Dysregulation of MicroRNA-34a Expression in Head and Neck Squamous Cell Carcinoma Promotes Tumor Growth and Tumor Angiogenesis Bhavna Kumar1,2, Arti Yadav2, James Lang1,2, Theodoros N. Teknos1,2, Pawan Kumar1,2* 1 Department of Otolaryngology-Head and Neck Surgery, The Ohio State University, Columbus, Ohio, United States of America, 2 The Ohio State University Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, United States of America Abstract Background: MicroRNAs (miRs) are small non-coding RNAs that play an important role in cancer development where they can act as oncogenes or as tumor-suppressors. miR-34a is a tumor-suppressor that is frequently downregulated in a number of tumor types. However, little is known about the role of miR-34a in head and neck squamous cell carcinoma (HNSCC). Methods and Results: miR-34a expression in tumor samples, HNSCC cell lines and endothelial cells was examined by real time PCR. Lipofectamine-2000 was used to transfect miR-34a in HNSCC cell lines and human endothelial cells. Cell- proliferation, migration and clonogenic survival was examined by MTT, Xcelligence system, scratch assay and colony formation assay. miR-34a effect on tumor growth and tumor angiogenesis was examined by in vivo SCID mouse xenograft model. Our results demonstrate that miR-34a is significantly downregulated in HNSCC tumors and cell lines. Ectopic expression of miR-34a in HNSCC cell lines significantly inhibited tumor cell proliferation, colony formation and migration. miR-34a overexpression also markedly downregulated E2F3 and survivin levels. Rescue experiments using microRNA resistant E2F3 isoforms suggest that miR-34a-mediated inhibition of cell proliferation and colony formation is predominantly mediated by E2F3a isoform. In addition, tumor samples from HNSCC patients showed an inverse relationship between miR-34a and survivin as well as miR-34a and E2F3 levels. Overexpression of E2F3a completely rescued survivin expression in miR-34a expressing cells, thereby suggesting that miR-34a may be regulating survivin expression via E2F3a. Ectopic expression of miR-34a also significantly inhibited tumor growth and tumor angiogenesis in a SCID mouse xenograft model. Interestingly, miR-34a inhibited tumor angiogenesis by blocking VEGF production by tumor cells as well as directly inhibiting endothelial cell functions. Conclusions: Taken together, these findings suggest that dysregulation of miR-34a expression is common in HNSCC and modulation of miR34a activity might represent a novel therapeutic strategy for the treatment of HNSCC. Citation: Kumar B, Yadav A, Lang J, Teknos TN, Kumar P (2012) Dysregulation of MicroRNA-34a Expression in Head and Neck Squamous Cell Carcinoma Promotes Tumor Growth and Tumor Angiogenesis. PLoS ONE 7(5): e37601. doi:10.1371/journal.pone.0037601 Editor: Irina V. Lebedeva, Enzo Life Sciences, Inc., United States of America Received August 16, 2011; Accepted April 26, 2012; Published May 22, 2012 Copyright: 2012 Kumar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by National Institutes of Health/National Cancer Institute grant CA133250 (PK) and Joan’s Fund Research Grant (BK and PK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pawan.kumar@osumc.edu Introduction Head and neck squamous cell carcinoma (HNSCC), which includes cancers of oral cavity, oropharynx, larynx, and hypo- pharynx accounts for approximately 600,000 new cases every year and is sixth leading cancer by incidence worldwide [1]. The most important risk factors identified so far are tobacco use and alcohol consumption, which seem to have a synergistic effect [2]. In recent years, the incidence of oropharyngeal cancers, particularly in the western world, has markedly increased, which may be related to increase in oral and oropharyngeal human papillomavirus (HPV) infections [3]. Despite advancements in surgical and other therapeutic regimens, 5 year survival rates for head and neck patients have stayed around 50% during the last two decades [4]. Patients that survive with surgery and/or chemo-radiation treatment (CRT) often live with significant cosmetic and functional defects. The limited information available on the molecular carcinogenesis of HNSCC and the genetic and biological heterogeneity of the disease has hampered the development of novel therapeutic strategies. Cancer is a complex genetic disease in which deregulated cell growth arises due to defects in major pathways that are fundamental for normal homeostasis [1]. Evidence is emerging that alterations in the expression of microRNAs (miRs) may play a key role in cancer development and progression [5]. MicroRNAs (miRs), first described about 18 years ago in Caenorhabditis elegans [6], are small non-coding RNAs that act in concert to regulate expression of myriad target Proteins. Today more than 17000 microRNA’s in over 153 species have been identified (miRBase Sequence Database - Release 17; www. miRbase.org), of which around 1400 are in humans. Most miRs are evolutionarily conserved and often found in clusters [7]. In mammals, mature miRs are recognized by specific proteins to form RNA-inducing silencing complex (RISC) and associate PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e37601