BY LOOKING BACK WE CAN SEE THE WAY FORWARD
On the cusp of the 20th century, Beatson1 reported the first case of an oophorectomy as a treatment for breast cancer. The procedure rapidly gained traction as the only method of producing any hope of causing breast tumor regression. Boyd2 subsequently gathered the reports of all known cases of oophorectomy to treat breast cancer around Britain and discovered a 30% response rate.
The discovery that the ovaries contained a substance that caused responses in reproductive organs3 is key to focusing on estrogen action and breast cancer. Allen and Doisy3 ovariectomized mice, thereby stopping the estrous cycle. They extracted pig ovaries and inoculated the mice. The vaginal epithelium changed, and mice became more receptive to males. They named their new extracted material “estrogen” (Latin for “frenzy”) and the Allen-Doisy test was born to identify all further estrogens and eventually synthetic antiestrogens. Parenthetically, one of the authors (V.C.J.) used the Allen-Doisy test throughout his PhD (1969–1972) to quantify the estrogenicity of newly synthesized nonsteroidal antiestrogens.4 The explosion of new synthetic nonsteroidal estrogens in the 1930s5 now turned attention to finding a practical use for the synthetic estrogens in medicine.
During the 1940s, Haddow et al.6 discovered that carcinogenic polycyclic hydrocarbons caused tumor regression in laboratory animals. Naturally these compounds could not be used in patient cancer care, but they reasoned that the new polycyclic synthetic estrogens could be used in patients. Prostate and breast cancer had a 30% response rate in patients. Today, we know that prostate cancer regresses because of estrogen's action at the hypothalamopituitary axis to prevent gonadotrophin secretion. The elucidation of the mechanism of estrogen action to treat breast cancer through apoptosis had to wait until the development of animal models to study breast cancer in the 1980s.7
To advance the clinical utility of high does synthetic estrogen treatment, Haddow8 organized a clinical trial with a dozen centers administered by the Royal Society of Medicine (Section of Oncology, of which he was the head).
During his Karnofsky lecture he stated:
When the various reports were assembled at the end of that time, it was fascinating to discover the rather general impression, not sufficiently strong from the relatively small numbers in a single site, became reinforced to the point of certainty; namely, the beneficial responses were 3 times more frequent in women over the age of 60 years than in the women under that age; that estrogen may, on the contrary, accelerate the course mammary cancer in younger women, and that their therapeutic use should be restricted to cases 5 years beyond the menopause. Here was an early example of the advantages which may accrue from cooperative clinical trials.8
A similar conclusion was noted by Stoll9 through a review of his lifelong experience with 407 postmenopausal patients with stage IV breast cancer treated with high-dose estrogen (Table 1).
TABLE 1 -
Objective Response Rates in Postmenopausal Women With Metastatic Breast Cancer Using High-Dose Estrogen Therapy
|Age Since Menopause
|Postmenopausal 0–5 y
A total of 407 patients were classified on the basis of the time from menopause.9
High-dose synthetic estrogen therapy became the standard of care for stage IV breast cancer patients until the approval of tamoxifen in the United Kingdom (1973) and the United States (1977). This clinical decision was based on fewer adverse effects observed with tamoxifen as the clinical response rate was the same for tamoxifen and estrogen at approximately 30%.10,11 During the 30 years that high-dose estrogen use was the standard of care, there was no progress in understanding the anticancer action of high-dose estrogen in long-term estrogen-deprived (LTED) patients (5 years postmenopause). By contrast, if surgical removal of organs that synthesize estrogen precursors or hormones that stimulate estrogen synthesis caused breast cancer regression, then some breast tumors depended on estrogen to grow.12 This made the mechanism of action of tamoxifen, a nonsteroidal antiestrogen, self-evident once the estrogen receptor (ER) was identified in estrogen target tissues and approximately two-thirds of human breast cancers.13
LONG-TERM ADJUVANT TAMOXIFEN THERAPY AS A LIFESAVING STRATEGY IN ER-POSITIVE BREAST CANCER PATIENTS
Once the translational research strategy of long-term adjuvant therapy14 was proven in the Oxford Overview Analysis,15 the major challenge, for translational research, was to create models to discover mechanisms to develop new medicines. The previous DMBA-induced rat mammary carcinoma model was inappropriate. By contrast, the human-derived MCF-7 cell line was relevant as it is an ER-positive breast cancer cell line and transplantable into ovariectomized athymic (immune deficient) mice. Subsequently, tamoxifen-resistant tumors can be retransplanted for years, that is, the actual time course for the treatment of human disease. Several surprises were in store.
Tamoxifen-stimulated MCF-7 tumors started to grow after approximately 6 months of tamoxifen therapy, so tamoxifen was not killing the MCF-7 cells. The unique feature was the discovery that tamoxifen was the first treatment for cancer to cause the growth of resistant breast cancers cells.16 Indeed, either tamoxifen or estradiol caused ER-positive breast cancers to grow.16,17 Early clinical case reports documented a withdrawal response of tumor regression for metastatic breast cancer that occurred on stopping selective ER modulators (SERMs), and the drug was cleared from the patient.18,19 However, in the laboratories after tamoxifen-stimulated tumors had been transplanted for 5 years, estrogen no longer stimulated tumor growth, but killed tumor cells with rapid tumor regression. Haddow8 was speaking to us from his Karnofsky lecture
The extraordinary extent of tumor regression observed in the 1% of postmenopausal cases (with estrogen) has always been regarded as of a major theoretical importance, and it is a matter for some disappointment that so much of the underlying mechanism continues to illude [elude] us.
