Abstract 511: HPV mediates HNSCC carcinogenesis by exosomal exclusion of tumor suppressive miRNAs

Abstract

Abstract Background: Exosomes are small vesicles between 40-150 nm in diameter that are secreted by various cell types, including tumor cells. Exosomes of cancer cells are known to be involved in tumor pathology and have altered expression of microRNAs (miRNAs). However, the effect of HPV-16 (Human Papilloma Virus-16) infection on miRNA content of exosomes released by infected HNSCC cells has not yet been explored. Materials & Methods: Exosomes were isolated from media of cultured HPV positive 93VU-147T and HPV negative UPCI:SCC 116 HNSCC cells and were characterized by Transmission Electron Microscopy. Presence of exosomal marker CD63 was confirmed using western blotting. Small RNA seq of exoRNA of HPV positive and HPV negative cells was carried out. For validation of identified miRNAs, HPV16 E6E7 was cloned into pcDNA3.1(-ve) vector and was transfected into SCC116 cells and stable cells were selected using G418. Differentially expressed miRNAs were validated in cells and exosomes using Real Time PCR. Results: Seven miRNAs 6724-5p, 6087, 4497, 4792, 4508, 199a-3p and 199b-3p were found to be significantly upregulated in HPV positive HNSCC exosomes as compared to HPV negative HNSCC exosomes from NGS data. Upregulation of these miRNAs was further confirmed in exosomes derived from HPV positive 93VU-147T cells and SCC116 E6E7 cells as compared to HPV negative cells (SCC 116 (3.1-ve) (vector only transfected SCC 116) cells. Expression of these miRNAs was significantly downregulated in 93VU14T and SCC116 E6E7 (HPV +ve) cells as compared to HPV negative cells. Three of these miRNAS (4792, 199a-3p and 199b-3p) are known tumor suppressor miRNAs. miRNA 6724-5p, a novel miRNA was found to be down regulated the most in HPV +ve cells. We identified CBX6, PDK2 and FOXK1 which are known to be involved in carcinogenesis, as its target genes through Targetscan. Expression of these genes was found to be significantly upregulated in HPV positive 93VU-147T cells and SCC116 E6E7 cells as compared to HPV negative cells indicating tumor suppressive role of miRNA 6724-5p in carcinogenesis. Conclusion: Our results suggest that HPV may mediate HNSCC carcinogenesis by exosomal exclusion of tumor suppressive miRNAs. Citation Format: Madhu Khullar, Varinder Singh, Nilambra Dogra, Malay Sannigrahi, Naresh Panda. HPV mediates HNSCC carcinogenesis by exosomal exclusion of tumor suppressive miRNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 511.