Hemochromatosis enhances tumor progression via upregulation of intracellular iron in head and neck cancer.

INTRODUCTION: Despite improvements in treatment strategies for head and neck squamous cell carcinoma (HNSCC), outcomes have not significantly improved; highlighting the importance of identifying novel therapeutic approaches to target this disease. To address this challenge, we proceeded to evaluate the role of iron in HNSCC. EXPERIMENTAL DESIGN: Expression levels of iron-related genes were evaluated in HNSCC cell lines using quantitative RT-PCR. Cellular phenotypic effects were assessed using viability (MTS), clonogenic survival, BrdU, and tumor formation assays. The prognostic significance of iron-related proteins was determined using immunohistochemistry. RESULTS: In a panel of HNSCC cell lines, hemochromatosis (HFE) was one of the most overexpressed genes involved in iron regulation. In vitro knockdown of HFE in HNSCC cell lines significantly decreased hepcidin (HAMP) expression and intracellular iron level. This in turn, resulted in a significant decrease in HNSCC cell viability, clonogenicity, DNA synthesis, and Wnt signalling. These cellular changes were reversed by re-introducing iron back into HNSCC cells after HFE knockdown, indicating that iron was mediating this phenotype. Concordantly, treating HNSCC cells with an iron chelator, ciclopirox olamine (CPX), significantly reduced viability and clonogenic survival. Finally, patients with high HFE expression experienced a reduced survival compared to patients with low HFE expression. CONCLUSIONS: Our data identify HFE as potentially novel prognostic marker in HNSCC that promotes tumour progression via HAMP and elevated intracellular iron levels, leading to increased cellular proliferation and tumour formation. Hence, these findings suggest that iron chelators might have a therapeutic role in HNSCC management.

MicroRNA-193b enhances tumor progression via down regulation of neurofibromin 1.

Despite improvements in therapeutic approaches for head and neck squamous cell carcinomas (HNSCC), clinical outcome has remained disappointing, with 5-year overall survival rates hovering around 40-50%, underscoring an urgent need to better understand the biological bases of this disease. We chose to address this challenge by studying the role of micro-RNAs (miRNAs) in HNSCC. MiR-193b was identified as an over-expressed miRNA from global miRNA profiling studies previously conducted in our lab, and confirmed in HNSCC cell lines. In vitro knockdown of miR-193b in FaDu cancer cells substantially reduced cell proliferation, migration and invasion, along with suppressed tumour formation in vivo. By integrating in silico prediction algorithms with in vitro experimental mRNA profilings, plus mRNA expression data of clinical specimens, neurofibromin 1 (NF1) was identified to be a target of miR-193b. Concordantly, miR-193b knockdown decreased NF1 transcript and protein levels significantly. Luciferase reporter assays confirmed the direct interaction of miR-193b with NF1. Moreover, p-ERK, a downstream target of NF1 was also suppressed after miR-193b knockdown. FaDu cells treated with a p-ERK inhibitor (U0126) phenocopied the reduced cell proliferation, migration and invasion observed with miR-193b knockdown. Finally, HNSCC patients whose tumours expressed high levels of miR-193b experienced a lower disease-free survival compared to patients with low miR-193b expression. Our findings identified miR-193b as a potentially novel prognostic marker in HNSCC that drives tumour progression via down-regulating NF1, in turn leading to activation of ERK, resulting in proliferation, migration, invasion, and tumour formation.

Lin28b promotes head and neck cancer progression via modulation of the insulin-like growth factor survival pathway.

