BREAST CANCER RESEARCH LABORATORY

Breast cancer represents the neoplastic disease most frequently diagnosed in American and northern European women. While its incidence continues to rise, the mortality rate from breast cancer has remained almost unchanged for the past five decades, becoming only second to lung cancer as a cause of cancer-related death. The failure to eradicate breast cancer is due to a combination of: 1) a lack of identification of a specific etiologic agent and of the precise time of initiation of this disease, and 2) a lack of understanding the mechanisms responsible for cancer initiation and progression. The comparative analysis of epidemiological and experimental data has provided important information on the influence of breast development on the susceptibility of this organ to undergo neoplastic transformation, as well as on the protective role of pregnancy. The importance of breast development is highlighted by the fact that this is one of the few organs of the body that is not completely developed at birth, reaching full differentiation only under the stimulus of the hormones of pregnancy and lactation. After puberty, the breast is composed of lobular structures that reflect different stages of development. In the young nulliparous woman's breast, the most undifferentiated structure, the lobule type 1 (Lob 1), or terminal ductal lobular unit (TDLU), predominates. Lob 1 evolves to Lob 2 under the cyclic hormonal influences of the ovary. During pregnancy, Lob 1 and Lob 2 rapidly progress to Lob 3, then to Lob 4 during the last trimester of pregnancy and during lactation. After weaning, Lob 4 progressively regress to Lob 3 and Lob 2; when menopause supervenes, they acquire the appearance of Lob 1.

Lob 1 and Lob 2 in the breast of young nulliparous women have a higher proliferative activity than that of Lob 3, which are found in the breast of both nulliparous and parous women. The development of the breast is influenced by a myriad of hormones and growth factors. A major role is played by ovarian steroid hormones, mainly estrogens, which are considered to promote the proliferation of both the normal and the neoplastic breast epithelium. This effect is considered to be mediated by at least three mechanisms: 1) a receptor-mediated mechanism via the interaction of the ligand with a nuclear receptor, 2) an autocrine/paracrine loop mechanism, and 3) a negative feedback mechanism in which estrogens remove the effect of one or several inhibitor factors present in the serum. The level of expression of the traditionally studied estrogen receptor a (ERa) and progesterone receptor (PgR), like the proliferative activity of the mammary epithelium, decreases with the differentiation of the mammary lobules. However, the cells that proliferate and those that contain ER and PgR are not the same. They are, however, closely juxtaposed indicating that there are two different cell populations; one that is the target of steroid hormones, and the other that is capable of proliferating under a yet unidentified stimulus. We hypothesize that the hormone receptor positive cells synthesize a growth factor(s) in response to estrogen and/or progesterone stimulation; this factor(s) acts on the receptor negative cells, which then respond by proliferating. These receptor negative cells, thereby, constitute the true stem cells.

The correlation of lobular architecture and morphology with rate of cell proliferation has provided an important developmental basis for understanding the pathogenesis of breast cancer; ductal carcinoma, the most frequent breast malignancy, originates in the Lob 1, which have both a high receptor content and a high rate of cell proliferation. Pregnancy reduces the number of Lob 1, as well as the receptor content and rate of cell proliferation of the mammary epithelium. We postulate that the lower breast cancer incidence associated with parity is mediated by the induction of differentiation of immature ductal structures, i.e., Lob 1 to Lob 3 and Lob 4. The focus of our research is understanding the mechanisms that regulate the susceptibility of the breast to undergo neoplastic transformation through the following studies: 1) identifying what genes are involved in the process of breast differentiation, 2) determining the role of steroid hormone receptors in the induction of breast cell proliferation and differentiation, 3) determining the influence of estrogens in the transformation of human breast epithelial cells, and 4) elucidating the role played by cell immortalization in neoplastic transformation.

To determine what specific genes are associated with breast differentiation, we utilized the differential display (DD) technique. Using this technique, we analyzed gene expression in primary cultures of human breast epithelial cells (HBECs) obtained from Lob 1 and Lob 3, in mortal S130 HBECs, and in the following cell lines: immortal MCF-10F, BP1 and BP1E cells, which are cells transformed with the carcinogen benz(a)pyrene (BP); D3-1 cells, which are cells transformed with 7,12-dimethylbenz(a)anthracene (DMBA); breast carcinoma MCF7, T47D, and BT20 cells; and, primary breast carcinoma cells (adjacent normal tissues were used, in this case, as controls). DD and reverse Northern analysis revealed that twenty genes were preferentially expressed in Lob3 cells. Two of the clones were found by sequence analysis to be alternatively-spliced isoforms of a novel serpin. The level of expression of this serpin was high in two primary cultures of HBECs and in normal breast tissues, moderate in S130 cells, low in MCF-10F, BP1, and BP1E cell lines, and absent in MCF7, T47D, and BT20 breast carcinoma cell lines, as well as breast tumor tissues.

