Apolipoprotein E in Head and Neck Cancer Invasion
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Head and neck squamous cell carcinomas (HNSCC) are the sixth most common malignancy and have a poor patient prognosis. This has been attributed to their tendency to invade into the surrounding normal tissue. Our goals for this thesis work were to identify a novel gene that impacts HNSCC invasion, and to determine how it affects invasion at the molecular level.;To identify novel genes involved in invasion, RNA was extracted from HNSCC tumors from the oral cavity (OSCC) that were scored for the histological parameter worst pattern of invasion (WPOI), a parameter significantly associated with local recurrence and overall survival. Two microarray platforms were used to compare WPOI 5 (more invasive) and WPOI 3 (less invasive) tumors. We identified 97 genes that were overexpressed at least 1.5-fold in the more invasive tumors. Forty-five genes were assessed using in vitro invasion assays. Significant reductions in invasion were seen for knockdown of 15 of the 45 genes. The gene whose knockdown had the strongest effect on invasion was Apolipoprotein E (APOE). Matrix degradation and mature invadopodia were decreased with APOE knockdown. APOE knockdown resulted in increased cellular cholesterol, and decreased phospho-ERK1/2, phospho-JNK, and phospho-cJun. AP-1 activity was decreased in a luciferase reporter assay. Expression of the AP-1 target MMP7 was also decreased. To identify additional downstream signaling molecules affected by APOE knockdown, we used RNA sequencing to assess global gene expression changes in OSCC cell lines. Using Ingenuity Pathway Analysis to analyze the differentially expressed genes, we found that our initial observation of deactivation of ERK, JNK, and cJun/AP-1 were supported by the RNA sequencing, as well as additional pathways that we had not initially observed.;In conclusion, our results support that APOE is an important gene in head and neck cancer invasion due to its role in regulation of cellular cholesterol and subsequent effects on cellular signaling and AP-1 activity. AP-1 regulates the expression of MMP7, as well as other invasion genes. The RNA sequencing studies helped to support these findings as well as highlight other potential signaling molecules and pathways that could be studied for their affect on invasion.
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