Squamous cell carcinoma of the head and neck (HNSCC) is the 6th most common cancer in the developed world, affecting nearly 44,000 patients each year in the US, which results in ~11,000 deaths. The vast majority of these malignancies involve neoplastic lesions in the oral cavity, lip, and pharynx. HNSCC remains among the most fatal and morbid of cancers at any anatomic site. Overall, fewer than 50% of HNSCC patients are alive 5 years after diagnosis, and those who do survive undergo severe functional and esthetic compromise secondary to their treatment. Like most cancers, HNSCC results from a series of discrete, irreversible and sequential alterations in genes that control cell growth and differentiation, together with genetic aberrations promoting invasion and metastasis. The goal of our program is scientific excellence in addressing the devastating problem of oral cancer. In particular, we aim to elucidate the genetic changes that contribute to the evolution of oral neoplasia, and to use this knowledge to develop molecular markers of disease progression and novel therapeutic approaches for oral malignancies. Gene discovery in oral squamous cell carcinoma through the head and neck cancer genome anatomy project (HN-CGAP): Confirmation by microarray analysis: The near completion of the human genome project and the recent development of novel, highly sensitive high-throughput techniques have now afforded the unique opportunity to perform a comprehensive molecular characterization of normal, precancerous, and malignant cells. As part of these efforts, the Head and Neck Cancer Genome Anatomy Project (HN-CGAP) was establish as a joint program between NCI and NIDCR aimed to identify the nature of those genes expressed during HNSCC development. Recently, we have used the bioinformatic tools in the CGAP website to analyze the database from the HN-CGAP libraries derived from microdissected normal and HNSCC tissues contributed by our ongoing effort. Differential expression of genes between normal and tumor tissues were further confirmed using an oral cancer specific cDNA microarray. A very high number of genes were discovered from these libraries, and highly expressed known genes uniquely expressed in normal or HNSCC libraries were also identified. Their contribution to HNSCC progression can now be investigated. The results from the oral cancer specific microarray suggest that some of these genes are highly expressed in this particular tumor type. These efforts, together with other multi-institutional genomic and proteomic initiatives are expected to contribute to the complete understanding of the molecular pathogenesis of HNSCCs, thus helping to identify new markers for the early detection of preneoplastic lesions and novel targets for pharmacological intervention in this devastating disease. Proteomic approaches to understand oral cancer: We have also made a concerted effort to investigate protein expression profiles during HNSCC tumor development. In collaboration with the NCI, we have demonstrated the feasibility of utilizing antibody arrays for the study of protein expression in laser-capture microdissection (LCM)-procured material. Towards this end, a novel method that involves the labeling of total cellular proteins by biotinylation, their exposure to antibodies arrayed on special membranes, and their detection by enzyme-conjugated stretptavidin, was developed. This study provided the first indication that antibody arrays can be used to detect the status of growth promoting and cell cycle regulatory molecules in microdissected clinical samples. It also helped identify a number of unexpected alterations in the Stat and MAP kinase-signaling pathways in the otherwise phenotypically normal tissue adjacent to neoplastic lesion. Potent antitumor activity of UCN-01 in carcinomas of the head and neck: Our team has continued its drug evaluation effort, which is aimed to developing novel therapies for improving the quality of life and life expectancy of oral cancer patients. In this regard, altered and deregulated cyclin dependent kinase (cdk) activity is now believed to play a major role in the pathogenesis of HNSCC, thus providing a suitable cellular target for therapeutic intervention. UCN-01 (7-hydroxy-staurosporine), is a protein kinase C and cdk modulator, which exhibits antiproliferative and antitumor properties in some experimental tumor models, thus representing a potential candidate to test in HNSCC. In collaboration with the Developmental Therapeutic Program (DTP), NCI, and the Molecular Therapeutic Unit, we found that UCN-01 displays potent antiproliferative properties (IC50~of 17-80 nM) in HNSCC cells. Cell cycle analysis revealed that UCN-01 treatment of HNSCC cells for 24 h leads to a G1 block with a concomitant loss of cells in S and G2/M and the emerging sub-G1 cell population, confirmed to be apoptotic by TUNEL analysis. Antitumor properties of UCN-01 were also assessed in vivo, by treating HNSCC xenografts with UCN-01 for 5 consecutive days. Total sustained abolition of tumor growth (p<0.00001) was obtained with only one cycle of UCN-01 treatment. TUNEL staining of xenograft samples revealed a higher incidence of apoptosis in treated tissues when compared with control. Thus, UCN-01 exhibits both in vitro and in vivo antitumor properties in HNSCC models, and may represent a suitable drug candidate for treating HNSCC patients. Endothelial-specific retroviral transduction of Kaposi?s Sarcoma Virus oncogenes: A novel mouse model for Kaposi?s Sarcoma: Kaposi?s Sarcoma (KS) is the most common cancer arising in HIV-infected patients and the most frequent oral neoplasm in immunosuppressed patients. KS has also emerged as one of the most prevalent cancers among children and adult men in the developing world. The Kaposi?s Sarcoma Associated Herpesvirus (KSHV; HHV-8) has been recently identified as the infectious cause of Kaposi?s Sarcoma. Of interest, compelling evidence now supports a critical role for the oral cavity as the primary source of infectious HHV-8 in both immunocompetent and immunosuppressed patients. Indeed, mucosal shedding of this virus may represent the principal mode of viral transmission. Unfortunately, despite extensive investigation into its molecular etiology, KS remains a devastating and incurable disease. The molecular characterization of the KSHV genome has revealed the presence of numerous potential oncogenes. To begin addressing their contribution to the development of the endothelial cell-derived KS tumor, we developed a novel transgenic animal model expressing the avian retroviral receptor, TVA, under the TIE2 promoter. This enabled endothelial cell-specific infection in vivo using avian leukosis virus expressing candidate KSHV oncogenes, including Kaposin, vFlip, vGPCR, vIRF-1, vCyclin, and vBCL-2. Remarkably, retroviral transduction of one gene, vGPCR, was sufficient to induce angioproliferative tumors that strikingly resemble human KS, suggestive of a critical role for vGPCR in initiating KS tumor development. As GPCRs have proven to be effective targets for pharmacological intervention, these results also suggest that the vGPCR may represent an ideal target for the development of pathogenesis-based therapies against KSHV.
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