BREAST CANCER PREVENTION

The incidence of breast cancer is steadily increasing in most Western countries and in societies that are recently becoming westernized. While the reasons for this increase are uncertain, epidemiologic and clinical evidence indicates that endocrine and reproductive influences play major roles in this phenomenon. Nulliparity is one of the most firmly established risk factors for breast cancer, while early full term pregnancy and multiparity confer a significant protection. Of interest is the fact that women from different countries and ethnic groups exhibit a similar degree of parity-induced protection from breast cancer, regardless of the endogenous incidence of this malignancy. This observation indicates that the reduction in breast cancer risk associated with early first full-term pregnancy does not result from extrinsic factors specific to a particular environmental, genetic, or socioeconomic setting, but rather from an intrinsic effect of parity on the biology of the breast.

Our studies of chemically induced mammary carcinogenesis in rodents have allowed us to test the influence of pregnancy-induced differentiation of the mammary gland on the susceptibility of the mammary epithelium to carcinogenesis. Results demonstrate that mammary cancer in rodents can be induced only in the young nulliparous females, while the differentiated gland of parous animals is resistant to chemically induced carcinogenesis. This effect is mimicked by chorionic gonadotropin (CG), a glycoprotein hormone first secreted by the fertilized egg and later on by the placenta, as demonstrated by the inhibition of cancer initiation and progression via the administration of this hormone to virgin animals. The hormonal treatment results in a dose-related reduction in tumor incidence and number of tumors per animal, a phenomenon mediated by an inhibition of cell proliferation, an increase in the DNA repair capabilities of the mammary epithelium, a decrease in binding of the carcinogen to the DNA, and an activation of genes controlling programmed cell death.

Work performed in our laboratory is aimed at determining whether the inhibitory effect of human CG (hCG) on tumor progression is 1) a direct effect of the hormone on tumor cells effected by the binding of hCG to a specific lutropin-choriogonadotropin receptor (LH-CG-R), 2) mediated by the induction of the synthesis of tumor suppressor factors, such as inhibin, or 3) activation or repression of specific genes.

To date, the main role ascribed to hCG is the maintenance of the ovarian corpus luteum of pregnancy. In the ovary, hCG acts on thecal, granulosa, luteal, and interstitial cells through the LH-CG-R, a Gprotein coupled receptor that hCG shares with the pituitary hormone, LH. The LH-CG-R is, in turn, induced in the granulosa cells by the pituitary follicle-stimulating hormone (FSH). hCG inhibits proliferation, activates specific genes and gene products in mammary epithelial cells in vitro, and inhibits mammary carcinogenesis in ovariectomized rats. These observations lead us to postulate that hCG acts directly on the mammary epithelium through a specific receptor.

Three basic procedures were selected for the detection of hCG receptors in the mammary gland: 1) Light microscopy autoradiography for the detection of radioiodinated hCG (¹²⁵I-hCG) binding sites, 2) immunocytochemical detection of constitutively expressed LH-CG-R utilizing a polyclonal antibody raised against recombinant LH (r-LH-R), and 3) immunocytochemical detection of binding sites of hCG utilizing specific anti-b-hCG antibodies.

For the autoradiographic study, the binding sites of ¹²⁵I-hCG in the mammary glands and ovaries of virgin unprimed Sprague-Dawley rats were compared with those of ¹²⁵I-FSH and ¹²⁵I-LH. The radio-iodinated hormones were injected intravenously to five groups of rats; two animals from each group were sacrificed at 30 minutes, 1, 2, 4, and 6 hours after injection. Mammary glands and ovaries were processed for histopathological analysis. Deparaffinized sections were coated with photographic emulsion, and developed after two-week exposure in the dark. Cells exhibiting a minimum of 4 silver grains on the plasma membrane were considered to be positive for the specific receptor tested. The binding of ¹²⁵I-hCG, ¹²⁵I-FSH, and ¹²⁵I-LH to the ovarian follicles served as the positive control. Binding of the three hormones was observed by 30 minutes after injection, and remained bound up to 6 hours. Both ¹²⁵I-hCG and ¹²⁵IFSH bound more strongly to the basal layers of granulosa cells of the large antral and preovulatory follicles, while ¹²⁵I-LH bound more frequently to thecal cells. Only ¹²⁵I-hCG was found to be bound to the mammary gland epithelium; silver grains were observed on the luminal border of epithelial cells lining lobules and terminal ducts. The semiquantitative evaluation of the content and distribution of hCG receptors by autoradiography and the comparison of these results with those obtained by immunocytochemical detection of the receptor with the polyclonal antibody rLH-R, and with specific anti-b-hCG antibodies is ongoing in our laboratory.