MCF-10F cells displayed seven cDNA bands that differed from the parent mortal S130 cells. Sequence analysis revealed 99% homology to the EF-hand calcium-binding protein, S100P (placental). This clone was overexpressed in MCF-10F, BP1E, D3-1, and T47D cells, and in three invasive ductal carcinomas when compared with the levels found in S130 cells, other primary HBECs, and normal adjacent tissues, respectively. Immunohistochemical studies revealed that this protein was strongly expressed in ductal hyperplasia and ductal carcinomas, both in situ and invasive, but was absent in normal tissues. Our results indicate that the expression of this novel serpin was a differentiation-associated event, which was repressed during in vitro cell transformation and lost during in vivo neoplastic progression. The overexpression of S100P, on the other hand, occurred as an early event during cell immortalization, progressively increasing with neoplastic progression.

The recent molecular cloning of ERb provides a possible alternative pathway of estrogen signaling; little is known, however, concerning the role of ERb in the development of human breast cancer. In the present study, expression of ERa and ERb mRNA was determined in a series of chemically transformed HBECs, as well as in various normal and malignant breast cancer cell lines. We observed a very low level of ERb expression in S130 and MCF10-F cells. BP1, D3, and BP1-ras cells, which are derived from MCF-10F cells treated with the carcinogens BP, DMBA, and BP plus ras transfection, respectively, exhibited elevated levels of ERb expression. An even higher level of ERb expression was detected in the more transformed BP1-E, D3-1 and D3-1-ras cell lines.

These results indicate that ERb is not constitutively expressed in normal or immortalized HBECs, but can be induced by chemical carcinogens. In addition, the level of expression of the receptor increases proportionally with the increased expression of transformation phenotypes, i.e., the more transformed cells showed higher levels of ERb expression, regardless of the chemical carcinogen initially utilized for inducing cell transformation. These results suggest that the expression of ERb may contribute to the initiation and progression of chemical carcinogen-induced neoplastic transformation.

Estrogen has long been recognized as the most important endocrine factor contributing to the development of human breast cancer, yet there is little information available concerning the carcinogenicity of estrogen in human breast epithelial cells. In the present study, the cell-transforming potentials of 17b-estradiol (E2), a natural estrogen, and diethylstilbestrol (DES), a synthetic estrogen, were determined in the estrogen and progesterone receptor negative MCF10F cells. Carcinogen-treated and control MCF-10F cells served as positive and negative controls, respectively. MCF-10F cells can be reproducibly transformed by the chemical carcinogen BP, validating our model of in vitro cell transformation for determining the transforming potential of estrogens in HBECs. Treatment of MCF-10F cells with BP and with estrogens, especially DES, enhanced the colony forming efficiency of MCF-10F cells in agar methocel; this result indicates that, in HBECs, estrogens along with a carcinogen induce similar phenotypic changes indicative of cell transformation. Our data provide the first experimental evidence that normal HBECs can be transformed in vitro by estrogen treatment. As expected from our previous finding that neither a nor b subtypes of ERs were expressed in these cells, we observed no effects of estrogens on the growth of MCF10F cells. From this, it is reasonable to conclude that DES-induced neoplastic transformation is due to genotoxic effects independent of ERs. Studies are currently in progress to evaluate the growth properties and tumorigenic potentials of those isolated colonies, as well as the transforming potential of estrogens under different treatment regimens.

Primary cultures of HBECs have a limited life span when cultured under standard conditions. The number of population doublings can be increased by lowering the conventional concentration of calcium (Ca⁺⁺) in the medium from 1.05 mM (Hi-Ca⁺⁺) to 0.04 mM (Low-Ca⁺⁺). The expression of telomerase activity in cancer cells is thought to be one of the multiple pathways that lead to cancer development and progression. The present study was designed to determine whether Ca⁺⁺ concentration regulates telomerase activity as well. Telomerase activity was determined using telomere repeat amplification protocol (TRAP) assays in primary cultures of the S#254, S#256, S#257, MCF-10F, BP1, and BP1-Tras cell lines; all cells were cultured under Hi-Ca⁺⁺ and Low-Ca⁺⁺ conditions. Telomerase activity was not detected in any of the HBEC primary cultures, regardless of the Ca⁺⁺ concentration in the medium. MCF-10F cells exhibited high telomerase activity when cultured in Low-Ca⁺⁺ medium; the activity was reduced when cultured in Hi-Ca⁺⁺ medium indicating that telomerase activity in these cells is Ca⁺⁺-dependent. BP1 and BP1-Tras cells, on the other hand, had a high telomerase activity under both Ca⁺⁺ concentration conditions. These data indicate that the immortalization and transformation of HBECs is associated with telomerase activation. Our study demonstrates for the first time that extracellular Ca⁺⁺ down-regulates telomerase activity in the normal immortalized MCF-10F cells, but has no effect on their transformed derivatives.

