Neoplastic transformation of cells is characterized by loss of control of cell proliferation and failure of the cells to undergo differentiation. Central to the development of neoplasia are genetic events involving both activation of oncogenes and inactivation of tumor suppressor genes. The retinoic acid receptors (RARs) are important in regulation of cell growth and differentiation via their role as mediators of retinoid action. While several studies suggest that alterations in at least one retinoic acid receptor gene, RAR alpha. may play a role in the development of acute promyelocytic leukemia, the potential role that alterations in RARs may play in other human tumors has not been systematically evaluated. The objective of this proposal is to test the hypotheses that alterations in RARs may contribute to human carcinogenesis and further that the RARs may act as tumor suppressor genes in human cancer. Since several studies indicate that the molecular alterations observed in tumors are at least partly organ specific, tumors derived from two different organs will be examined. Primary squamous cell carcinomas (SCCs) of the head and neck and their metastases will be studied because of the important role of retinoids in squamous cell differentiation and because of the positive effects of retinoid treatment in some cases of squamous cell malignancies. SCC cell lines will also be examined. A collection of non-small cell lung carcinomas (NSCLC) composed of squamous cell carcinomas and adenocarcinomas will also be studied. These tumors exhibit frequent loss of heterozygosity at chromosome 3p21-25, where the RAR beta gene resides. All tumors will be examined for gross alterations and changes in expression of the 3 RARs with Southern and Northern techniques. All tumors will also be screened for mutations in each RAR using a reverse transcriptase PCR/Single Stranded Conformation Polymorphism (SSCP) protocol. Mutations detected by SSCP screening will be identified by direct sequencing of PCR products. The effects of any identified mutant receptors will be characterized with respect to ligand binding, mutant RAR-RXR dimerization, and transactivation capabilities. Mutant receptors will also be tested to determine whether they act in a dominant-negative fashion on the retinoic acid-induced differentiation of P19 embryonal carcinoma cells. Lastly, antisense expression techniques will be used to test the hypothesis that the RARs may act as tumor suppressor genes in vitro. The effects of functional elimination of each RAR on the phenotypes of two different immortalized human cell lines, a keratinocyte and a SCC cell line (non-tumorigenic in nude mice), will be studied with respect to morphological transformation, ability to grow in soft agar, to elicit tumors in nude mice, and to invade a basement membrane material (Matrigel) in vitro. These descriptive and mechanistic studies should provide important information as to the potential role of RARs in human carcinogenesis and the molecular changes associated with squamous cell carcinogenesis. They may also ultimately provide insights on the potential use of retinoids as therapeutic agents in the treatment of human cancer.