These studies were designed to determine whether the suppression of mammary carcinomas induced by the chemical carcinogen 7,12 dimethylbenz(a)anthracene (DMBA) by hCG was associated with the synthesis of inhibin. For these purposes, virgin rats received 8 mg DMBA /100 gram body weight; 20 days later they were injected daily with 100IU/hCG for 40 days (DMBA plus hCG group). Age-matched untreated, hCG-, and DMBA plus saline-treated rats were used as controls. Mammary tissues were collected at the time of DMBA administration and at 5, 10, 20, and 40 days of hCG injection for histopathological analysis and mRNA isolation and probing. None of the animals of the Control and hCG groups developed mammary tumors, while microscopic lesions, i.e., intraductal proliferations (IDP) and ductal carcinomas in situ (DCIS), as well as palpable tumors were detected in both DMBA- and DMBA+hCG-treated groups. DMBA alone induced the highest number of IDP, DCIS and palpable tumors, while hCG treatment significantly reduced the development of all these lesions. The mammary glands of hCG and DMBA+hCG treated animals exhibited elevated expression of inhibin A and B (1.5 to 3.4 fold). Positive immunoreactivity for inhibin a and b subunits was observed at 5 days of hCG treatment, increasing progressively for reaching its maximal expression on day 20, in association with lobular differentiation of the mammary gland. Our findings indicate that inhibin production is associated with both mammary gland differentiation and tumor regression.

Treatment of virgin rats with hCG induces differentiation of the mammary gland and inhibits the progression of DMBA-induced mammary carcinomas; both phenomena are associated with an increased synthesis of inhibins, which are heterodimeric proteins that are structurally related to the transforming growth factor-b (TGF-b) family. The present study was designed to determine whether, in addition of inhibins, early response genes were involved in the inhibition of tumor growth induced by hCG. To this end, 45-day-old virgin Sprague rats received 8 mg DMBA/100 gram body weight; 20 days later they were injected daily with 100 IU/hCG for 40 days. Age-matched untreated, hCG-, and DMBA plus saline-treated rats were used as controls. Total and polyadenylated RNAs were collected from the mammary glands at the time of DMBA administration, at 5, 10, 20, and 40 days of hCG injection, and 20 days post-treatment. The mammary glands of hCG-treated animals exhibited elevated expression of inhibin A and B mRNA (1.5 to 3.4 fold) from 5 to 20 days post hCG treatment. A marked induction of c-myc and c-jun expression was found at 20 days post hCG treatment, but no significant changes were found in the levels cfos expression. The present data indicate that hCG inhibits mammary tumor development through the induction of inhibin synthesis and activation of early response genes.

We have previously shown that roscovitine, an olomoucine-related purine analog and a selective inhibitor of cyclin-dependent kinases (cdks), inhibited the proliferative activity of human breast epithelial cells in vitro. The purpose of the present study was to identify the cellular processes and targets affected by roscovitine treatment in the estrogen receptor-negative MDA-MB-231 human breast carcinoma cells. Treatment of the cells with 10 mg/ml roscovitine daily for 24 to 240 hours revealed that the compound inhibited DNA synthesis, induced cell death and irreversibly inhibited the proliferative activity of the cells. Morphological analysis of roscovitine-treated cells by light and fluorescence microscopy demonstrated that this cyclin-dependent kinase (cdk) inhibitor induced cell shrinkage, chromatin condensation, reorganization of actin microfilament architecture and extensive detachment of cells from the cell culture substratum. These cellular events are all known to be associated with apoptosis. Collectively, the data generated from this study suggest that roscovitine induced apoptosis in the estrogen receptor-negative MDA-MB-231 human breast cancer cells. Since the efficacy of many anticancer drugs depends on their ability to induce apoptotic cell death, modulation of this parameter by roscovitine may provide a new chemopreventive and chemotherapeutic strategy for the clinical management of hormone-resistant breast cancers.

Human breast cancer has been associated with genomic alterations (GA) such as microsatellite instability (MSI) and/or loss of heterozygosity (LOH) of several chromosomes. To determine whether GA in chromosome 11 plays a role in the initiation and/or progression of breast cancer, we determined the incidence of MSI and LOH in DNA obtained from 69 breast specimens containing ductal hyperplasia (DHP), carcinoma in situ (CIS), or invasive carcinoma (INV). Four polymorphic microsatellite markers, INT-2 (11q13.1-13.4), D11S614 (11q21-23.3), D11S940 (11q23.1-23.3), and D11S912 (11q24-25), were tested for allelic losses or instability. Forty-five percent of the cases showed GA in one or more markers, which were found in DHP, CIS and INV. D11S912 presented the highest frequency of both MSI and LOH in comparison with the other markers tested, having a higher frequency of MSI in CIS and of LOH in INV lesions. No significant correlation was found between the incidence of GA at D11S912 and clinico-pathological parameters such as age of patients at the time of surgery, tumor size and lymph node status. Our findings suggest that this telomeric region of chromosome 11 might encode gene(s) of significance in the progression of cancer of the breast.

