ALTERATION OF microRNA PROFILES IN SQUAMOUS CELL CARCINOMA OF THE HEAD AND NECK CELL LINES BY HUMAN PAPILLOMAVIRUS

Squamous cell carcinoma of the head and neck (SCCHN) ranks sixth among cancers worldwide.1 Many of these cases are associated with heavy consumption of alcohol and/or tobacco use, which over time induce mutations in essential genetic pathways that regulate the cell cycle. However, human papillomavirus (HPV) type 16 DNA has been found in up to 30 percent of these cancers, most often in the oropharynx region, and such cases of SCCHN are often found in individuals without the risk factors of alcohol and tobacco use.1, 2 The HPV-positive SCCHN subset has increased in the past 10 years.2 Because of this demographic shift and distinct clinical behavior, the association and relevance of HPV in SCCHN is under intense investigation. Characteristics of HPV-associated SCCHN are very different from HPV-negative SCCHN, causing disputes whether these cancers should be classified as distinct tumors.3 HPV-positive oral tumors often exhibit loss of cell cycle control proteins, including pRb and cyclin D1, whereas these two proteins are commonly overexpressed in HPV-negative oral tumors.1, 3 One of the most common tumor suppressor proteins, p53, is mutated in up to half of oral cancers, but is very rarely mutated in HPV-positive SCCHN, and tumors with a high viral load have a better prognosis compared to tumors with a low viral load or tumors that are HPV-negative.1, 3 Patients with HPV-positive oral tumors have a better response to chemotherapy, radiation, and surgery,3 and have evidence of immune activation against viral antigens,4 despite having frequent metastasis to regional lymph nodes.3 The biological basis for the differential behavior of HPV-positive SCCHN is not understood. Micro (mi) RNAs are small, ∼22 nt long, chromosome-encoded single-stranded RNAs that are commonly associated with negative regulation of gene expression.5 MiRNAs are transcribed and exported to the cytoplasm where further processing takes place, and the mature miRNA strand is incorporated into the RNA-induced silencing complex (RISC).5 The miRNA guides the RISC to the 3′ untranslated region of its target mRNA where, depending upon the degree of complementarity, the miRNA either translationally represses the mRNA or targets it for degradation.5 MiRNA dysregulation has been implicated in many different types of human cancers.6, 7 Previous reports have shown altered miRNA profiles in head and neck cancers compared to the normal oral tissue.8-11 MiRNAs with high expression in the tumors compared to the normal oral tissue included miR-21, while miR-125b was downregulated.8, 10, 11 Basal miRNA expression in nine head and neck cancer cell lines found that 33 miRNAs were expressed at a high level and 22 miRNAs were expressed at a low level.12 Interestingly, one of these cell lines, UM-SCC47, is HPV-16-positive.13 In all nine cell lines, let-7a, miR-16, miR-21, and miR-205 were highly expressed, and miR-342, miR-346, and miR-373* were expressed at low levels.12 Although these studies show alterations in miRNA levels in head and neck cancer, they do not address the role of HPVs. Since the number of cases of HPV-16-positive SCCHN have been increasing in the past 10 years,2 and the characteristics of HPV-positive and HPV-negative SCCHN support distinction between these cancers,3 we sought to analyze the miRNA profiles in HPV-positive and HPV-negative SCCHN cell lines. In this study, we demonstrate that miRNA expression profiles in HPV-16-positive SCCHN cells are distinctly different from those in HPV-negative SCCHN cells and in normal oral keratinocytes (NOKs) that have been immortalized by activation of h-TERT. Using human foreskin keratinocytes expressing either the HPV-16 E6 or E7 oncogene, we also demonstrate that expression of the E6 oncogene results in upregulation of miR-363 and downregulation of miR-181a, mR-218 and miR-29a. Furthermore, siRNA knockdown of HPV-16 E6 in the HPV-positive SCCHN cell line SCC2 reduced expression of miR-363.