Recent studies have found that EPS8, a mediator of growth factor signaling to the cytoskeleton, may upregulate expression of the FoxM1B transcription factor and aurora A kinase, both of which have been linked to oncogenic activity. Cell lines transfected with EPS8 and FoxM1B, and appropriate controls, were generated and analyzed by MTT proliferation assays and flow cytometry for relative rates of cell proliferation as well as to determine the percentage of cells in different phases of the cell cycle. qRT-PCR and western blots confirmed higher levels of EPS8, FoxM1B and Aurora A kinase in the overexpressing cell lines. To investigate the role of PI3K-dependent signaling in EPS8-mediated upregulation of FoxM1B and its targets, studies were carried out usingLY294002, an inhibitor of PI3K. In cells overexpressing EPS8, treatment with LY294002resulted in decreased expression of FoxM1B and Aurora A kinase, indicating that PI3Ksignaling mediates EPS8-dependent upregulation of FoxM1B and Aurora A kinase. The study suggests that EPS8 deregulates cell growth by affecting the expression of common regulators of cell cycle progression, in part through PI3K, a known pro-oncogenic kinase.

Head and neck squamous cell carcinoma (HNSCC) is a type of cancer that begins in the epithelial cells that line the mucosal surfaces of the head and neck, including the oral cavity, pharynx, larynx, paranasal sinuses, nasal cavity, and salivary glands. Head and neck cancer is the sixth most common type of cancer with a 5-year survival rate of 60% for all cases. Over the past few years, a subset of cells with stem-like properties, called cancer stem cells, are believed to have tumor-initiation capabilities and are responsible for maintaining on-going tumor growth. Previous data from our lab suggested that cells grown in suspension, called spheroids, may have stem cell like properties. We employed a model system where a primary HNSCC cell line, HN4, was used to set up spheroids. We found that expression of EPS8 and its downstream targets, FOXM1 and CXCL5, was increased in HN4 spheroids. In addition, we measured the expression of Nanog, as it is a transcription factor involved in the self-renewal of human embryonic stem cells. We also used a metastatic HNSCC cell line, HN12, to see how it compared to spheroids. We wanted to investigate the hypothesis that activation of Src potentiates EPS8 function to deregulate downstream signaling pathways. We used a small molecule tyrosine kinase inhibitor, Dasatinib, on HN4 spheroids and HN12 cells. We found that when Src is inhibited, EPS8 expression is decreased in HN4 spheroids and it also interferes with spheroid formation. The results of the current study were also able to show that the proliferation capability of HN12 cells is greatly diminished when treated with Dasatinib, due to G1 arrest in the cell cycle. When we measured for FOXM1, which is a cell cycle regulator, we found the levels were reduced in Dasatinib treated cells, preventing the cells from completing mitosis. With all of the data taken together, it suggests that Src does in fact play a role in regulating the downstream signaling pathways of EPS8, and its inhibition leads to the loss of cell proliferation. Additional studies need to be performed to discover whether Src inhibition will stop the proliferation of cancer stem cells, which are believed to be more resistant to cytotoxic therapies.

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common type of cancer in the western hemisphere with a five-year survival rate of only 50% for patients with a localized tumor, which decreases significantly to as low as 5% for those patients with tumors that have metastasized to distant sites of the body. It has been found that both mutant p53 and epidermal growth factor receptor (EGFR) signaling pathways function to increase the expression of CXCL5, which has been identified as a key mediator in the process of tumor metastasis. Previous data from our lab suggested that the p53 homolog, p63, may function as a negative regulator of CXCL5 and that mutant p53 may inhibit this molecule to elevate CXCL5 expression levels. In the current study we utilized an model system in which the H179L p53 mutant was expressed in HN4 cells to investigate the hypothesis that mutant p53 enhances expression of CXCL5 by both interfering with p63 function and cooperating with EGFR/EPS8 signaling, leading to increased cell proliferation and motility. The results of the current study indicate a role for mutant p53 in head and neck squamous cell carcinoma proliferation, migration and tumorigenicity, possibly through enhancement of CXCL5 expression. We were able to show that mutant p53 expression caused an increase in the expression of this chemokine in addition to increasing proliferation and migration of the cells compared to the vector control. Additionally, we showed that p63 protein is a negative regulator of CXCL5 that is downregulated in the cells expressing mutant p53, which suggests that through direct interaction, mutant p53 may function to inhibit p63 function as well as target it for degradation. These results support the hypothesis that GOF mutant p53 enhances expression of CXCL5 by interfering with p63 function in cancer cells. The results of the current study results also showed that upon treatment with EGF, HN4 cells expressing mutant p53 express elevated levels of CXCL5; and that the mutant p53-expressing HN4 cells cooperate with EGFR/EPS8 signaling to further deregulate chemokine expression. These data taken together suggest there are complex interactions taking place between mutant p53, p63, EGFR signaling, and CXCL5 to regulate the biological processes that promote tumor progression that could lead to metastasis. Additional studies are needed to further elucidate the molecules involved in the mutant p53 mechanism that promotes tumorigenesis.

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