Abstract 3100: MicroRNA profiling reveals miR-205 upregulation is associated with head and neck squamous cell carcinoma and modulates E2F1 signaling.
Authors: Howard Jason D. , Cheng Haixia , Ratner Elena , Fertig Elana J. , Perez Jimena , Quon Harry , Considine Michael , Ochs Michael , Weidhaas Joanne , Chung Christine H. 
Published: August 2014

Introduction: Based on our previous screen of miRNAs in head and neck squamous cell carcinoma (HNSCC), we sought to functionally evaluate candidate transcripts that could modify Rb/E2F signaling. Thus, we chose to investigate miR-205 as a putative HNSCC oncogene and regulator of E2F1 protein expression.

Methods: miRNAs were isolated from 29 newly diagnosed HNSCC tumors (HPV-positive: 14, HPV-negative: 15) and 4 normal mucosa samples using the PureLink RNA Isolation Kit (Invitrogen). miRNA was analyzed using the ABI Megaplex protocol without pre-amplification (Applied Biosystems). Reactions were run using the miRNA Reverse Transcription Kit and Megaplex RT Human Pool A primers, then analyzed by TaqMan Low-Density Array (TLDA) cards. miRNA data was normalized to MammU6 expression, with undetectable transcripts assigned a Ct value of 40. Empirical Bayes moderated t-statistics were used to assess miRNA differential expression and the Benjamini-

Hotchberg correction was applied to these p-values to account for multiple hypothesis testing. miR-205 expression was then transiently modified (Dharmacon) in an HPV-positive and -negative HNSCC cell line. These cells were characterized by Western blot and assayed for changes in proliferation utilizing two- and three-dimensional growth assays.

Results: HNSCC miRNA expression was characterized by a general upregulation of individual transcripts and miRNA families compared to normal mucosa. This difference in expression was independent of HPV-status, with only two miRNAs demonstrating differential expression between HPV-positive and -negative tumors: miR-449a and miR-129-3p. miR-205, a transcript upregulated in both subtypes, was able to modulate proliferation and E2F1 expression levels in 93VU147T (HPV+), but not UM-SCC-15 (HPV-). Further Western blot analysis concluded modulations in Rb-phosphorylation status and apoptotic signaling factors may explain these E2F1-mediated effects. Conclusions: While significant differences are evident in the miRNA profiles of HNSCC compared to normal mucosa, a striking degree of similarity exists between HPV-positive and -negative miRNA deregulation. A functional evaluation of miR-205 determined this transcript is capable of modulating HNSCC growth in the genetic context of p16 expression and proapoptotic signaling.

Citation Format: Jason D. Howard, Haixia Cheng, Elena Ratner, Elana J. Fertig, Jimena Perez, Harry Quon, Michael Considine, Michael Ochs, Joanne Weidhaas, Christine H. Chung. MicroRNA profiling reveals miR-205 upregulation is associated with head and neck squamous cell carcinoma and modulates E2F1 signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3100. doi:10.1158/1538-7445.AM2013-3100