Novel in Vitro Oral Cancer Gene Identification Assay

Objectives: Oral squamous cell cancer (OSCC) is characterized by dysregulation of genetic pathways allowing epithelial cells to proliferate, invade, and metastasize. The specific genetic disruptions vary from tumor to tumor. Distinguishing which alterations are the result of cancer and which cause cancer is difficult. Early tumor genotyping might allow interventions to be tailored to the genetic makeup of a patient's particular cancer. OSCC is often detected in late stages of tumor progression and surgery results in disfigurement and loss of function. Five-year survival rates for OSCC have not improved in the last 40 years. These facts define the need to understand the genetically diverse pathways that drive OSCC. Sleeping Beauty (SB) insertional mouse mutagenesis can be used for forward genetic screens for genes involved in cancer development and we hypothesize, for OSCC also. Methods: We employed a murine SB expression system in which the transposase (SB11) is knocked into the Rosa26 locus. We have bred Rosa26-SB11 transgenic mice with a mutagenic transposon vector (T2/Onc) for the purpose of either disrupting tumor suppressor genes or activating oncogenes in wild-type mice or in cancer-predisposed p19ARF-null mice. SB transposition has already proved to accelerate sarcomas or induce lymphomas in adult mice in vivo. We hypothesize that oral keratinocytes from mice in which SB transposition is occurring will show signs of transformation in vitro. We have isolated oral keratinocytes from SB co-transgenic mice. Results: An epithelial phenotype could be verified by cytokeratin immunohistochemistry. Interval assessment experiments are currently being performed to detect tumorigenic transformation using morphology, doubling time, loss of contact inhibition, anchorage independent growth (soft agar assay and spheroid cultures), and in vivo tumor formation. Conclusions: A keratinocyte culture system from the oral cavities of SB transgenic mice could provide a new tool for identifying driver mutations in OSCC. NIDCR T32 DE007288 12-16