MOLECULAR PATHOLOGY OF TUMOR PROGRESSION

Using human and mouse tumor models, we are studying the cellular and molecular events that characterize gradual malignant changes. Focusing on cell cycle-regulating genes such as cyclins, cyclin-dependent kinases and E2F-1, we have observed that abnormalities in these genes are associated with the development and/or progression of ductal adenocarcinomas of the breast, squamous cell carcinomas (SCC) of skin and carcinomas of the upper aerodigestive tract. In addition, early abnormalities in cell cycle regulation have been studied in premalignant conditions of the oral epithelia, which coincide with an increase in microvessel density and vascular endothelial growth factor (VEGF) expression, indicating that angiogenesis is an early event in oral epithelial carcinogenesis. In a mouse lipoxygenase knock-out model, we have observed that angiogenesis is induced by in situ dysplastic precursor lesions and is probably regulated by 12p-lipoxygenase (12-LO).

We have evaluated E2F-1 in several head and neck tumors and found that this cyclin cycle-regulating gene (CCRG), which is the best known terminal transcription factor of the cyclin-cdk-Rb pathway, is overexpressed in several SCC cell lines and primary tumors of high grade phenotype. Similarly, using immunohistochemistry, we found that E2F-1 could be a useful tool to grade tumors. Although this was not totally clear with our present series of SCC of the head and neck (more are being accrued and analyzed), it proved to be a very good marker in breast ductal carcinomas. We studied the expression of this gene product in 133 specimens, including normal breast (NB), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) of different grades and stages in order to investigate the feasibility of its use as a marker. Although, E2F-1 was present to some extent in all specimens, we found that DCIS had higher E2F-1 expression than NB and that this further increased in IDC. The percentage of E2F-1 positive cells increased from 2% in normal ductal epithelium to 6% in DCIS and to 15% in IDC. In addition, high-grade tumors, as well as advanced stage disease, correlated with higher expression of E2F-1. In an in vitro experiment, we found that a similar difference existed between a non-tumorigenic breast cell line and two widely used breast carcinoma cell lines. The breast carcinoma cell lines, T-47D and MCF7, had more E2F-1 positive cells than the non-tumorigenic cell line, MCF-10F, as assayed by immunohistochemistry. Western blot analysis also showed that carcinoma cells expressed more E2F-1 protein than the non-carcinoma cell line.

This study indicates that deregulation of E2F-1 may be involved in the development of breast IDC because E2F-1 expression was significantly higher in IDC and DCIS than in NB. In addition, E2F-1 expression could also be involved in tumor progression since the increased E2F-1 score correlated with known prognostic predictors of breast cancer, such as histologic grade, stage, metastasis status and estrogen receptor/progesterone receptor (ER/PR) levels. Thus, E2F-1 is a promising candidate to become a new prognostic and/or predictive marker of breast cancer and eventually of SCC of head and neck.

The clinical assessment of angiogenesis has been linked to the risk of metastasis and overall survival in many tumors including head and neck SCC (HNSCC). VEGF, an endothelial-specific mitogen overexpressed in most tumors, is felt to be a key regulator of angiogenesis. This study evaluates VEGF expression and microvessel density during the progression from normal epithelium to invasive cancer of the tongue.

Paired tissue sections of tongue specimens containing normal epithelium, mild dysplasia, carcinoma in situ (CIS), and/or invasive cancer (T1 primary tumors) from fifty different patients who had received no prior therapy for their lesion were immunostained for VEGF protein and factor VIII-related antigen. VEGF staining within all pathologic lesions and adjacent normal epithelium (if present) was scored using a semiquantitative scale. Quantitative assessment of factor VIII staining was used to evaluate microvessel density in peritumoral or intratumoral stroma, and in the subepithelial plexi of all pathologic lesions and adjacent normal epithelium present within each specimen. Statistical significance was identified using Dunnett's two-tailed t-test and the Kruskal-Wallis test.

Normal epithelium contained few capillaries in the subepithelial plexus (87.7±21.0 vessels/mm²). In marked contrast, there were significant differences in the number of capillaries associated with dysplastic epithelium (mild dysplasia and CIS; 147.0± 21.7 and 161.7± 28.4, respectively) that persisted in the stroma surrounding invasive cancers (163.6± 37.5). Intense staining for VEGF was not seen in normal epithelium, but there was a shift towards strong expression of VEGF within dysplastic epithelium, CIS, and invasive cancer (p=0.0001, Kruskal-Wallis test). A strong relationship existed between VEGF expression and subepithelial MVD (p=0.001, Kruskal-Wallis test).

