Despite advances in treatment, the five-year survival rate for patients with head and neck squamous cell carcinoma (HNSCC) has shown little improvement in recent decades. MicroRNAs regulate multiple processes involved in carcinogenesis and cancer progression and have been identified as potential diagnostic and prognostic markers. We conducted microRNA expression profiling of HNSCC patient and paired normal samples to identify microRNAs with significantly altered expression. We identified miR375 as the most consistently downregulated miRNA in tumor samples. Patients in the lowest quartile of miR-375 tumor:normal (T:N) expression showed significantly decreased disease-specific survival, increased incidence of locoregional recurrence and distant metastasis.
We evaluated possible mechanisms by which miR-375 expression would cause altered HNSCC invasion. Stable transductant cell lines with increased expression of miR-375 were generated from HNSCC cell lines utilizing lentiviral constructs expressing either the precursor form of miR-375 or an empty vector control. We observed that HNSCC cells with increased miR-375 expression exhibited significantly reduced cell invasion in vitro. We also observed that elevated miR-375 expression suppressed matrix degradation as well as the frequency of mature invadopodia. In addition, higher miR-375 expression led to significant reductions in the secreted levels and mRNA expression levels of specific proteases.
We utilized a quantitative proteomic strategy, stable isotope labeling with amino acids in cell culture (SILAC) and reverse-phase liquid chromatography mass spectrometry (RPLC-MS) to compare differences in protein abundance between UMSCC1 expressing high or low levels of miR-375. The top candidates that emerged were L-plastin and vimentin. We identified runt-related transcription factor 1 (RUNX1) as a miR-375 target candidate that regulates vimentin and L-plastin expression. We also showed that knockdown of L-plastin and vimentin reduced cell invasion.
In summary, we have identified miR-375 as potential prognostic marker of poor outcome and metastasis in HNSCC. We demonstrated that increased miR-375 expression levels in HNSCC cells suppressed cell invasion in part through reduced invadopodial function. These effects may occur through suppression of RUNX1, which can drive expression of vimentin and L-plastin.
|Advisor:||Prystowskv, Michael B.|
|School Location:||United States -- New York|
|Source:||DAI-B 77/02(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology, Oncology|
|Keywords:||HNSCC, Invadopodia, Invasion, MicroRNA-375, SILAC-Mass Spec|
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