The Effect of the Activation of the Wnt16 Gene in Head and Neck Squamous Carcinomas

Title: The Effect of the Activation of the Wnt16 Gene in Head and Neck Squamous Carcinomas

Fellow: Jared Mark Alswang

Preceptor: Dr. Antonio Jimeno

Background: Head and neck cancers are a group of cancers that primarily originate in the squamous cells of the head and neck and in the salivary glands. In 2012, 52,000 Americans were diagnosed with a head and neck cancer, about three percent of all cancers in the United States. The average survival rate of head and neck cancers is about 50%, making them some of the deadliest types of cancers. In this project the relationship between head and neck squamous cell carcinomas (HNSCC) and the wnt signaling pathway was researched. The wnt-signaling pathway plays an integral role in the regulation of cell-to-cell interactions and is associated with stem cell control. Researchers have identified wnt proteins as proto-oncogenes and have correlated wnt’s misregulation with deleterious cell growth, responsible for malignant and benign tumor production alike.

Material and Methods: To observe the effect of the activation of the wnt16 gene on cancerous stem cells, the wnt16 gene was virally inserted into CUHNO67 cancer cells, so that certain characteristics of the cell line could be compared to that of a Parental No DNA cell line and a Parental Empty cell line. Using RT-PCR’s and western blots, the level of expression of certain genes and proteins was detected in cells with an activated wnt-signaling pathway. Sphere formations, a sign of stem cell proliferation, were tested by performing a non-adherent cell culture. A matrigel invasion assay was used to test the ability of wnt16-activated cells to invade through a membrane. The difference in drug resistance to Docetaxel, Erlotininb, Rapamycin, and ZSTK474 between cell lines was tested using an SRB assay.

Results: In preliminary western blots, we were able to demonstrate that the activation of wnt16 correlated with an increase in expression of the P-LRP6 protein and a decrease in expression of LRP6 and SNAIL proteins. RT-PCR results indicated that the activation of the wnt16 gene caused on average a 250% increase in SOX2 expression (p=0.114), a 60% decrease in Zeb1 expression (p=0.027), a 45% decrease in SNAI1 expression (p=0.006), and a 55% decrease in Axin2 expression (p=0.018) compared to the Parental No DNA control. The wnt16 cell line produced on average 27 more spheres per image than the Parental No DNA control and 45 more spheres per image than the Parental Empty control. But because of the large range of sphere sizes observed in each cell line, the overall average sphere sizes were not significantly different between the two controls and the wnt16 cell line. In the invasion assay, there was a consistent pattern indicating that the wnt16 activated cells invaded the membrane less frequently than the controls, however, due to the variance in data between the trials the statistical significance of these results are questionable. There was no statistically significant difference in drug resistance in the SRB assays.

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