). miRNA was analyzed using the ABI Megaplex protocol without pre-amplification (Applied Biosystems). Reactions were run using the ABI miRNA Reverse Transcription Kit and Megaplex RT Human Pool A primers, then loaded onto TaqMan Low-Density Array (TLDA) cards. TLDAs were analyzed using an ABI7900HT Real Time PCR machine using the default ABI TLDA protocol. A tissue microarray (TMA) of 153 cores from an additional 51 HNSCC tumors was stained with a phospho-specific AKT (pAKT) antibody (Ser473, Cell Signaling Technology), and digitally quantified with TMAJ software (Johns Hopkins Univ.). After cores were omitted due to lack of tumor or unknown HPV-status, staining was analyzed from 123 cores from 45 tumors and evaluated using a two-sample t-test assuming equal variances.

Results: The number of differentially expressed miRNAs were as follows: 36 in HPV (+) compared to NME; 29 in HPV (−) compared to NME; 40 shared between the tumor sets compared to NME; and one (miR-449a) between both tumor sets after multi-variable corrections. An miRNA lower in both HPV (+) and HPV (−) tumors with respect to NME was miR-124, a putative regulator of PIK3CA. To contrast functional differences in PI3KCA signaling with HPV status, a TMA of 45 HNSCC tumors was examined by IHC for pAKT expression. The mean pAKT staining was higher in HPV (+) tumors compared to HPV (−) cohorts (p = 0.003). Interestingly, lower pAKT levels correlated with tumor recurrence in HPV (+) tumors (p = 2.5×10−8), while no statistical difference was noted in HPV (−) tumors (p = 0.24).

Conclusions: There are significant differences in the miRNA profiles of tumors compared to NME, suggesting deregulation of miRNAs may play an important role in the development of HNSCC. Our data suggest the downstream effects of miR-124 regulation may differ in the context of HPV status, and that PI3K/AKT signaling may influence the observed difference in HPV-related tumor clinical outcomes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5061. doi:10.1158/1538-7445.AM2011-5061