William L. McGuire

Estrogen Receptors in Human Breast Cancer

Specific quantitative techniques have been used to measure the cytoplasmic estradiol-binding protein (EBP) in human mammary carcinoma tissue specimens. Sucrose gradient centrifugation reveals EBP to sediment at 8S and 4S. Variable quantities of non-specific estradiol binding occurs in the 4S region of the sucrose gradient necessitating controls to insure specificity of the estradiol protein interaction. Using dextran-coated charcoal to separate bound from free estradiol Scatchard analysis finds the dissociation constant of the estradiol EBP interaction to be approximately 2.6x10(-10) M, indicative of the very high affinity of the ligand for the EBP. Quantitation of EBP sites in 64 primary and metastatic human breast tumors demonstrates a continuous spectrum of values from 0 to 612 fmol per mg of cytoplasmic protein. Specific 8S binding in the sucrose gradient centrifugation was not detected in specimens containing less than 9.0 fmol EBP per mg cytoplasmic protein. Since data from animal breast tumors and preliminary evidence from human breast tumors indicates an excellent correlation between the presence of abundant tumor EBP and endocrine-induced breast cancer regressions, precise quantitation of EBP in all human primary tumors may prove to be an excellent prognosticator of endocrine therapy in metastatic breast cancer.

Progesterone receptors and human breast cancer

SummaryEstrogen receptor protein is known to be an important prognostic factor for patients with breast cancer. The presence of estrogen receptor correlates with response to endocrine therapy in patients with metastatic disease and is associated with prolonged disease-free and overall survival in patients with primary disease. But the correlation between estrogen receptor positivity and endocrine dependence is not perfect. Approximately 40% of estrogen receptor positive tumors fail to regress with endocrine therapy. It has been hypothesized that another protein, progesterone receptor, may be a more effective marker of endocrine responsiveness since progesterone receptor is the end product of estrogen action. We have examined the relationship between progesterone receptor and response of advanced breast cancer tumors to hormonal manipulations. Promising retrospective results indicate the need for new, prospective clinical trials to further define the prognostic value of progesterone receptor for these tumors. We have also analyzed the disease-free intervals of patients with primary disease and found that progesterone receptor was more important than estrogen receptor for predicting time to recurrence. We suggest that both estrogen receptor and progesterone receptor be routinely measured in all breast cancer tumors, and that the results of these assays will help the physician individualize therapy for breast cancer patients.

Progesterone antagonists: Tumor-inhibiting potential and mechanism of action☆

A new approach for the treatment of breast cancer could be the use of progesterone antagonists. These compounds were originally developed for the inhibition of progesterone-dependent processes and have been shown to be effective in inhibition of nidation and interruption of pregnancy. Although the roles of progesterone and the progesterone receptor in control of cell growth remain unclear, it was found in progesterone receptor positive mammary carcinoma cell lines that the antiprogestin, Mifepristone, had an inhibitory effect on cell growth and a growth-inhibiting action on the DMBA-induced mammary carcinoma of the rat. We have shown that the progesterone antagonists, Onapristone and ZK 112993, which possess a reduced antiglucocorticoid activity compared to Mifepristone, exert a strong tumor-inhibiting effect in a panel of hormone-dependent mammary tumor models. The effects of these compounds were in some systems superior to those of tamoxifen or high dose progestins and comparable to ovariectomy. Although prerequisites for their antiproliferative potency are an affinity to the progesterone receptor as well as a sufficient number of available receptors in the tumors, the strong tumor inhibiting potential of the antiprogestins cannot be explained by a classical anti-hormonal mechanism. Surprisingly, the antitumor activity is evident in spite of elevated serum levels of ovarian and pituitary hormones. It was established by morphometric procedures that treatment with Onapristone triggers differentiation of the mitotically active polygonal tumor epithelial cell towards secretory active glandular structures and acini. All our quantitative light and electron microscopic data indicate that the antitumor action of antiprogestins is accompanied by the initiation of terminal differentiation leading to (apoptotic) cell death. Finally, our flow cytometry studies revealed an accumulation of the tumor cells in the G0G1 phase of the cell cycle, which may result from induction of differentiation since a differentiation-specific G1 arrest has already been proposed for other stem cell systems. It can be concluded from these data that the progesterone receptor antagonists differ in their mode of action from compounds used in established endocrine treatment strategies for mammary carcinoma. The ability of progesterone antagonists like Onapristone to reduce the number of cells in S-phase may offer a significant clinical advantage, since it is established that the S-phase fraction is a highly significant predictor of disease-free survival among axillary node-negative patients with diploid mammary tumors.

