Carol Wright

Enzymatic Control of Estrogen Production in Human Breast Cancer: Relative Significance of Aromatase versus Sulfatase Pathways

One-third of the cases of breast cancer in postmenopausal women are hormone-dependent and the lesions regress upon treatment with antiestrogens or inhibition of estrogen biosynthesis. In these patients, estrogens are synthesized in extraglandular tissues from adrenal precursors and re-enter plasma to produce estrone levels of 52 +/- 6.5 pg/ml (mean +/- SEM) and estradiol concentrations of 13.1 +/- 0.7 pg/ml. However, the fact that the levels of estrogen in breast tumor tissue are an order of magnitude higher than plasma levels suggested the possibility of in situ estrogen production. To address this possibility, we measured several enzymes involved in estradiol biosynthesis in human tumors. Forty-eight of 61 tumors contained aromatase (estrogen synthetase) activity ranging from 5-80 pg/gm protein per hour. By comparison, the levels of estrone sulfatase were 10(6) higher, ranging from 0.8-125 micrograms/gm protein per hour. Because the sulfatase enzyme was of lower affinity (i.e., Km = 27 microM) than that of aromatase (i.e., 0.027 microM), the amount of estrogen formed under conditions of similar substrate concentrations was compared and found to be 10-fold higher via the sulfatase enzyme. In 41 additional tumors, the 17 beta-hydroxysteroid dehydrogenase enzyme, catalyzing the conversion of estrone to estradiol, was uniformly present. To test the biologic relevance of the estrone sulfate to estrone to estradiol pathway, estrogen-dependent nitrosomethylurea rat mammary tumors were grown in soft agar in the presence of estrone sulfate. Concentrations of estrone sulfate of 10(-6) microM significantly (p less than 0.01) stimulated colony formation in this system in which 75.5-98.6% of estrone sulfate was converted to estrone and 0.2 to 6% to estradiol. These data support the hypothesis that mammary carcinomas can synthesize estradiol in situ from circulating estrogen precursor and that local conversion is biologically important. On the basis of comparative data, the estrone sulfate to estrone to estradiol pathway is quantitatively more important than that involving androstenedione to estrone to estradiol.

Estrone sulfate: A potential source of estradiol in human breast cancer tissues

SummaryLocal formation of estradiol in human breast tumors could provide a more important source of estrogen than is delivered from plasma. Prior studies have suggested that estrone is primarily synthesized from estrone sulfate. The enzyme 17β-hydroxysteroid dehydrogenase (HSD) would be required to convert estrone to estradiol.This study characterized HSD in 1000 × g supernatants from human breast tumors. Estradiol synthesis was linearly related to tissue concentration or time over the range studied. Cofactor requirements varied with estrone concentration. High and low affinity sites were found in 50% of tissues studied, while the remainder contained only low affinity sites. Screen assays showed measurable activity in all 42 samples tested. This activity ranged from 0.73−>100 nmol estrone synthesized/g protein/hr, with a median activity of 5.9 nmol/g/hr.We evaluated the biological relevance of the sulfatase-HSD pathway by testing the ability of estrone sulfate to stimulate colony formation in soft agar cultures of nitrosomethylurea-induced rat mammary tumors. The maximally effective concentration ranged from 10−7 to 10−4 M. Significant stimulation of colony formation was observed in 7 of 8 experiments. The estrone sulfate stimulation pattern was similar to that previously observed with estradiol. Of the3H-estrone sulfate added to the dishes, 20–98% was recovered as estrone and 0.2–6% as estradiol. These studies suggest that the requisite enzymes are present in human breast tumors for conversion of estrone sulfate to estradiol, and that this pathway may be biologically significant.

Polyamines and the synthesis of estradiol-regulated growth factors in rat mammary cancer in culture

We have recently provided evidence to suggest that the polyamine pathway plays an essential role in the expression of the growth-promoting effect of estradiol (E2) regulated growth factors in the N-nitrosomethylurea (NMU) induced rat mammary tumor culturedin vitro in the soft agar clonogenic assay. To further explore the interaction between the polyamine pathway and autocrine control of tumor growth by E2, we tested whether, in our system, polyamines play a role in the synthesis of E2-regulated growth factors. Conditioned medium (CM) obtained from tumors treated with E2 and the polyamine biosynthesis inhibitor α-difluoromethyl-ornithine (DFMO) (1 mM) no longer exhibited the colony-stimulating effect which was consistently observed with E2-CM. Such growth promoting activity was restored in a dose-dependent fashion with CM obtained from tumors treated with E2, DFMO, and increasing concentrations of spermidine (from 1 to 100µM). Conditioned medium obtained from tumors treated with DFMO with and without spermidine in the absence of E2 had no discernible effects on colony formation. The colony stimulating effect of the CM employed could not be accounted for by the contaminating presence in the media of E2, DFMO, or polyamines. These results indicate that, in our system, the polyamine pathway plays an important role in the synthesis of E2-regulated growth factors.