VEGF overexpression may participate in a change from premalignant disease to invasive cancer of the oral cavity. This overexpression is paralleled, or even preceded, by an increase in microvessel density in the vicinity of precursor lesions. Angiogenesis represents an early event in the carcinogenesis of HNSCC that does not appear to be related to tumor size or malignant potential.

p53 mutation frequency in head and neck tumors is increased by exposure to tobacco smoke, but studies have not examined whether any environmental or dietary exposures reduce p53 mutations. Many studies have shown a reduction in risk of head and neck cancer with high intake of fruits and vegetables, and use of vitamin supplements. The mechanism of this protective effect may be related to antioxidant defenses against DNA damage. We examined the effect of vitamin supplement use, in conjunction with other exposures, on the frequency of p53 gene mutations in head and neck tumors.

Tumors from 135 patients with head and neck cancer were analyzed for p53 mutations by single-stranded conformation polymorphism analysis and DNA sequencing. Information on vitamin supplement use and other exposures was obtained by questionnaire. We compared the frequency of p53 mutations according to vitamin use and other variables.

p53 mutations were found in 35.6% of tumors. Regular use of vitamin supplements corresponded to a 64% decrease in the risk of p53 mutation (odds ratio, 0.36; 95% CI, 0.17 to 0.80; P=0.01). Use of vitamin C was strongly protective for p53 mutation (odds ratio, 0.27; 95% CI, 0.09 to 0.84; P=0.02), as was use of any antioxidant vitamin A, C or E (odds ratio, 0.23; 95% CI, 0.08 to 0.65; P=0.005). Cumulative smoking exposure modified the association between vitamin supplement use and p53 mutation frequency. Among those who smoked for less than 30 years, vitamin use profoundly reduced risk of p53 mutation (odds ratio 0.04; 95% CI, 0.00 to 0.31; P=0.001). Among longer-term smokers (>=30 years), the reduction in mutation frequency was not statistically significant (odds ratio 0.89; 95% CI, 0.34 to 2.30; P=0.82).

We observed a strong reduction in p53 mutation frequency in head and neck tumors with regular use of vitamin supplements. To our knowledge, this is the first published report of a protective effect of vitamin supplements on p53 mutation. Because of the more aggressive phenotype of tumors with p53 mutations, these results may have implications for chemoprevention

Arachidonic acid (AA) metabolites are heavily involved in numerous reactive mechanisms of disease such as inflammation, immune responses, tissue repair and proliferation. More specifically they have been associated with tumor development and, particularly, with tumor promotion. One of the major pathways of AA metabolism is via 12-LO. This pathway results in the formation of 12-hydroxyeicosatetraenoic acid (12-HETE). One member of the 12-LO family is the platelet type 12LO (P-12-LO), which is localized in platelet, megakaryocytes, epidermal cells, and some tumor cell lines.

Skin tumor promoters are known to increase the 12-HETE levels in skin, and together with other AA metabolites, is considered to be partially or totally responsible for the inflammatory and hyperplasiogenic effects of tumor promoters. In order to determine more precisely the role of P-12-LO in the process of skin carcinogenesis, we used mice in which the P-12-LO gene was disrupted by gene targeting (P-12LO-/- mice; Johnson et al., Proc. Natl. Acad. Sci. 98:3100, 1998). Sixteen wild type and twelve p-12LO -/- mice were treated with a two stage carcinogenesis protocol using DMBA as the initiator and TPA as the promoter. After 35 weeks, the number of papillomas per mouse was approximately 5 in both groups of mice. Conversely, the number of squamous cell carcinomas per mouse was 0.75 and 0.08 in the wild type and knock out groups, respectively. Immunohistochemical analysis of P12LO showed an absence of expression in the P-12LO-/- mice and variable expression in the tumors of the wild type animals. In addition, increased expression of P-12LO was seen in mast cells of the dermis in tumor-bearing wild type animals. Other studies using explants of aortic endothelium indicated that the in vitro growth ability of endothelial cells of wild type animals was markedly enhanced in serum-free medium as compared with the endothelial explants from knock out animals. Taken together, these results indicate that the absence of P12LO produced a 10-fold decrease in the incidence of squamous cell carcinomas of the skin. This enzyme could exert its function not only by producing AA metabolites that influence inflammatory processes in the dermis and proliferation of epithelial cells, but could also mediate, either directly or indirectly, the process of tumor angiogenesis and affect conversion of papillomas into carcinomas. On-going studies using a complete carcinogenesis protocol seem to support this evidence.

