Cutaneous melanoma is an increasingly common skin cancer characterized by aggressive metastatic growth and poor prognosis. The mechanisms behind melanoma progression are not fully understood, but emerging evidence suggests that a group of newly discovered small regulatory RNAs, named microRNAs (miRNAs), plays an important role. miRNAs are ~ 22 nucleotide single strand non-coding RNAs that post-transcriptionally regulate gene expression by binding to target messenger RNAs (mRNAs), leading to mRNA degradation and translation inhibition. Abnormal expression of miRNAs has been observed in human malignancies and is associated with tumorigenesis. The main goals of this thesis are to investigate miRNA dysregulation in melanoma and to identify potential miRNAs involved in melanoma pathogenesis. Initially, the expression of 470 miRNAs was profiled in 8 metastatic melanoma and 8 benign nevus tissue samples. We discovered unique miRNA expression profiles and identified differentially expressed miRNAs in melanomas as compared to nevi. miR-193b was one of the most significantly downregulated miRNAs in melanoma, and its function and regulatory targets were unknown. Subsequently, in vitro functional studies revealed that ectopic expression of miR-193b in melanoma cells drastically repressed cell proliferation and migration. Although it does not directly induce apoptosis in melanoma cells, miR-193b does sensitize these cells to ABT-737-mediated cell death. In concert with functional studies, gene expression analysis and in silico target prediction were performed to globally screen for mRNA targets of miR-193b. We identified eighteen genes as candidates in that they were downregulated by miR-193b and contained predicted miR-193b binding sites. Based on their known biological functions, three genes were particularly interesting: cyclin D1 (CCND1), myeloid cell leukemia sequence 1 (Mcl-1), and stathmin 1 (STMN1). CCND1 and Mcl-1 are two well-known melanoma oncogenes, and we validated their role in cell proliferation and apoptosis respectively. Furthermore, using similar approach, we were the first to identify STMN1 as a novel melanoma oncogene. We demonstrated that CCND1, Mcl-1, and STMN1 were directly regulated by miR-193b. During melanoma progression, reduced expression of miR-193b may promote cell proliferation, migration and survival. Taken together, this thesis describes the dysregulation of miRNAs in melanoma and demonstrates that miR-193b functions as a tumor suppressor. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2012-05-31 15:27:01.707

Despite improvements in treatment strategies for head and neck squamous cell carcinoma (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), and iron in HNSCC. We performed global profiling on 51 primary HNSCC compared to 4 normal laryngeal epithelial tissues, and identified 38 differentially expressed miRNAs between cancer vs. normal patient tissues. Functional validation confirmed a tumour promoting phenotype for miR-106b and miR-375. Integrating these findings with global miR profiling of HNSCC revealed two significantly over expressed miRNAs in HNSCC cell lines and patient samples: miR-193b and miR-205. Knockdown of miR-205 and miR-193b in HNSCC cell lines significantly decreased cell proliferation and colony formation. Moreover, NF1 was identified as a target of miR-193b. Downstream targets of NF1 including active-RAS and p-ERK were also suppressed after miR-193b knockdown. Finally, HNSCC patients with high levels of miR-193b experienced a lower disease-free survival than patients with low miR-193b expression. The second approach we took to better understand the biology of HNSCC was to examine the involvement of iron in the disease. In a panel of HNSCC cell lines, hemochromatosis (HFE) was one of the most overexpressed genes involved in iron regulation. Knockdown of HFE in HNSCC cell lines significantly decreased intracellular iron levels, resulting in a significant decrease in HNSCC cell proliferation, DNA synthesis, and Wnt signalling. When iron was re-introduced back into the cell after HFE knockdown, these cellular changes were reversed, indicating that iron was mediating this phenotype. Concordantly, HNSCC cells treated with an iron chelator ciclopirox olamine (CPX) significantly reduced proliferation and clonogenic survival. Finally, patients with high HFE expression experienced a reduced survival compared to patients with low HFE expression, corroborating the oncogenic role of HFE in HNSCC. In summary, using two independent methods, we have identified two potential prognostic biomarkers for HNSCC, namely miR-193b and HFE. Characterization of these two molecules, exposed critically dysregulated pathways driving disease progression. Specifically, the miR-193b~NF1 axis uncovered a novel mechanism of RAS and p-ERK activation in HNSCC; similarly, HFE exposed a novel tumour promotion role of iron in this disease.

Despite improvements in treatment strategies for head and neck squamous cell carcinoma (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), and iron in HNSCC. We performed global profiling on 51 primary HNSCC compared to 4 normal laryngeal epithelial tissues, and identified 38 differentially expressed miRNAs between cancer vs. normal patient tissues. Functional validation confirmed a tumour promoting phenotype for miR-106b and miR-375. Integrating these findings with global miR profiling of HNSCC revealed two significantly over expressed miRNAs in HNSCC cell lines and patient samples: miR-193b and miR-205. Knockdown of miR-205 and miR-193b in HNSCC cell lines significantly decreased cell proliferation and colony formation. Moreover, NF1 was identified as a target of miR-193b. Downstream targets of NF1 including active-RAS and p-ERK were also suppressed after miR-193b knockdown. Finally, HNSCC patients with high levels of miR-193b experienced a lower disease-free survival than patients with low miR-193b expression. The second approach we took to better understand the biology of HNSCC was to examine the involvement of iron in the disease. In a panel of HNSCC cell lines, hemochromatosis (HFE) was one of the most overexpressed genes involved in iron regulation. Knockdown of HFE in HNSCC cell lines significantly decreased intracellular iron levels, resulting in a significant decrease in HNSCC cell proliferation, DNA synthesis, and Wnt signalling. When iron was re-introduced back into the cell after HFE knockdown, these cellular changes were reversed, indicating that iron was mediating this phenotype. Concordantly, HNSCC cells treated with an iron chelator ciclopirox olamine (CPX) significantly reduced proliferation and clonogenic survival. Finally, patients with high HFE expression experienced a reduced survival compared to patients with low HFE expression, corroborating the oncogenic role of HFE in HNSCC. In summary, using two independent methods, we have identified two potential prognostic biomarkers for HNSCC, namely miR-193b and HFE. Characterization of these two molecules, exposed critically dysregulated pathways driving disease progression. Specifically, the miR-193b~NF1 axis uncovered a novel mechanism of RAS and p-ERK activation in HNSCC; similarly, HFE exposed a novel tumour promotion role of iron in this disease.