We have shown that introduction of the human chromosomes 11 and 17 into the chemically transformed HBEC line, BP1E, partially or completely suppresses the expression of transformed phenotypes, and that chromosome 17 alone reverses microsatellite instability (MSI) on 17p. In this study, we confirmed allelic retention of the donor chromosomes in the microcell hybrids by microsatellite DNA polymorphic analysis. Two out of six informative markers, D11S911 at ql3-23 and DRD2 at q23.1, of donor chromosome 11 were present in microcell hybrid BP1E-11neo cells. On chromosome 17, a total of 27 informative markers were analyzed. For microcell hybrid BPIE-17neo cells, positive retention of the donor alleles was found only in the region of 17q24.3-25.2 (D17S515) and 17q25.2 (Dl7S785). In addition, the MSI-positive markers on 17p, including D17S849 (pl3.3), D17S1566 (p13.3), D17S796 (pl3.1-13.3), D17S513 (pl3.1), TP53 (pl3.1), D17S786 (pl3.1), D17S1852 (pl3.1), D17S945 (pl3-12), D17S793 (p13.1-11.2), D17S520 (p12), D17S71 (p12-11.2) and D17S1176 (p11.2), were reversed to normal in BP1E-17neo cells, but not in BP1E-11neo cells. In summary, we determined that several specific regions of the donor chromosomes 11 or 17 were retained in microcell hybrids, BPIE-11neo and BPIE-17neo cells, respectively. These observations indicate that these regions may play a functional role in the suppression of transformed phenotypes.

We have demonstrated that MCF-10F, BP1, and BP1E exhibit increased telomerase activity. Since the transfer of chromosomes 11 or 17 partially or completely inhibited the expression of transformation phenotypes in BP1E cells, we determined whether this inhibition affected telomerase activity. Telomerase activity was completely lost in the microcell hybrid BP1E-17neo cells, whereas it remained positive in BP1E-11neo cells, as well as in BP1 and in BP1E cells transfected with control vector, pSV2neo. The demonstration that chromosome 17 specifically repressed telomerase activity suggests that this might represent an important mechanism for the reversion of immortalization and transformation phenotypes.

BARBISAN, L.F., RUSSO, J., MELLO, M.L.S. Nuclear and nucleolar image analysis of human breast epithelial cells transformed with the c-Ha-ras oncogene. Ann. Cell. Pathol. 16:193-199, 1998.

HU, Y.F., LAU, K.M., HO, S.M., RUSSO, J. Increased expression of estrogen receptor beta in chemically transformed human breast epithelial cells. Int. J. Oncol. 12:1225-1228, 1998.

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

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., RUSSO, I.H. Antiproliferative effect of synthetic reverastrol on human breast epithelial cells. Int. J. Oncol. 12:865-869, 1998.

MGBONYEBI, O.P., RUSSO, J., RUSSO, I.H. Roscovitine inhibits the proliferative activity of immortal and neoplastic human breast epithelial cells. Anticancer Res. 18:751-756, 1998.

MGBONYEBI, O.P., RUSSO, J., RUSSO, I.H. Roscovitine induced cell death and morphological changes indicative of apoptosis in MDA-MB-231 breast cancer cells. Cancer Res. (in press).

RUSSO, J. Differentiation and pathogenesis of breast cancer. Women and Cancer (1):14-21, 1998.

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

RUSSO, J., RUSSO, I.H. Development of the human breast. In Encyclopedia of Reproduction, edited by E. Knobil and J. D. Neill. Academic Press, New York, Vol. 3, pages 71-80, 1998.

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, J., HU, Y.F., AO, X., GRILL, C., RUSSO, I.H. Critical appraisal of estrogens as carcinogenic agents. IV European Congress on Menopause, edited by M.H. Birhhauser and H. Rozenbaum. Editions Eska, Paris, pp. 279-287, 1998.

RUSSO, I. H., RUSSO, J. Role of pregnancy and chorionic gonadotropin in breast cancer prevention. IV European Congress on Menopause, edited by M.H. Birhhauser and H. Rozenbaum. Editions Eska, Paris, pp. 133-142, 1998.

RUSSO, J., AO, X., GRILL, C., RUSSO, I.H. Pattern of distribution 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. The cellular basis of breast cancer susceptibility. Oncol. Res. (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 approaches to breast cancer prevention. J. Cell. Biochem. (in press).

RUSSO, J., RUSSO, I.H. Human chorionic gonadotropin in breast cancer. In Endocrine Oncology, edited by S. Ethier. Humana Press (in press).

RUSSO, J., SANTEN, R. Breast physiology. In Textbook of Endocrinology, edited by DeGroot (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., 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).

VIDAL, B.C., RUSSO, J., MELLO, M.L.S. Feulgen-DNA content and chromatin texture of benzo (a) pyrene-transformed human breast epithelial cells as assessed by image analysis. Exp. Cell Res. 244:77-82, 1998.

WEI-ZEN, W., PAULEY, R. LICHLYTER, D., SOULE, H., SHI, W.P., CALAF, G., RUSSO, J., JONES, R.F. Neoplastic progression of breast epithelial cells a molecular analysis. Br. J. Cancer 78:198-204, 1998.

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.

^§   Fox Chase researcher

^a   Y.-J. Huang: Present address--Dept. of Pathology, Temple University School of Medicine, Philadelphia, PA 19122

^b   I. D.C.G. Silva: Present address--UNIFESP-Escola Paulista de Medicina, Disciplina de Ginecologia, São Paulo, Brazil

^c   K.M. Lau, S.M. Ho: Tufts University School of Medicine, Boston, MA 02153

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

Fox Chase Cancer Center

Scientific Report 1998