John L. Clifford - US grants

Squamous cell carcinoma (SCC) is the most clinically aggressive form of non-melanoma skin cancer and in spite of aggressive treatment, deeply invasive SCC lesions recur at a high rate (30 percent). Therefore there is an urgent need for the development of improved therapeutic and preventive treatments for this disease. Retinoids are important modulators of epithelial differentiation and proliferation. Retinoids are effective in the treatment and prevention of epithelial cancers, including cutaneous SCC. However the mechanism for this effect is not well understood. In order to better design chemopreventive therapies for cutaneous SCC, more information about the mechanism of retinoid action in this system is needed. Retinoids exert their effects primarily through nuclear receptors, retinoic acid receptors (RARalpha, beta and gamma) and retinoid X receptors (RXRalpha, beta and gamma), members of the steroid hormone receptor superfamily. Retinoid receptor loss has been correlated with malignancy in several systems, and one recent study has demonstrated a suppression of RARalpha, RARgamma and RXRalpha expression in SCCs of patients. This study is based on the hypothesis that retinoid receptor loss contributes to the malignant phenotype by rendering epithelial cells resistant to retinoids, which wold normally suppress carcinogenesis. To test this hypothesis, HaCaT cells, a spontaneously immortalized, nontumorigenic, keratinocyte- derived cell line was chose. These cells express the two predominant retinoid receptors normally found in skin, RARgamma and RXRalpha, and retain the same differentiation characteristics and response to retinoids as normal keratinocytes. The HaCaT cells, which are a model for premalignant cells, will be used to accomplish the following specific aims: 1.) To create RARgamma null, RXTalpha null and RARgamma/RXRalpha double null cells by homologous recombination-mediated gene targeting. 2.) To determine whether loss of receptor expression results in an altered cell phenotype, specifically with regard to tumorigenic potential, differentiation capacity and response to alterations in retinoic acid (RA) levels. 3.) To reexpress the missing receptor (RARgamma or RXRalpha) in the knockout cell lines by stable transfection in order to determine whether the putative phenotype observed is the direct result of the genetic lesion. A somatic cell gene targeting approach has been successfully by the principle investigator to analyze retinoid receptor function in F9 embryonal carcinoma cells. It is expected that the generation of receptor null HaCaT cells will provide an equally powerful tool for understanding the role of retinoid receptors in the development of cutaneous SCC, as well as their role in epidermal differentiation.

[unreadable] DESCRIPTION (provided by applicant): Project Summary: Transitional cell carcinoma of the bladder (TCC) ranks 4th in incidence of all cancers in the developed world, yet the mechanisms of its origin and progression remain poorly understood. There are also few useful diagnostic or prognostic biomarkers for this disease. The long-term aim of this proposal is to determine molecular mechanisms involved in early and late steps in the development of TCC. In addition we hope to identify new biomarkers for detection, diagnosis and prognosis of TCC. To accomplish this we will use a mouse model for bladder cancer in which the SV40 large T antigen (SV40 T) is expressed under the control of the uroplakin promoter, which restricts expression to the bladder urothelium. These mice (UPII- SV40T) develop TCC, which is preceded by a condition resembling human CIS. The hypothesis underlying this proposal is that there are differences in gene expression profiles and gene regulatory networks between invasive TCC and CIS and between CIS and distant normal appearing urothelium, and that these changes are indicative of the malignant state. This hypothesis is based on the multistep carcinogenesis concept. An extension of our hypothesis is that CIS is an intermediate step between normal urothelium and invasive TCC and that there should be changes in gene expression corresponding to this intermediate state. The specific aims are: 1. Identify the genes and gene regulatory networks that are differentially expressed between the normal urothelium of nontransgenic and UPII-SV40T mice, CIS and TCC, using Affymetrix GeneChip(r) Arrays. The relevence of individual genes to human TCC will be validated by assessing expression of the human homologs in patient TCC and normal urothelium specimens. In silico analysis of the microarray data, which includes generating gene expression profiles (clustering analysis) and the development of promoter models based on the promoter sequences of the differentially expressed genes will be performed. 2. Assess the function and potential relevance to bladder carcinogenesis of these genes in vivo by stable overexpression, or siRNA-mediated suppression, of the validated genes in human bladder-derived cell lines. Relevence: Completion of these studies should advance our understanding of the molecular events underlying TCC development. In addition, the identification of genes differentially expressed between normal urothelium, CIS and TCC will provide much needed diagnostic and prognostic markers for this disease. [unreadable] [unreadable] [unreadable]