J. sreroid Biochem. Vol. 24. No. I. pp. 147-154. 1986 0022-473 1186 $3.00 + 0.00 Printed in Great Britain Pergamon Press Ltd AUTOCRINE AND PARACRINE GROWTH REGULATION OF HUMAN BREAST CANCER MARC E. LIPPMAN, ROBERT B. DICKSON, ATTAN KASID. EDWARD GELMANN, NANCY DAVIDSON, MARY MCMANAWAY, KAREN HUFF, DIANE BRONZERT, SUSAN BATES, SANDRA SWAIN and CORNELIUS KNABBE Medical Breast Cancer Section, Medicine Branch, National Cancer Institute, Bethesda, MD 20205, U.S.A. Summary-Previous work from our laboratory has demonstrated that human breast cancer (BC) cells in culture can be stimulated by physiologic concentrations of estrogen. In an effort to further understand this process, we have examined the biochemical and biological properties of proteins secreted by human BC cells in vim. We have developed a defined medium system which simultaneously allows the collection of factors secreted by the BC cells. facilitates their purification and allows for an unequivocal assay of their effect on other BC cells. By both biochemical and radioimmunoassay procedures, MCF-7 cells secrete large quantities of IGF-I-like activity. The cells contain receptors for IGF-I and are stimulated by physiologic concentrations of IGF-I. Multiple additional peaks of growth stimulatory activity can be obtained by partial purification of conditioned media from human BC cells by sequential dialysis, acid extraction and Biogel P60 chromatography. These peaks are induced up to 200-fold by physiologic concentrations of estrogen. Several of these peaks cross-react in a radioreceptor assay with EGF and are thus candidates for transforming growth factors. Monoclonal antibodies (MCA) have been prepared which react with secreted proteins from the MCF-7 cells. One of these MCAs binds to material from MCF-7 and ZR-75-1 hormone-dependent BC cells only when these two lines are treated with estrogen but reacts with conditioned medium from several other hormone-independent cell lines in the absence of estrogen stimulation. This MCA is currently undergoing further characterization and evaluation of its biological potency. We conclude that with estrogen stimulation. hormone-dependent human BC cells secrete peptides which when partially purified can replace estrogen as a mitogen. Their role as autocrine or paracrine growth factors and their effects on surrounding nonneoplastic stroma may suggest a means of interfering with tumor proliferation. Data derived from epidemiologic and clinical obser- vations as well as experimental evidence data has established the central role of estrogens in the genesis of human breast cancer [I]. When estrogenic stimu- lation does not occur (for example, in the case of primary ovarian failure), the incidence of breast cancer is only 1% of that in normal women. Normal breast development requires physiologic concen- trations of estradiol. For nearly a century it has been known that some human breast cancers will regress following some for of endocrine therapy; however, by the time breast cancer has reached the stage of overt metastatic disease it retains the hormone-dependent phenotype of the normal breast approx. one-third of the time. The proportion of clinically-evident hormone-dependent primary breast cancers is un- known. As tumors evolve (particularly in the face of selective pressures exerted by systemic therapy), an increasing number become hormone independent. Multiple lines of investigation all suggest that es- trogens are critically responsible for continued growth of these hormone-dependent breast cancers. Removal of sources of estrogens such as ovary and adrenal or therapy with antiestrogens results in worthwhile clinical responses. Response to these en- docrine therapies is highly associated with the pres- ence of specific cellular receptors for estrogens-a point in favor of a direct interation of hormone with tumor. Administration of physiologic doses of es- trogens to castrate or postmenopausal women can stimulate tumor proliferation in zko. While all of these data clearly demonstrate that estrogens are mitogens for human breast cancer (BC) they do not elucidate the exact mechanism of their action nor even prove that estrogens act directly on tumor cells to stimulate their growth. In this review of some of our recent work we will present some information which supports the follow- ing three hypotheses: I. Estrogens directly interact with human breast cancer (BC) cells and can directly alter gene expression at the level of messenger RNA concentrations. This results in increased ac- tivity of many enzymes involved in cell growth as well as increased activities of other intracellular proteins such as progesterone receptor. II. One aspect of these effects on gene expression induced by estrogens results in alterations in specific proteins secreted by hormone- responsive BC cells. Some of these secreted proteins serve as autocrine and paracrine growth regulatory activities with multiple 147 sa 24,1&K