Career Development Program
Project Two: Role of PKCepsilon in Establishing an Aggressive Phenotype in HNSCC
Quintin Pan, Ph.D.
In the United States, head and neck squamous cell carcinoma (HNSCC) accounts for 38,000 new cases and an estimated 14,000 deaths per year. Surgery and radiotherapy are highly effective in the treatment of stage I and II disease; however, over 70% of patients present with locoregionally advanced stage III and IV disease. In spite of therapy, locoregional disease recurs in 60% of patients and metastatic disease develops in 15-25% of patients, causing a major decline in quality and length of life. Fewer than 30% of HNSCC patients are free of disease after three years, and the 5-year survival rates have remained largely unchanged in the past three decades. Therefore, there is a need to identify genes that are responsible for inducing an aggressive HNSCC phenotype prone to relapse and metastasize to allow for better clinical detection and management. Recent evidence has shown that protein kinase Ce (PKCε), a member of a family of serine/threonine protein kinases, is a transforming oncogene. To date, there is only a limited literature on the role of PKCε in HNSCC development and progression. Our preliminary data provides evidence that PKCε is involved in establishing an aggressive, highly metastatic and chemoresistant phenotype in HNSCC. In this proposal, we will functionally characterize the role of PKCε in HNSCC and translate its biology into clinical utility. Our hypothesis is that detection of PKCε in HNSCC will identify highly aggressive phenotypes at the time of primary diagnosis and allow tailored treatment and perhaps targeted intervention to prevent the development of recurrence and metastases. Specifically, we will (a) determine the clinical utility of PKCε as a novel biomarker of aggressive head and neck cancers in a large cohort of patients with clinical and treatment information, (b) determine the role of PKCε in regulating the neoplastic and chemotherapy resistant phenotype of head and neck cancers by independently overexpressing and inhibiting PKCε in immortalized human oral keratinocytes and in head and neck squamous carcinoma cells, respectively and (c) screen and identify small molecule inhibitors targeted against the unique C2 domain of PKCε.
Specific Aim 1: To determine the clinical utility of PKCε as a novel biomarker of aggressive head and neck cancers in a large cohort of patients with clinical and treatment information.
Our working hypothesis is that PKCε protein levels will identify aggressive HNSCC prone to relapse and metastasis, which will aid clinicians in tailoring treatment and offering a novel target of intervention. We have obtained high-density tissue microarrays from other members of this SPORE grant, namely Drs. Bradford and Chepeha, and are in the process of optimizing a monoclonal PKCε antibody for tissue staining.
Specific Aim 2: To determine the role of PKCε in regulating the metastatic and chemotherapy resistant phenotype of head and neck cancers by independently overexpressing and inhibiting PKCε in immortalized human oral keratinocytes and in head and neck squamous carcinoma cells.
Our working hypothesis is that high PKCε levels are associated with a more invasive and chemoresistant phenotype in HNSCC. We screened a panel of fifteen HNSCC cell lines obtained from Dr. Carey and identified two cell lines, UMSCC11A and 36, with high endogenous PKCε protein levels. Using RNA interference technology, we generated stable PKCε negative UMSCC11A and 36 clones. The PKCε negative UMSCC11A and 36 clones were significantly less invasive and motile than the control-transfected clones or parental wildtype cells. The proliferation rates of PKCε negative UMSCC11A and 36 clones were similar to control-tranfectants and parental cells. Moreover, we found that PKCε negative UMSCC11A and 36 clones were significantly more sensitive to cis-platinum. These exciting results indicate that PKCε is intimately involved in promoting a metastatic and chemoresistance phenotype in head and neck cancer and more importantly, inhibition of PKCε, by itself, is sufficient to alter the aggressive phenotype.
At present, we are in the process of designing in vivo experiments to determine if our in vitro findings will translate to in vivo animal models. We will determine the tumorigenic and metastatic potential of these PKCε negative UMSCC11A and 36 clones using the floor of the mouth orthotopic model of head and neck cancer and the pulmonary colonization metastasis model. Moreover, we will ascertain whether the PKCε negative UMSCC11A and 36 clones are more responsive to systemic cis-platinum therapy in vivo. These studies as outlined will provide further evidence to support the validation of PKCε as a novel therapeutic target for head and neck cancer.