Role of MicroRNA-363 in Human Papillomavirus-Associated Squamous Cell Carcinoma of the Head and Neck

Squamous cell carcinoma of the head and neck (SCCHN) ranks sixth among cancers worldwide. Despite recent advances in the detection and therapy of SCCHN, the five-year survival rate remains low. Human papillomavirus (HPV) has been detected in a new class of SCCHN that has emerged in younger patients without the common risk factors of alcohol and/or tobacco. HPV-positive and HPV-negative SCCHN have different clinical characteristics, leading some to classify them as distinct diseases. High-risk HPVs have been implicated in a number of cancers including cervical, anogenital, and head and neck cancers. Micro (mi) RNAs are a recently discovered class of endogenously encoded small RNAs that most commonly function as negative regulators of gene expression. MiRNA expression profiles are often altered in cellular stress conditions, including cancers and viral infections. We hypothesized that HPV disrupts cellular miRNA expression in HPV-positive SCCHN and contributes to the development of this cancer. \ud MiRNA microarray analysis of HPV-positive and HPV-negative SCCHN cell lines revealed the dysregulation of several miRNAs, including the upregulation of miR-363 and downregulation of miR-181a, miR-218, and miR-29a. Exogenous expression of the HPV-16 E6 oncogene in normal primary human keratinocytes showed similar changes in the above miRNAs, implicating HPV E6 in this up/downregulation. Tissues from SCCHN patients in the western Pennsylvania area showed an HPV-positivity rate of 59 percent. HPV-positive SCCHN cases were seen only in the tonsil and base of tongue, and patients with HPV-positive SCCHN were diagnosed on average nine years younger than those with HPV-negative SCCHN. Additionally, miR-363 was upregulated in HPV-16-positive SCCHN tissues compared to the HPV-negative SCCHN tissues.\ud We utilized gene expression data and spectral counting proteomics to narrow the possible cellular targets of miR-363 in SCCHN. As confirmed via luciferase assays, miR-363 targets MYO1B, a myosin protein involved in cell motility. Functional assays showed that overexpression of miR-363 in HPV-negative SCCHN cells or siRNA knockdown of MYO1B in HPV-positive SCCHN cells independently reduced cell migration. Furthermore, the addition of miR-363 to HPV-negative SCCHN cells also caused an increase in cell proliferation and colony formation. \ud Based on our studies, we envision that high-level expression of the E6 oncogene in HPV-positive SCCHN results in an increase in miR-363 levels. Increased expression of miR-363 may then reduce MYO1B levels, thereby reducing the migratory ability of these cells. Furthermore, high levels of miR-363 may also promote cellular proliferation and colony formation. \u