Prognostic factors in primary breast cancer

SummaryEstrogen receptor (ER) has been well documented as an important predictor of long disease-free intervals and survival for patients with primary breast cancer (1). In advanced breast cancer it has been hypothesized that the presence of progesterone receptor (PR) might be a more sensitive marker for predicting response to endocrine therapy (2,3). We have recently found that PR was more important than ER in predicting diseasefree survival for a group of patients with stage-II breast disease that was treated according to a randomized protocol (Clark et al., submitted for publication).This report examines the generality of that result by extending our analysis to include patients from other institutions. The additional patients were not treated according to a rigid clinical protocol, but rather received treatments in much the same way as the majority of breast cancer patients in a community practice. Of interest was whether the relationship between steroid receptors and disease-free survival that we have previously reported would hold with this new group of patients with different demographic and treatment profiles. The relationships between steroid receptor levels and both disease-free and overall survival were examined in detail.

Estrogen control of progesterone receptor induction in human breast cancer: role of nuclear estrogen receptor.

We have provocative data which suggest that the nuclear estrogen-receptor complex interacts with DNA, that this interaction is required for appropriate receptor turnover or processing, and that processing may be essential for induction of a specific protein by estrogen. If the receptor is improperly inserted into DNA, as for instance when it is bound by nafoxidine, processing fails and the biological effect is blunted.

Hormones and breast cancer

The study of steroid hormone receptors in human breast cancer provides insight into tumor biology and forms the rationale for many therapeutic modalities. Clinical assays for estrogen and progesterone receptors improve the selection of patients who may benefit from hormonal therapy. In addition, receptor content in breast cancer tissue is useful for determining prognosis. Together with other new prognostic factors, hormone receptors help to distinguish among patients with primary breast cancer who are at risk for early recurrence.

A role for progesterone in breast cancer.

Because of the cyclic changes of blood estrogen and progesterone levels that occur in females and the interrelationships among these hormones in regulating target tissue development and growth, it was inevitable that progesterone would be studied for its effect on breast cancer. Although progesterone itself is not a carcinogen, it may be a potent target-specific cocarcinogen for induction of mammary tumors by viral or chemical agents.l As such, the hormone has been implicated both in tumor enhancement and in tumor suppression. That progesterone plays a role in stimulating tumor growth is suggested by the pioneering studies of Huggins et al.2-4 They showed that pregnancy promoted the growth of dimethylbenzanthracene (DMBA)-induced rat mammary tumors. Administration of progesterone to intact rats accelerated the appearance of tumors, increased the numbers of tumors, and augmented the growth rate of established tumors. If DMBA-induced mammary tumor-bearing rats are ovariectornized and simultaneous lesions are placed in the median eminence to increase prolactin release, the tumor grows at an accelerated pace for only 10-12 days and then regresses, even though prolactin levels remain e l e ~ a t e d . ~ . ~ The ovarian factor responsible for maintaining tumor growth under these circumstances has not been identified, but the following experiments suggest the importance of progesterone. Pregnancy stimulates the growth of DMBA-induced mammary tumors, while parturition and weaning are followed by regression of a large number of these tum o r ~ . ~ , ~ , ~ The tumor growth-promoting factor of pregnancy is probably placental l a ~ t o g e n , ~ while prolactin has been implicated as being responsible for the maintenance of tumor size and growth during lactation, because tumors regress if suckling is prevented. I 0 * l I The true situation is more complex, however, because ovariectomy blocks the stimulatory effects of endogenous or exogenous prolactin on tumor growth, and injection of progesterone removes this block.I0 One interpretation would be that prolactin stimulation of tumors under these circumstances is dependent on progesterone or, alternatively, that the high levels of circulating progesterone found in the lactating rat, which are under prolactin control,I2 are responsible for the tumor growth. This does not necessarily mean that progesterone alone is responsible for maintaining rat mammary tumor growth, because in these experiments the animals had both high prolactin levels and intact adrenal glands. On the other hand, they do suggest that progesterone plays an important physiologic role in stimulating tumor growth. In contrast to the stimulatory effects of progesterone described above, progesterone can induce rat mammary tumor regression or prevent tumor appearance when combined with moderate to large doses of estrogen.2,13 In humans, the percentage of breast tumor regressions to a progesterone-estrogen combina-

