The Contribution of "Alternative Approaches" to Understanding Steroid Hormone Action

Elwood V. Jensen

Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521

Address all correspondence and requests for reprints to: Elwood V. Jensen, Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521. E-mail: elwood.jensen{at}uc.edu.

ABSTRACT

In the 47 yr since the first evidence for a steroid hormone receptor was presented at an international congress to an audience of five persons, the concept of "alternative approach" has played an important role in providing new understanding. By asking not what does an estrogenic hormone do to cellular processes in responsive tissues but what do these cells do to the hormone, it was shown that rat uterus contains a characteristic protein with which the hormone associates to promote growth. In the following decade, it was established that this substance is a true receptor, involved in hormonal action. Furthermore, estradiol was found not to undergo a chemical change as it exerts its effect. Finally, estrogenic action was established as a two-step process in which association with the hormone converts the receptor from an inactive to an active form that can bind tightly in the nucleus to modify transcription. These findings, obtained by studying the fate of the hormone itself, disproved the then accepted concept that estrogens interact with enzyme systems, and opened a new field of research. Soon various laboratories identified intracellular receptors for all classes of steroid hormones, the action of which involves a similar two-step process. Several laboratories then attempted, without success, to obtain antibodies to these receptors by conventional techniques. The unconventional approach of gradient ultracentrifugation, using radioactive estradiol as a marker for the receptor, gave a means of recognizing the soluble immune complexes formed with estrogen receptor and provided the first antibodies to any steroid hormone receptor, permitting its cloning. Finally, the knowledge that estrogens act through a receptor suggested that measuring the receptor content of an excised tumor specimen could identify, in advance, two thirds of the human breast cancers that are not estrogen dependent. These patients will not benefit from endocrine ablation or antiestrogen treatment and should be placed directly on chemotherapy. This is now standard clinical practice.

IN SURVEYING THE stages in understanding the action of what are now known as nuclear hormone receptors, one is struck by the frequency with which important progress has resulted from the utilization of an "alternative approach" to the problem being addressed. I first became aware of this concept in 1947 when, as postdoctoral student at the Swiss Federal Institute of Technology (Eidgenössische Technische Hochschule or ETH) in Zürich, I had occasion to pay a visit to Zermatt and gaze on the regal Matterhorn (Fig. 1Go). Although I had no previous experience in mountain climbing, the enchantment of this stately peak overcame reason, and I allowed myself to join another American student, Kyle Packer, an experienced climber who had already employed a Swiss guide, in making an assault on the peak. Although, as a novice, it was the most difficult physical challenge that I have encountered, we succeeded in reaching the summit (1). I was puzzled as to why the Matterhorn was the last major peak in Europe to be climbed, for it could not be that hard for the experts.



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Fig. 1. View of the Matterhorn from Zermatt in 1947

Northeast face is to the left and provided the key to the easiest way to climb. Individuals are a student, Kyle Packer, about to begin the climb, and Mary C. Jensen, deceased in 1982.

 
In subsequent reading about the history of the Matterhorn, I learned that essentially all early attempts to scale this peak were made from the French or Italian sides, because the northeast face, as seen from Zermatt, appears to be an impossibly sheer wall of rock (Fig. 1Go). In the early 1860s, a young English engraver named Edward Whymper studied the physiogamy of the Matterhorn and concluded that the northeast face may not be as sheer as it appears in photographs. In 1865, he, and six companions, attained the summit, and today even a novice can do so by following their route. This lesson concerning an "alternative approach" persisted into later studies in hormone action, three useful examples of which are described in this Perspective.

1. RECOGNITION OF RECEPTORS FOR STEROID HORMONES

By the 1950s, most of the basic actions of the estrogenic hormones were recognized, such as their stimulation of the growth and function of tissues of the female reproductive tract. But the biochemical processes involved were not entirely clear. It was assumed that the hormone must have been acting on enzyme systems, because this was the great era of enzymology, but the initial step in this process was obscure. The generally accepted hypothesis was that the 17-hydroxyl group of estradiol underwent enzymatic oxidation using one coenzyme and the resulting estrone was reduced using another, thereby transferring a hydrogen atom from reduced nicotinamide adenine dinucleotide (NADH) to reduced nicotinamide adenine dinucleotide phosphate (NADPH), a coenzyme involved in many biosynthetic processes. However, this mechanism could not explain the strong estrogenic action of diethylstilbestrol. Coming into the field from the area of organic chemistry, where one generally knows the nature of both the reactants and the products, it seemed as if valuable information could be obtained by an alternative approach. Thus, rather than ask the question "what does the hormone do to the tissue?" which was being studied everywhere, we asked "what does the tissue do with the hormone?" as it exerts its physiological action.

