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Purpose: The objective of this study is to investigate the significance of microRNAs (miRNA) in patients with locally advanced head and neck squamous cell carcinoma (HNSCC).Experimental Design: A global miRNA profiling was done on 51 formalin-fixed archival HNSCC samples using quantitative reverse transcription-PCR approach, correlated with patients' clinical parameters. Functional characterization of HNSCC-associated miRNAs was conducted on three HNSCC cell lines. Cell viability and proliferation were investigated using MTS and clonogenic assays, respectively; cell cycle analyses were assessed using flow cytometry.Results: Thirty-eight of the 117 (33%) consistently detected miRNAs were significantly differentially expressed between malignant versus normal tissues. Concordant with previous reports, overexpression of miR-21, miR-155, let-7i, and miR-142-3p and underexpression of miR-125b and miR-375 were detected. Upregulation of miR-423, miR-106b, miR-20a, and miR-16 as well as downregulation of miR-10a were newly observed. Exogenous overexpression of miR-375 in HNSCC cell lines reduced proliferation and clonogenicity and increased cells in sub-G1. Similar cellular effects were observed in knockdown studies of the miR-106b-25 cluster but with accumulation of cells in G1 arrest. No major difference was detected in miRNA profiles among laryngeal, oropharyngeal, or hypopharyngeal cancers. miR-451 was found to be the only significantly overexpressed miRNA by 4.7-fold between nonrelapsed and relapsed patients.Conclusion: We have identified a group of aberrantly expressed miRNAs in HNSCC and showed that underexpression of miR-375 and overexpression of miR-106b-25 cluster might play oncogenic roles in this disease. Further detailed examinations of miRNAs will provide opportunities to dissect the complex molecular abnormalities driving HNSCC progression. Clin Cancer Res; 16(4); 1129–39

Purpose: Despite recent improvements in local control of head and neck cancers (HNC), distant metastasis remains a major cause of death. Hence, further understanding of HNC biology, and in particular, the genes/pathways driving metastasis is essential to improve outcome.Experimental Design: Quantitative reverse transcriptase PCR (qRT-PCR) was used to measure the expression of miR-375 and metadherin (MTDH) in HNC patient samples. Targets of miR-375 were confirmed using qRT-PCR, Western blot analysis, and luciferase assays. Phenotypic effects of miR-375 reexpression and MTDH knockdown were assessed using viability (MTS), clonogenic survival, cell migration/invasion, as well as in vivo tumor formation assays. The prognostic significance of miR-375 or MTDH in nasopharyngeal carcinoma (NPC) was determined by comparing low versus high expression groups.Results: MiR-375 expression was significantly reduced (P = 0.01), and conversely, MTDH was significantly increased (P = 0.0001) in NPC samples. qRT-PCR, Western blots, and luciferase assays corroborated MTDH as a target of miR-375. Reexpression of miR-375 and siRNA knockdown of MTDH both decreased cell viability and clonogenic survival, cell migration/invasion, as well as in vivo tumor formation. NPC patients whose tumors expressed high levels of MTDH experienced significantly lower survival and, in particular, higher distant relapse rates (5-year distant relapse rates: 26% vs. 5%; P = 0.005).Conclusions: Dysregulation of miR-375 and MTDH may represent an important oncogenic pathway driving human HNC progression, particularly distant metastases, which is now emerging as a major cause of death for HNC patients. Hence, targeting this pathway could potentially be a novel therapeutic strategy by which HNC patient outcome could be improved. Clin Cancer Res; 17(24); 7539–50. ©2011 AACR.