Role of polyamines in the growth of hormone-responsive experimental breast cancerin vivo

SummaryWe have provided evidence for a critical role of polyamines in the growth of the hormone-responsive N-nitrosomethyl-urea (NMU)-induced rat mammary tumorin vitro. The present experiments were designed to test whether polyamines are involved in the growth of this experimental tumorin vivo. To test this hypothesis, groups of rats bearing NMU-induced mammary cancers were randomly allocated to receive no treatment or escalating doses of the polyamine biosynthesis inhibitor α-difluoromethyl-ornithine (DFMO) (0.5%, 1%, 2%, 3% in drinking water). DFMO inhibited tumor growth in a dose-dependent fashion and consistently reduced tumor putrescine level. To evaluate the time dependency of this effect, additional groups of rats received either no treatment or 2% DFMO for 3, 7, 14, and 21 days. At all times DFMO suppressed tumor putrescine level as well as spermidine to spermine ratio. Finally, exogenous administration of putrescine (200 mg/kg/i.p./day × 21 days) given concomitantly with DFMO restored tumor growth, partially repleted tumor putrescine level, and raised the spermidine to spermine ratio to control levels. Putrescine, given alone, had no significant effect on either tumor polyamine levels or tumor growth. Except for modest weight loss, no major toxicity was encountered. These results indicate that polyamines play an important role in the growth of the NMU rat mammary tumorin vivo. The interaction between polyamines and hormones in supporting NMU mammary tumor growthin vivo remains to be elucidated.

Role of polyamines in the synthesis of prolactin-regulated growth factors by experimental breast cancer in culture

SummaryThe present experiments were designed to test whether polyamines play an essential role in the synthesis of growth factors induced by ovine prolactin (oPRL), using the N-nitrosomethylurea (NMU)-induced rat mammary tumor cultured in the soft agar clonogenic assay. Conditioned media (CM) obtained from tumors treated with oPRL and the polyamine biosynthesis inhibitor α-difluoromethylornithine (DFMO) (1 mM) no longer exerted the colony-stimulating effect which was observed with oPRL-CM. Such growth-promoting activity was restored with conditioned media obtained from tumors treated with oPRL, DFMO, and increasing concentrations of spermidine from 1 to 500µM. The colony-stimulating effect of the CM employed could not be accounted for by the contaminating presence in the media of oPRL, DFMO, and polyamines. These results indicate that in our system polyamines play an important role in the synthesis of oPRL-regulated growth factors.

Polyamines and estrogen control of growth of the NMU-induced rat mammary tumor

SummaryRecentin vitro evidence suggests that polyamines play an important role in the growth of the N-nitrosomethyl-urea (NMU)-induced rat mammary tumor, and that they may be involved in mediating the effect of estrogens on tumor growth. In support of this hypothesis, we here show that inhibition of polyamine biosynthesis with α-difluoromethyl-ornithine (DFMO) blocks the mitogenic effect of estradiol-17β (E2) added to NMU-mammary tumors grown in soft agar in the presence of the antiestrogen tamoxifen (Tam). Exogenous polyamine administration reversed the inhibitory effect of DFMO and restored E2 action. Administration of polyamine inhibitors to NMU-tumor-bearing rats induced significant inhibition of tumor growth, although tumor ornithine decarboxylase (ODC) was not consistently suppressed. Under our experimental conditions, such treatment did not potentiate the antitumor effect of Tam. Tam alone was found to suppress tumor ODC, suggesting a possible involvement of the polyamine pathway in its antitumor action. These data suggest that the polyamines may play an important role in the hormonal control of the growth of this experimental breast cancer.

Assessment of mitogenesis of the hormone-responsive NMU rat mammary tumor grown in culture in soft agar, using3H-thymidine incorporation into DNA

SummaryWe have recently shown that the soft agar clonogenic assay is suitable for growing the hormone-responsive N-nitrosomethylurea (NMU) rat mammary tumor and for evaluating the factors affecting its growth. In order to improve our assessment of tumor mitogenesis beyond simple colony counting, we have validated and characterized in our system the use of3H-thymidine (3H-ThD) incorporation of DNA. In time-course studies, we observed that in most tumors an initial peak of3H-ThD uptake occurred on day 1 after plating followed by a decline in counts on day 2 and a second peak on day 3.3H-ThD incorporation was markedly diminished on day 4 and 6 when maximal colony formation occurred. In experiments where NMU rat mammary tumors were plated at different cell concentrations, we observed that increasing the number of cells plated resulted in a parallel increase in counts on day 1 and colonies on day 6. Overall, a highly significant correlation was observed between3H-ThD uptake and colony formation (r = 0.86,p<0.001). These results demonstrate that the3H-ThD incorporation assay is a fast and effective method for assessing mitogenesis of hormoneresponsive NMU mammary tumors grown in the soft agar clonogenic assay.

Species-specificity of estradiol regulated growth factors in breast cancer.

Recent evidence indicates that autocrine/paracrine mechanisms may mediate the mitogenic effect of estradiol (E2) both in human and experimental breast cancer. However, the species-specificity of E2-regulated growth factors with regard to their biologic action has not been evaluated. To test this issue, we examined, in the soft agar clonogenic assay, the colony-stimulating activity in human breast cancers of conditioned media obtained from rat mammary carcinomas exposed to E2 (rat E2-CM). Of 22 primary human breast cancers plated in soft agar in the absence of serum, 18 (82%) successfully grew with a mean colony number of 62.4 +/- 9.8 (S.E.M.) (range 14-193). Rat E2-CM significantly stimulated colony formation in 10/18 (56%) human breast cancers to 155 +/- 11% (S.E.M.) of control. E2 administration (10(-9) M) in these tumors had a virtually identical overall effect (154 +/- 13% of control colony number). In the remaining eight tumors (44%), neither rat E2-CM nor E2 had, in general, a significant colony-stimulating effect. The growth-promoting action of rat E2-CM and E2 was not influenced by the hormone receptor status of the tumor. These results suggest that E2-regulated growth factors may not be species-specific, at least with regard to their colony-stimulating effects in soft agar.