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. 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 molecular changes that contribute to the evolution of oral neoplasia, and to use this knowledge to develop markers of disease progression and novel therapeutic approaches for oral malignancies. Genomic and proteomic approaches to understand oral cancer: Our laboratory has used a number of novel approaches to investigate gene and protein expression profiles in HNSCC. We began by demonstrating the successful use of laser capture microdissection (LCM) to procure specific cell populations from heterogenous clinical samples of HNSCC, and showed that LCM-procured material can be used effectively to extract RNA, DNA, and proteins. We have recently focused on nasopharyngeal carcinoma (NPC), a major public health problem in Southeast Asia that also affects ~2,000 new patients each year in the US. Specifically, we conducted a genome-wide transcriptome analysis of NPC by probing high density cDNA microarrays with fluorescent-labeled amplified RNA derived from LCM cells procured from normal nasopharyngeal epithelium, areas of metaplasia-dysplasia, and carcinoma from Epstein Bar virus (EBV)-associated NPCs. This approach enabled the identification of genes differentially expressed in each cell population. Furthermore, this analysis identified numerous genes whose overexpression can help explain the aggressive clinical nature of this tumor type, including genes indicating cell cycle aberrations and invasive-metastatic potential, and a decrease in genes involved in apoptosis, cell structure, and tumor suppression. Ultimately, the aberrant expression and activity of molecules present in HNSCC cells are responsible for their malignant behavior. Thus, we have also made a concerted effort to investigate protein expression profiles during HNSCC tumor development. For example, we have examined the activity of tissue remodeling proteases in HNSCC in collaboration with PTRU, and conducted a direct proteome-wide analysis of LCM-procured normal and tumor tissues using a combination of liquid chromatography (LC) and tandem mass spectrometry (MS). In collaboration with William Hancock (Barnett Institute, MA), techniques for protein extraction and global proteolysis of whole cell lysates, followed by the separation of complex peptide mixtures by reverse phase LC and analysis by MS followed by tandem MS sequencing of selected peptides were optimized. In an initial analysis of clinical samples from HNSCC patients, amininoacid sequence information was obtained from 94-105 proteins per tissue set. Further development of these techniques may allow proteome-wide profiling of HNSCC tissues. Persistent activation of the Akt pathway in HNSCC: A potential target for UCN-01. The serine/threonine protein kinase Akt is a key regulator of normal and cancerous growth and cell fate decisions. Recently, we have observed that mouse models for chemically-induced SCC exhibit elevated Akt activity, and that Akt activation represents an early event during papilloma formation, which precedes SCC. Prompted by this observation, we have examined the status of activation of Akt in different stages of SCC development in mice and in HNSCC patients. By immunohistochemical analysis using phosphorylation-state specific antibodies, we have demonstrated that Akt activation corrrelates closely with the progression of mouse skin SCC, and observed that activation of Akt is a frequent event in human HNSCC. Interestingly, recently available evidence indicated that the novel chemotherapeutic agent UCN-01 blocks PDK1, an immediate upstream kinase that regulates Akt. Indeed, we provided evidence that the Akt signaling pathway may represent a biologically relevant target for UCN-01, at concentrations safely achievable in clinically relevant situations. Development of conditional animal models for squamous carcinogenesis: With the assistance of GTF, NIDCR, we have generated transgenic mice carrying the tetracycline-inducible system (tet-on receptor) targeted to the basal layer of stratified epithelium using the cytokeratin 5 promoter. In this system, transgenes of interest, including candidate oncogenes, can be expressed under the control of a tetracycline-responsive promoter. As a proof of principle, we have examined the ability of active ras genes to promote SCC when conditionally expressed. Although prior animal models suggested that active ras alone promotes benign papilloma formation with a variable rate of delayed carcinoma conversion, the use of this system, which targets a cell compartment that includes the epidermal stem cells, was sufficient to promote the rapid (10-20 days) formation of malignant SCC in the skin and oral tissues of animals treated with doxycycline. Using this model, we are now positioned to test the transforming potential in vivo of activated alleles of newly discovered signaling molecules, alone or in animals engineered to be deficient in relevant tumor suppressor genes. Current and future use of this model system may help to unravel the mechanisms responsible for SCC, and aid in the search for alternative oral cancer treatments. The Kaposi?s sarcoma associated herpesvirus vGPCR: Role in direct and paracrine neoplasia. KS is the most common cancer arising in HIV-infected patients and the most frequent oral neoplasm in immunosuppressed patients. The KSHV (HHV-8) has been recently identified as the infectious cause of KS, and the oral cavity may represent the primary source of infectious KSHV in both immunocompetent and immunosuppressed patients. The molecular characterization of the KSHV genome has revealed the presence of numerous potential oncogenes. To study systematically the transforming potential of KSHV-encoded proteins in vivo, we generated transgenic mice carrying the receptor for the avian leukosis virus, tv-a, targeted to the endothelial cells, and used these animals using highly infective avian retroviruses carrying candidate KSHV oncogenes. Surprisingly, among the numerous KSHV genes tested, vGPCR was the only gene that caused the formation of KS-like lesions, and we obtained evidence that cells expressing vGPCR cooperate with cells expressing latent genes to promote tumorigenesis. Collectively, these results implicated vGPCR in both the initiation and promotion of Kaposi?s sarcomagenesis. Which mitogenic and survival pathways are utilized by vGPCR to induce tumorigenesis is under current investigation. Currently available evidence indicates that vGPCR stimulates Akt in vivo, and that Akt plays a key role in both the direct and paracrine neoplasia induced by vGPCR. Indeed, Akt activation was found to be a hallmark of human KS, and thus may represent suitable targets for the development of pathogenesis-based therapies against KS.
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