IADR Abstract Archives
The Role of Fibronectin and Btbd7 in Oral Squamous Cell Carcinoma
Objectives: Our laboratory previously established that the extracellular matrix protein fibronectin (FN) and its downstream signaling effector Btbd7 promote transient normal epithelial cell migration during development. Therefore, to help bridge the gap between cell migration during development and invasive migration during cancer, we investigated the role of FN-binding integrins and Btbd7 in oral squamous cell carcinoma (SCC). Specifically, we investigated whether the FN receptor α5β1 integrin and Btbd7 are required for oral SCC invasion and migration.
Methods: A classic Matrigel-coated transwell invasion assay was used to evaluate normal human oral keratinocyte, SCC-25, and SCC-9 for their invasive capabilities, while a complementary collagen spheroid assays was used to evaluate migratory invasive behaviors. We also used an immunolocalization approach to examine the expression of FN, α5β1 integrin, and Btbd7.
Results: We evaluated the invasive capabilities of two oral SCC lines, both isolated from the tongue, compared with normal human oral keratinocytes. SCC-9 cells, but not SCC-25 cells, proved significantly more invasive than normal oral keratinocytes. To further evaluate the difference between the non-invasive SCC-25 cells and invasive SCC-9 cells, we investigated the expression patterns of FN in each of these lines. Immunofluorescence demonstrated that confluent SCC-9 cells assembled large FN bundles, whereas SCC-25 cells did not. Additionally, the SCC-9 cells exhibited co-localization of FN and its receptor, the α5β1 integrin. Inhibition of α5β1 integrin function inhibited not only SCC-9 invasion in the transwell assay, but also SCC-9 migration from spheroids in collagen. Because Btbd7 has been shown to be activated downstream of FN, we tested its role in invasiveness by depleting Btbd7 protein levels in SCC-9 cells with siRNAs. Reduction of Btbd7 levels resulted in substantial inhibition of cell invasion through the transwells.
Conclusions: Together, these results indicate that the FN α5β1 integrin complex and Btbd7 play roles in SCC-9 cell invasion. Future work will seek to determine a direct causal link between FN and Btbd7 in these cells.
Methods: A classic Matrigel-coated transwell invasion assay was used to evaluate normal human oral keratinocyte, SCC-25, and SCC-9 for their invasive capabilities, while a complementary collagen spheroid assays was used to evaluate migratory invasive behaviors. We also used an immunolocalization approach to examine the expression of FN, α5β1 integrin, and Btbd7.
Results: We evaluated the invasive capabilities of two oral SCC lines, both isolated from the tongue, compared with normal human oral keratinocytes. SCC-9 cells, but not SCC-25 cells, proved significantly more invasive than normal oral keratinocytes. To further evaluate the difference between the non-invasive SCC-25 cells and invasive SCC-9 cells, we investigated the expression patterns of FN in each of these lines. Immunofluorescence demonstrated that confluent SCC-9 cells assembled large FN bundles, whereas SCC-25 cells did not. Additionally, the SCC-9 cells exhibited co-localization of FN and its receptor, the α5β1 integrin. Inhibition of α5β1 integrin function inhibited not only SCC-9 invasion in the transwell assay, but also SCC-9 migration from spheroids in collagen. Because Btbd7 has been shown to be activated downstream of FN, we tested its role in invasiveness by depleting Btbd7 protein levels in SCC-9 cells with siRNAs. Reduction of Btbd7 levels resulted in substantial inhibition of cell invasion through the transwells.
Conclusions: Together, these results indicate that the FN α5β1 integrin complex and Btbd7 play roles in SCC-9 cell invasion. Future work will seek to determine a direct causal link between FN and Btbd7 in these cells.