PRECLINICAL CHEMOPREVENTION: BIOMARKERS OF CANCER RISK AND CHEMOPREVENTIVE RESPONSE

Advancements in the field of chemoprevention continue to be hindered by the unavailability of accurate and sensitive biomarkers of cancer susceptibility and disease progression. Such biomarkers are anticipated to facilitate the identification of asymptomatic individuals at high risk for cancer, and decrease the prohibitive cost and duration of both preclinical and clinical investigations. The ideal biomarker of cancer risk is an early alteration during the premalignant phase of neoplasia which predicts with high sensitivity and specificity the future progression and invasive potential of malignant cells.

The goal of this laboratory is to develop efficacious regimens for the clinical prevention of cancer. Translational studies continue to focus on the chemoprevention of colon, lung and oral cancer. Areas of research include: 1) the identification of effective chemopreventive agents and their mechanisms of action, 2) the establishment of biomarkers of cancer susceptibility and surrogate endpoints of carcinogenesis, and 3) the identification of populations at increased risk for cancer.

Mutations in the APC gene, the gatekeeper of colorectal cancer, are associated with the initiation of colorectal adenomas. One of the important tumor suppressor activities of APC is the regulation of bcatenin degradation. Mutations in the APC gene can cause b-catenin to accumulate in the cytoplasm and translocate to the nucleus. Increased nuclear localization of b-catenin accompanies the progression of colorectal adenomas to carcinomas. Sulindac sulfone (FGN-1) is an irreversibly oxidized derivative of the non-steroidal anti-inflammatory drug, sulindac, which lacks cyclooxygenase inhibitory activity. Its ability to inhibit chemically induced colon tumors, and cause regression of precancerous colonic lesions in familial adenomatous polyposis patients, has been demonstrated by others. The purpose of this study was to determine the effect of FGN-1 on the cellular distribution and expression of bcatenin in human colon carcinoma cells bearing mutated APC.

Subconfluent cultures of SW-480 human colon adenocarcinoma cells were treated with either 480 mM FGN-1 or vehicle (DMSO). Cells were harvested 48 hours posttreatment and either formalin fixed for immunohistochemistry or processed for immunoblotting. Western blot analyses and immunohistochemical staining were performed using polyclonal anti-b-catenin antiserum. Immunoreactive protein detected by Western blot was quantitated by densitometric scanning. Copyright restrictions prohibit discussion of the results from this study.

The AP-1 transcription factor comprises heterodimers of the fos and jun protein families, and has been implicated in the regulation of transcription by antioxidant inducers such as the chemopreventive agent oltipraz. To investigate the possibility that the GSTm class genes are regulated by AP-1, the basal expression of GST M1 and genes known to possess an antioxidant responsive element (ARE) was determined in two strains of mice bearing genetic alterations in the genes encoding c-fos and c-jun. The basal expression of mGST Ya and NAD(P)H:quinone oxidoreductase 1, genes regulated by AREs, was increased in cfos (-/-) homozygous knockout mice as compared to normal controls; although total GST activity was increased, immunoblots indicated little change in GSTm expression. Effects in cjun (+/-) heterozygous mice were much less pronounced than in c-fos knockouts; homozygous null c-jun mice are nonviable. Future experiments will focus on the effects of c-fos absence on the response of Phase II genes to antioxidant induction in the absence of c-fos. Cultured cells derived from c-fos (-/-) mice will be transfected with murine ARE/reporter constructs to determine the specific sequences sensitive to the c-fos null genotype.

Although the chemopreventive activity of oltipraz against a variety of tumor types has been well established, its mechanism of action remains to be elucidated. The transcriptional response to oltipraz exposure has been characterized in the mouse using an Atlas® multigene array. This array, containing 133 transcription factors and 455 other genes, was probed with labeled cDNA generated from the hepatic mRNA of vehicle- and oltipraz-treated ICR/Ha mice at 6 and 24 hours posttreatment. Due to copyright restrictions, the results from these analyses cannot be publicized at this time.

In contrast to men, the incidence of lung cancer among women has continued to increase over time. The contribution of genetic polymorphisms in the detoxication enzymes, cytochrome P450 1A1 (CYP1A1) and GST M1, to this increased risk was examined in the present study. Lung cancer patients (N=180) and healthy control subjects (N=138) completed a smoking questionnaire and donated a blood sample for genotypic analysis. No gender differences were observed among cancer patients in either age at presentation or histological diagnosis. Studies in progress aim to establish a genetic basis for the enhanced predisposition of female smokers to lung cancer and to identify a target population for smoking cessation and chemopreventive intervention. Copyright restrictions prohibit the inclusion of the results in this report prior to publication.

CLAPPER, M.L., ADRIAN, R.H., PFEIFFER, G.R., KIDO, K., COOPER, H.S., MURTHY, S. Depletion of colonic detoxication enzyme activity in mice with dextran sulfate sodium-induced colitis. Aliment. Pharmacol. Ther. 13:389-396, 1999.

LIU, S.C., SAUTER, E.R., CLAPPER, M.L., FELDMAN, R.S., LEVIN, L., CHEN, S.Y., YEN, T., ROSS, E., ENGSTROM, P.F., KLEIN-SZANTO, A.J.P. Markers of cell proliferation in normal epithelia and dysplastic leukoplakias of the oral cavity. Cancer Epidemiol. Biomark. Prev. 7:597-603, 1998.

LIU S-C., HU, Y., SAUTER, E.R., CLAPPER, M.L., CHEN, S.Y., LANFRANCHI, H.E., ENGSTROM, P.F., KLEINSZANTO, A.J.P. Image analysis of p53 and cyclin D1 expression in premalignant lesions of the oral mucosa. Quant. Cytol. Histol. (in press).

TOWNSEND, A.J., FIELDS, W.R., DOSS, A.J., CLAPPER, M.L., DOEHMER, J., MORROW, C.S. Modeling the chemoprotective functions of glutathione S-transferases in cultured cell lines by heterologous expression. Drug Metab. Rev. 31:43-69, 1998.

WILKINSON IV, J., RADJENDIRANE, V., PFEIFFER, G., JAISWAL, A.K., CLAPPER, M.L. Disruption of c-fos leads to increased expression of NAD(P)H:quinone oxidoreductase and glutathione S-transferase. Biochem. Biophys. Res. Comm. 253:855-858, 1998.

^§   Fox Chase researcher

^a   A. Flanigan, S. Murthy: MCP Hahnemann University, Philadelphia, PA 19102

^b   R. Venugopal, A. Jaiswal: Baylor College of Medicine, Houston TX 77030

^c   C. Dresler: SmithKline Beecham, 1500 Littleton Road, Parsippany, NJ 07054

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998