Lin28 is a developmentally regulated RNA binding protein which has recently emerged as key regulator in the biogenesis of the let-7 micro-RNA family. While the expression of Lin28b has been linked to advanced tumor stage, the precise molecular mechanism(s) by which Lin28b drives disease progression is still being unraveled. Herein, we generated a let-7-resistant Lin28b ORF, stably expressed in the FaDu head and neck cancer (HNC) cell line. FaDu-Lin28b cells exhibited enhanced tumor growth in vitro and in vivo. Global gene and micro-RNA expression analyses revealed significant enrichment in several pathways involved in cell migration, chromatin remodeling, and cellular stress response. Direct regulation of selected genes (HMGA2, CCND2, IGF1R, and IGF2BP2) via a let-7-Lin28b mechanism was validated. Notably, up-regulation of several genes in the IGF pathway in Lin28b-expressing cells was observed. Functional studies revealed significant increase in the survival of Lin28b-expressing cells when cultured under stress conditions, which was dependent on the presence of IGF1. Therefore, our data identified several novel gene targets for Lin28b-let7, and revealed a novel mechanism by which Lin28b promote tumorigenesis. Concordantly, clinical examinations of Lin28b, IGF2BP2 and IGF2 revealed a significant association between the expression of these genes with disease relapse, thereby corroborating the potential relevance for the Lin28b/IGF axis in HNC progression.

MiR-218 suppresses nasopharyngeal cancer progression through downregulation of survivin and the SLIT2-ROBO1 pathway.

Nasopharayngeal carcinoma (NPC) is an Epstein-Barr virus-associated malignancy most common in East Asia and Africa. Here we report frequent downregulation of the microRNA miR-218 in primary NPC tissues and cell lines where it plays a critical role in NPC progression. Suppression of miR-218 was associated with epigenetic silencing of SLIT2 and SLIT3, ligands of ROBO receptors that have been previously implicated in tumor angiogenesis. Exogenous expression of miR-218 caused significant toxicity in NPC cells in vitro and delayed tumor growth in vivo. We used an integrated trimodality approach to identify targets of miR-218 in NPC, cervical, and breast cell lines. Direct interaction between miR-218 and the 3'-untranslated regions (UTR) of mRNAs encoding ROBO1, survivin (BIRC5), and connexin43 (GJA1) was validated in a luciferase-based transcription reporter assay. Mechanistic investigations revealed a negative feedback loop wherein miR-218 regulates NPC cell migration via the SLIT-ROBO pathway. Pleotropic effects of miR-218 on NPC survival and migration were rescued by enforced expression of miR-218-resistant, engineered isoforms of survivin and ROBO1, respectively. In clinical specimens of NPC (n=71), ROBO1 overexpression was significantly associated with worse overall (P=0.04, HR=2.4) and nodal relapse-free survival (P=0.008, HR=6.0). Our findings define an integrative tumor suppressor function for miR-218 in NPC and further suggest that restoring miR-218 expression in NPC might be useful for its clinical management.

Comprehensive MicroRNA profiling for head and neck squamous cell carcinomas.

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-G(1). Similar cellular effects were observed in knockdown studies of the miR-106b-25 cluster but with accumulation of cells in G(1) 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.

Longitudinal cytokine expression during IMRT for prostate cancer and acute treatment toxicity.

PURPOSE: Proteomic profiling of patients undergoing intensity-modulated radiotherapy (IMRT) for prostate cancer can identify unique biomarkers that reflect acute toxicity in normal tissues. Our objectives were to measure inflammatory cytokine proteins during IMRT and assess the variability of individual proteomic signatures. EXPERIMENTAL DESIGN: Forty-two patients with intermediate-risk prostate cancer were recruited as follows: group 1, definitive IMRT (78 Gy in 39 fractions, n = 22), and group 2, IMRT postprostatectomy (66 Gy in 33 fractions, n = 20). Blood/urine samples were collected at baseline and weekly during IMRT. Acute toxicity was graded weekly during radiotherapy using CTC-AE v3.0 criteria. Multiplexed immunoassays were used to quantify cytokines including granulocyte macrophage colony-stimulating factor, IFN-gamma, tumor necrosis factor-alpha, interleukin (IL)-1alpha, IL-2, IL6, IL-8, IL-10, and IL-12p70. RESULTS: We observed positive correlations between cytokine expression between serum and plasma, but not between serum/plasma and urine. The Mann-Whitney test showed a significant increase in IFN-gamma and IL-6 during IMRT (P = 0.0077, 0.0035). Increasing IL-2 and IL-1 expression were associated with increased probability of acute gastrointestinal and genitourinary toxicity, respectively. CONCLUSIONS: Determination of radiation-response signatures is feasible using multiplexed immunoassays and is a promising predictive early biomarker of toxicity outcomes.