Our research group has assessed the global expression patterns of miRNAs in both tumor and normal samples from patients with head and neck squamous cell carcinoma (HNSCC);we identified miR-375 as the most consistently down-regulated miRNA in tumor samples when compared to paired normal samples. Patients in the lowest quartile of miR-375 expression had significantly decreased disease-specific survival, increased frequency of local regional recurrence and distant metastasis. I hypothesize that miR-375 suppresses the invasive phenotype of HNSCC through repression of the levels of specific protein targets. The highly significant association of lower miR-375 expression in patients with poor prognosis and distant metastasis led me to want to investigate how miR-375 expression causes alterations in the invasive properties of HNSCC. I have observed that precursor miR-375 transductants have reduced invasion in vitro. Furthermore, preliminary gene expression microarray analysis of the transductants has identified the down-regulated expression of genes having functions associated with cell motility and invasion;the expression of some of these genes also have been previously been correlated with HNSCC patient survival. This proposal addresses the invasive phenotype hypothesis with the following aims: 1) Utilize stable lentiviral transductants to investigate the effects of miR-375 on the invasive properties of HNSCC cell lines. Stable transductant cell lines will be used to assess whether miR-375 expression levels affects growth, invasion, chemotaxis and matrix degradation in vitro, as well as, three-dimensional embedded growth, three-dimensional invasion with inverted invasion and organotypic invasion assays. In addition HNSCC floor-of-mouth xenografts generated from UMSCC47 transductant cell lines will be used to evaluate the consequence of miR-375 expression levels on tumor growth, in vivo invasion and metastasis. 2) Identify specific miR-375 target genes and altered signaling pathways in HNSCC. Stable lentiviral transductants will be used to identify potential target genes of miR-375 by analyzing both RNA expression with whole genome microarrays and translationally repressed putative targets of miR-375 by SILAC (stable isotope labeling of cells in cell culture). Ingenuity Pathway Analysis will be used to identify cellular functions and signaling pathways that the potential targets of miR-375 are involved. The expression levels of the putative targets will be confirmed by qRT-PCR and/or western blot analysis. The interaction between miR-375 and the putative target mRNA transcripts will be confirmed by luciferase reporter assays. Our group has generated a database of mRNA microarray data and global proteomic data on primary tumors from patients along with clinical data on each sample. The disease relevance to any candidate miR-375 target identified in my analysis will be assessed by correlations of cell line data with patient-derived data from primary tumors. Functional validation of the phenotypic effects of the down-regulation of putative targets of miR-375 will be performed by siRNA knockdown and rescue of the targets in the transductant cells.
Head and neck squamous cell carcinoma is the sixth most common malignancy worldwide and the five-year survival rate for patients with HNSCC has shown little improvement over the last 30 years. The identification of prognostic biomarkers could lead to improved therapeutic strategies that may improve patient outcomes. From this project we anticipate gaining insight into how miR-375 expression levels can impact HNSCC patient outcome and incidence of distant metastasis through the exploration of its molecular mechanisms in HNSCC cell lines.