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Supplementary Data from Comprehensive MicroRNA Profiling for Head and Neck Squamous Cell Carcinomas

Purpose: Despite recent improvements in local control of head and neck cancers (HNC), distant metastasis remains a major cause of death. Hence, further understanding of HNC biology, and in particular, the genes/pathways driving metastasis is essential to improve outcome. Experimental Design: Quantitative reverse transcriptase PCR (qRT-PCR) was used to measure the expression of miR-375 and metadherin (MTDH) in HNC patient samples. Targets of miR-375 were confirmed using qRT-PCR, Western blot analysis, and luciferase assays. Phenotypic effects of miR-375 reexpression and MTDH knockdown were assessed using viability (MTS), clonogenic survival, cell migration/invasion, as well as in vivo tumor formation assays. The prognostic significance of miR-375 or MTDH in nasopharyngeal carcinoma (NPC) was determined by comparing low versus high expression groups. Results: MiR-375 expression was significantly reduced (P = 0.01), and conversely, MTDH was significantly increased (P = 0.0001) in NPC samples. qRT-PCR, Western blots, and luciferase assays corroborated MTDH as a target of miR-375. Reexpression of miR-375 and siRNA knockdown of MTDH both decreased cell viability and clonogenic survival, cell migration/invasion, as well as in vivo tumor formation. NPC patients whose tumors expressed high levels of MTDH experienced significantly lower survival and, in particular, higher distant relapse rates (5-year distant relapse rates: 26% vs. 5%; P = 0.005). Conclusions: Dysregulation of miR-375 and MTDH may represent an important oncogenic pathway driving human HNC progression, particularly distant metastases, which is now emerging as a major cause of death for HNC patients. Hence, targeting this pathway could potentially be a novel therapeutic strategy by which HNC patient outcome could be improved. Clin Cancer Res; 17(24); 7539–50. ©2011 AACR.

Introduction: Head and neck cancer (HNC) is a challenging disease due to its heterogeneity and complexity. Despite continued advances in therapeutic options, treatment-associated toxicities and overall clinical outcomes have remained disappointing. Even with radiation therapy (RT), which remains the primary curative modality for HNC, the most effective regimens achieve local control rates of 50-70%, with disease free survival rates of only 30-40% for patients with locally advanced HNSCC. Thus, the development of novel strategies to enhance tumor cell killing, while minimizing damage to the surrounding normal tissues, is critical for improving cure rates with RT. Methods: A siRNA-based high-throughput screen (HTS) was performed for the large-scale identification of novel genes that will selectively sensitize HNC cells to ionizing radiation (IR). The Dharmacon Protein Kinase and Druggable Genome siRNA Libraries were screened using FaDu cells (human hypopharyngeal squamous cell cancer). Radiosensitizing targets were subjected to functional validation studies and in vitro characterization of mechanisms for radiosensitization. In vivo validation studies including tumor formation assays and the treatment of established HNC xenograft models were also conducted. Results: The HTS identified 67 target sequences with potential radiosensitizing effects; the validity of the screen was corroborated by the identification of known radiosensitizing targets (e.g. ATM, ATR, AURKA). Targets reducing the surviving fraction by >50% at 2 Gy relative to their un-irradiated counterparts were selected for further evaluation. A key regulator of the heme biosynthetic pathway was thus identified as a novel tumor-selective radiosensitizing target. Down-regulation of the enzyme plus IR induced caspase-mediated apoptosis and cell cycle arrest in vitro, while delaying tumor growth in vivo. Radiosensitization appeared to be mediated via enhancement of tumor oxidative stress from perturbation of iron homeostasis and increased reactive oxygen species production. This radiosensitizing target was significantly over-expressed in HNC patient biopsies, wherein lower pre-RT mRNA levels correlated with improved survival, suggesting that this enzyme could also be a potential predictor for radiation response. Down-regulation of the enzyme also radiosensitized several different human cancer models, while sparing normal cells. Conclusion: We have successfully developed an RNAi-based radiosensitizer HTS, and uncovered a key regulator of heme biosynthesis as a potent sensitizer for RT, with potentially broad implications in the management of many human malignancies. Citation Format: Emma Ito, Shijun Yue, Eduardo H. Moriyama, Angela B. Hui, Inki Kim, Wei Shi, Nehad M. Alajez, Nirmal Bhogal, GuoHua Li, Alessandro Datti, Aaron D. Schimmer, Brian C. Wilson, Peter P. Liu, Daniel Durocher, Benjamin G. Neel, Brian O'Sullivan, Bernard Cummings, Rob Bristow, Fei-Fei Liu. An RNAi screen identifies a heme biosynthetic mediator as a novel radiosensitizing target for head and neck cancer [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SSY01-03