LAW, S.F., ZHANG, Y.Z., KLEIN-SZANTO, A.J.P., GOLEMIS, E.A. Cell cycle-regulated processing of HEF1 to multiple protein forms differentially targeted to multiple subcellular compartments. Mol. Cell. Biol. 18:3540-3551, 1998.

LIU, J.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 in premalignant lesions of the oral mucosa. Anal. Quant. Cytol. Histol. (in press).

MA, J., LI, F., KLEIN-SZANTO, A.J.P., GALLO, J.M. Modulation of angiogenesis by human glioma xenograft models that differentially express VEGF. Clin. Exp. Metast. 16:559-568, 1998.

MIKNYOCZKI, S.J., LANG D., HUANG, L., KLEIN-SZANTO, A.J.P., DIONNE, C., RUGGERI, B.A. Expression of neurotrophins and Trk receptors in human ductal pancreatic cancer. Int. J. Cancer 81:417-427, 1999.

RADJENDIRANE, V., JOSEPH, P., LEE, Y-H., GONZALEZ, F.J., KIMURA, S., KLEIN-SZANTO, A.J.P., JAISWAL, A.K. Knock-out of the DT diaphorase (NQ01) gene in mice leads to increased menadione toxicity. J. Biol. Chem. 273: 7382-7389, 1998.

SAUTER, E.R., NESBIT, M., WATSON, J.C., KLEIN-SZANTO, A.J.P., HERLYN, M. Vascular endothelial growth factor expression is a marker of tumor invasion and metastasis in squamous cell carcinomas of the head and neck. Clin. Cancer Res. 5:775-782, 1999.

SAUTER, E.R., KLEIN-SZANTO, A.J.P., MONTONE, K.T., GOODROW, T., BINDER, R., SEYKORA, J.T., HERLYN, M. UVB-induced epithelial and melanocytic changes in a xenograft model of human skin cancer. Mol. Carcinogen. 23: 168-174, 1998.

TANNO, S., OBARA, T., FUJII, T., MIZUKAMI, Y., NISHINO, N., URA, H., KLEIN-SZANTO, A.J.P., KOHGO, Y. Proliferative potential and K-Ras mutations in epithelial hyperplasia of the gall bladder in patients with anomalous pancreaticobiliary ductal union. Cancer 83(2): 267-275, 1998.

KLEIN-SZANTO, A.J.P., SAUTER, E.R., BABB, J.S., RIDGE, J.A., FRIEDMAN, C.D., WATSON, J.C. Early induction of angiogenesis by preinvasive lesions of the oral mucosa. In Head and Neck Oncology, edited by J. Alvarez Vincent. First World Congress. pp. 1215-1218, 1998.

WATSON, J.C., COOPER, H.S., BABB, J.S., SIGURDSON, E.R., KLEIN-SZANTO, A.J.P. Angiogenesis is an early event in the adenoma-carcinoma sequence in human colon cancer. Surg. Forum 50: 440-442, 1998.

JOSEPH, P., KLEIN-SZANTO, A.J.P., JAISWAL, A.K. Hydroquinones cause specific mutations and lead to cellular transformation and in vivo tumorigenesis. Brit. J. Cancer 78(3): 312-320, 1998.

KISIELEWSKI, A.E., XIAO, G.H., LIU, S.C., KLEIN-SZANTO, A.J., NOVARA, M., SINA, J., BLEICHER, K., YEUNG, R.S., GOODROW, T.L. Analysis of the FHIT gene and its product in squamous cell carcinomas of the head and neck. Oncogene 17(1):83-91, 1998.

§   Fox Chase researcher

^a   T.L. Goodrow: Present address-Merck Research Laboratories, West Point, PA 19486

^b   M. Brotzman, B. Trock: Molecular Epidemiology Program, Georgetown University Medical Center, Washington, DC 20007

^c   M. Shafarenko: Temple University, Philadelphia, PA 19122

^d   C. Funk, E. Johnson, J. Virmani: Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

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

Fox Chase Cancer Center

Scientific Report 1998