This work was designed to determine whether the presence of allelic imbalances (AI) such as MSI and LOH in chromosomes 2, 11, 13, and 17 in primary breast cancer could be used as prognostic indicators of patient survival. The DNA from individual breast cancers that were obtained from 29 patients who were followed for up to five years was analyzed for MSI and LOH. a panel of 24 markers located at chromosome 2 (TPO, D2S131, D2S144, D2S171, D2S177, D2S119, D2S123, D2S147 and D2S136), chromosome 11 (C-RAS, Int-2, D11S940, D11S912), chromosome 13 (D13S289, D13S260, D13S267, D13S218, D13S263, D13S155, and D13S162), and chromosome 17 (D17S513, TP53, D17S855, and D17S785) was utilized. The frequency of AI in the markers studied ranged from 30 to 55%; the highest frequencies were for D11S912, D2S171, TP53 and D17S855. Univariate analysis showed association between overall survival rate and AI in 9 out of the 24 markers tested. Five of the markers were located at the area of the mismatch repair gene (MMR)2 gene, two at 11p, one at 13q, and one at 17p. Using multivariable analysis, we observed that only pathological and clinical stage (defined as stage II or not) and AI at D2S171, D11S912, or D17STP53 generated significant predictive models for survival.

HUANG. Y., BOVE, B., WU, Y.L., RUSSO, I.H., YANG, X., ZEKRI, A., RUSSO, J. Microsatellite instability during immortalization and transformation of human breast epithelial cells in vitro. Mol. Carcinog. 24:118-127, 1999.

MGBONYEBI, O.P., RUSSO, J. and RUSSO, I.H. Roscovitine induced cell death and morphological changes indicative of apoptosis in MDA-MB-231 breast cancer cells. Cancer Res. 59:1903-1910, 1999.

RUSSO, I.H., RUSSO, J. Breast cancer prevention. BioMedicina 1:171-176, 1998.

RUSSO, J., HU, Y.F., RUSSO, I.H. Estrogens and breast cancer in humans. In Endocrine Disrupters of the Environment, edited by M. Metzler (in press).

RUSSO, J., HU, Y.F., YANG, X., HUANG, Y., SILVA, I., BOVE, B., HIGGY, N., RUSSO. I.H. Breast cancer multistage progression. Front. Biosci. 3:944-960, 1998.

RUSSO, J., HU, Y.F., AO, X., GRILL, C., RUSSO, I.H. Critical appraisal of estrogens as carcinogenic agents in the human breast. In Proc. IV. European Congress on Menopause, edited by M.H. Birkhauser and H. Rozenbaum. Editions ESKA, Paris, pp. 279-287, 1998.

RUSSO, J., AO, X., GRILL, C., RUSSO, I.H. Pattern of distribution of cells positive for estrogen receptor a and progesterone receptor in relation to proliferating cells in the mammary gland. Breast Cancer Res. Treat. (in press).

RUSSO, J., RUSSO, I.H. Molecular and cellular basis of breast cancer: Role of the estrogen receptor in carcinogenesis. J. Natl. Cancer Inst. (in press).

RUSSO, J., RUSSO, I.H. Hormonal approach to breast cancer prevention. J. Cell. Biochem. (in press).

RUSSO, J., HU, Y.F., AO, X., GRILL, C., RUSSO, I.H. Critical appraisal of estrogens as carcinogenic agents. Menopause Reviews (in press).

SALICIONI, A.M., RUSSO, I.H., RUSSO, J. Correlation between cell cycle regulators and the immortalization and transformation of human breast epithelial cell lines. Int. J. Oncol. 13:65-71, 1998.

SRIVASTAVA, P., RUSSO, J., MGBONYEBI, O.P., RUSSO, I.H. Growth inhibition and activation of apoptotic gene expression by human chorionic gonadotropin in human breast epithelial cells. Anticancer Res. 18: 4003-4010, 1998.

SRIVASTAVA, P., SILVA, I.D.C.G.; RUSSO, J., MGBONYEBI,O.P., RUSSO, I.H. Identification of genes differentially expressed in breast carcinoma cells treated with chorionic gonadotropin. Int. J. Oncol. 13:465-469, 1998.

SRIVASTAVA, P., RUSSO, J., RUSSO, I.H. Inhibition of rat mammary tumorigenesis by human chorionic gonadotropin is associated with increased expression of inhibin. Mol. Carcinog. (in press).

YANG, X., HUANG, Y., RUSSO, I.H., BALSARA, N.R., BARRETT, C., RUSSO, J. Functional roles of chromosomes 11 and 17 in the transformation of human breast epithelial cells in vitro. Carcinogenesis (in press).

RUSSO, I.H., RUSSO, J. Role of hormones in mammary cancer initiation and progression. Revista de la Federacion Latinoamericana de Mastologia 1:9-20, 1998.

RUSSO, I.H., RUSSO, J. Role of pregnancy and chorionic gonadotropin in breast cancer prevention. In Proc. IV. European Congress on Menopause, edited M.H. Birkhauser and H. Rozenbaum. Editions ESKA, Pa, pp. 133-142, 1998.

^§   Fox Chase researcher

^a   A-R. Zekri: National Cancer Institute and University of Cairo, Cairo, Egypt

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

Fox Chase Cancer Center

Scientific Report 1998