One problem with this alternative was that, because estradiol is active in such low doses, to be able to detect and identify products formed during the course of its action, one would need radioactively labeled steroid of much higher specific activity than had been known previously. This was achieved by catalytically reducing the double bond in 6-dehydroestradiol to give tritiated hormone that could be detected in amounts as low as one-trillionth of a gram (2). With this material, it was demonstrated (2, 3) that the tissues of the rat female reproductive tract take up and retain unchanged estradiol against a marked concentration with the blood (Fig. 2Go). Later it was shown (4) that this binding substance, with which estradiol associates without chemical change, is a true receptor involved its action, the first steroid hormone receptor to be recognized.



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Fig. 2. Radioactivity Content of Various Tissues of the Immature Rat after Administration of 0.09 µg of Tritiated Estradiol sc in Saline

Each point is the median of six animals (Ref. 2 ).

 
2. ANTIBODIES TO THE ESTROGEN RECEPTOR

After specific receptor proteins had been identified for all classes of steroid hormones, several laboratories attempted to prepare specific polyclonal and monoclonal antibodies to them by conventional techniques, but without success. It appeared to us that, because of their unusually low concentrations, antibodies to these receptors might need an alternative approach to permit detection of possibly soluble immune complexes. Addition of an antibody to a hormone-receptor complex would yield a larger entity, and, if the antibody did not displace the radioactive hormone needed as a marker, one could employ the technique of sucrose gradient centrifugation to detect a shift in the sedimentation peak of the estrogen-receptor complex. This approach worked very well. Not only could one identify polyclonal antibodies forming soluble immune complexes, but also one could show that two different monoclonal preparations recognize different epitopes on the receptor by the further shift of the immune complex peak when the second monoclonal antibody is added (Fig. 3Go). These antibodies were used by others in the first cloning of the estrogen receptor and provided immunochemical methods for measurement of estrogen receptors in tissues and tumors.



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Fig. 3. Recognition, by Sucrose Gradient Centrifugation, of the Reaction of Two Different Monoclonal Antibodies (D547 and D75) with Different Epitopes in the Estrogen Receptor Molecule

 
3. PREDICTION OF BREAST CANCER RESPONSE TO ENDOCRINE THERAPY

It is well established that about one third of human breast cancers retain the hormone dependency of the original breast tissue, so that depriving them of this support by endocrine ablation (ovariectomy, adrenalectomy, hypophysectomy), blocking estrogen biosynthesis (aromatase inhibitors), or blocking its action at the receptor level (antiestrogens) provide the treatments of choice as compared with chemotherapy. But for the two thirds of the patients whose tumors do not respond to endocrine manipulation, one needs a method for identifying them early so that the patients can be placed directly on chemotherapy.

In the past there were several attempts to correlate response of human breast cancers with various biochemical or pathological characteristics of the tumor, but the recognition that estrogens require specific receptor proteins for their actions provided a new approach to the early prediction of hormone dependency and the selection of optimal therapy for the patient. An early report in 1971 (5) indicated that remissions to endocrine therapy were rare in tumors that showed low or negligible estrogen receptor content, whereas most, but not all, cancers with substantial receptor levels showed remissions. These results were substantiated by a study with 160 patients (Fig. 4Go) reported in 1978 (6). It is obvious that, until one reaches a modest receptor level, indicating that nearly all the cells contain receptor, one sees few objective remissions, and that about two thirds of the cancers with high receptor content will show good remissions to endocrine therapy. Thus, estrogen receptor assay of excised breast tumor specimens predicts the correct therapy selection in 80% of the cases. Further research, perhaps by alternative approach, is needed to identify the estrogen receptor-positive cancers that still will not respond to endocrine therapy.



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Fig. 4. Relation of the Estrogen Receptor Content of 160 Excised Breast Cancer Specimens to the Response of the Patients to Estrogen Ablation, Mostly Adrenalectomy in Postmenopausal Women and Ovariectomy in Premenopausal Women

Green points indicate objective remissions and red indicate those who failed to show a true objective remission. Failure of those with modest receptor levels to respond to endocrine therapy is believed to reflect tumors that have a mixture of receptor-containing and non-receptor-containing cancers (Ref. 6 ).

 

Received for publication April 15, 2005. Accepted for publication April 19, 2005.

REFERENCES

  1. Jensen EV 1987 High point. Breast Cancer Res Treat 9:77–86[Medline]
  2. Jensen EV, Jacobson HI 1960 Fate of steroid estrogens in target tissues. In: Pincus G, EP Vollmer, eds. Biological activities of steroids in relation to cancer. New York: Academic Press; 161–174
  3. Jensen EV, Jacobson HI 1962 Basic guides to the mechanism of estrogen action. Recent Prog Horm Res 18:387–414
  4. Jensen EV 1965 Mechanism of estrogen action in relation to carcinogenesis. Proc Can Cancer Conf 6:143–165
  5. Jensen EV, Block GE, Smith, Kyser K, DeSombre ER 1971 Estrogen receptors and breast cancer response to adrenalectomy. Natl Cancer Inst Monogr 34:55–70[Medline]
  6. DeSombre ER, Greene GL, Jensen EV 1978 Estrophilin and endocrine responsiveness of breast cancer. In: McGuire WL, ed. Progress research and therapy. New York: Raven Press; 1–14