Prognosis in Breast Cancer

Only one-half of patients presenting with primary breast cancer are cured by local therapy. It is thus desirable to have a method to predict which patients are likely to have a recurrence so that systemic therapy can be instituted to delay, or even prevent, such recurrences. For many years, our laboratory in San Antonio has been measuring factors in human breast cancer patients which have been hypothesized to correlate with clinical outcome. Our efforts have dealt primarily with the prognostic factors listed in Table 1. We will briefly review each of these prognostic factors and provide the essential data which correlates each factor with clinical outcome. In this summary of San Antonio efforts, we will not attempt to review the literature or detail the work of others.

Progesterone receptors in breast cancer

A review of recent data which provides considerable support for the routine assay of estrogen receptors (ERs) and progesterone receptors (PgRs) in patients with metastatic breast cancer is reported. Assays for PgRs in breast cancer estrogen dependence of PgRs in experimental breast cancer cells and clinical correlation and PgRs are the main topics reviewed. It was shown that a relationship exists between presence of estrogen and presence of PgR. However this is not to say that estrogen-responsive tumor growth and estrogen-induced PgR are inexorably linked. At least 2 tumors grew in the absence of estrogen so that their growth may be considered autonomous whereas their PgRs declined and could thus be considered estrogen dependent. Data in experimental breast cancer suggest PgR is estrogen-dependent. Data indicate that estradiol acts directly and not as an intermediary hormone to induce PgR. It is emphasized that PgR induction is only 1 product of estrogen action. Some tumors grow in castrate rats so that growth can be considered estrogen autonomous while tumor PgR levels decline and could therefore be considered estrogen dependent. It is predicted that tumors regressive while on high dose estradiol would have elevated PgR levels. The studies strongly suggest that estrogen stimulation of PgR involves ERs. ERs and PgRs were measured in more than 500 breast tumor specimens. The results indicate that patients whose tumors lack both receptors have virtually no chance of responding to endocrine therapy and should receive combination chemotherapy.

Steroid hormone receptors and disease: breast cancer.

Summary The use of cytoplasmic estrogen receptor (ER) to help select patients with advanced breast cancer for endocrine therapy is well established and whenever possible should be part of the routine evaluation. Other potential uses of steroid receptors are emerging. The data suggest that an ER determination on the primary tumor may be very important in designing new adjuvant trials. Finally, the presence of progesterone receptor in a tumor signals the presence of a higher ER content and very likely a favorable response to endocrine therapy. Data from the authors own laboratory were supported in part by the NIH (CB 23862, CA 11378) and The American Cancer Society (BC-23G). I wish to thank Drs. Degenshein, King, Skinner, and Matsumoto for providing me with their data prior to publication, and Dr. J. P. Raynaud of Roussel Uclaf for providing the R5020 for the progesterone receptor studies.