In the laboratory, we develop the first reproducible, in vivo–transplantable, ER-positive breast cancer (MCF-7) tumor model that responded to estrogen with tumor regression.20,21 The tamoxifen-resistant model was expanded to a model using the SERM raloxifene22,23 once it was clear that raloxifene would be marketed to treat osteoporosis24 with the added advantage of reducing the risk of breast cancer at the same time.25 In addition, raloxifene was found to reduce the risk of breast cancer in high-risk postmenopausal women.26
The aromatase inhibitors were attracting attention in clinical trials in the early 1990s, so it was essential to develop appropriate LTED cells in vitro to study molecular mechanisms of estrogen-induced apoptosis. Although we developed cell lines in vitro,27,28 results were disappointing. These went back in the freezer for nearly a decade until the Santen group29 described the final stages of estrogen-induced apoptosis using an extrinsic mechanism. What they reported was as follows: (1) estrogen binds to the ER; (2) a week later, something happens; and (3) the extrinsic mechanism occurs via feedback to the cell membrane to trigger cell death.
Clearly, our in vivo model was not suited to document the subcellular steps leaving to apoptosis. The cells in our freezer were waiting to describe those mechanisms. The cloned cell line MCF-7:5C fully documented the mitochondrial pathway to trigger estrogen-induced apoptosis.30
THE PRACTICAL VALUE OF A LABORATORY INVESTIGATION OF ESTROGEN-INDUCED APOPTOSIS AND ITS MODULATION
There have been 2 practical benefits to creating models in the laboratory: (1) to decipher mechanisms of the modulation of breast cancer incidence in postmenopausal women in trials focused of the incidence of heart disease, that is, the Women's Health Initiative (WHI) and the Million Women Study (MWS); and (2) to discover mechanisms of estrogen-induced apoptosis with the goal of designing new estrogen-like molecules to treat patients who eventually fail long-term adjuvant aromatase inhibitor therapy.
THE WOMEN'S HEALTH INITIATIVE
The 30-year clinical trial (recruitment 1993–1998) referred to as the WHI consisted of 2 trials: women with an intact uterus who were randomized to either placebo (8102) or 2.5 mg daily medroxyprogesterone acetate (MPA) (8506) or 0.625 mg conjugated equine estrogen (CEE). In the second trial, hysterectomized women were randomized to placebo (5429) or 0.625 (CEE) (5310). The CEE plus MPA trial was stopped after 6.8 years in 2002 because of the expected increase in breast cancer. The CEE trial was stopped after 6.8 years of treatment because of the elevation of strokes.31,32
It is important to note that the mean age of screening to enter the 2 placebo-controlled trials was 63.3 years, that is, more than a dozen years after women would normally consider using hormone therapy at menopause. This gap was intentional to build in an increased risk of cardiovascular disease in older women. There was no benefit for women taking hormone therapy with regard to a cardiovascular endpoint.
A recent review33 fully documents the WHI and the conclusion of estrogen-induced apoptosis as the reason for prolonged decrease in breast cancer incidence in the women taking CEE. In addition, those women with a uterus took CEE/MPA for 7.2 years, at the anticipated rise in the incidence of breast cancer.
With regard to the breast cancer safety of combined CEE/MPA treatment for 7.2 years, it was concluded in 202034 (27 years after the start of combination therapies) that although there was a higher risk of breast cancer (hazard ratio, 1.28), there was no significant difference in breast cancer mortality (treatment: 73 deaths, control: 53 deaths). Bearing in mind that the trial included only 8506 women randomized with a uterus, the calculation should be revisited using the national statistics of women with a uterus on combination hormone replacement therapy (HRT). Only then can realistic claims be made on deaths from breast cancer. In addition, as most women who volunteered were more than a decade postmenopause, to ensure sufficient cardiovascular events would occur, the results for breast cancer are skewed as most women go on postmenopausal HRT at menopause, arbitrarily considered to be 50 years of age by most clinical trials groups.
The final breast cancer report of the WHI study34 had a sustained increase in breast cancer incidence for the CEE/MPA trials out to 22 years, and the CEE-alone trial had a sustained decrease in breast cancer incidence out to 22 years.
MECHANISMS OF ERT AND COMBINATION MPA + ERT THERAPY
The molecular mechanism of action of CEE to trigger apoptosis is summarized Figure 1, and the effect of MPA to neutralize estrogen-induced apoptosis is summarized in Figure 2. The molecular mechanisms on hormonal responses of LTED breast cancer have been studied and published in the refereed literature over the past 2 decades.42–45 The molecular mechanism of MPA to block estrogen-induced apoptosis has emerged with the demonstration that the glucocorticoid properties of MPA suppressed estrogen-induced inflammation critical to trigger apoptosis.36,38,40,41 This explains how MPA reverses the reduction of breast cancer incidence by CEE in the WHI.
THE MILLION WOMEN STUDY
The MWS was established to start recruitment between 1996 and 2001. Specific types of HRT were compared and contrasted, but unlike the WHI study where the average age of starting HRT was 63 years, the average age of the MWS was 50 years. There were several notable conclusions: results for different estrogens or progestin did not influence the incidence of breast cancer, but increased duration of treatment increased breast cancer. Women who had used HRT but did not develop breast cancer had the same relative risk as never-users. Only current users of HRT or tibolone had an increased risk of breast cancer.46 However, the most notable deviation between the WHI and the MWS was that in the WHI there was consistent and prolonged reduction of breast cancer in the WHI with estrogen alone34 that was not observed in the MWS.46
In the MWS, estrogen alone was consistently lower at increasing the relative risk of breast cancer compared with current user of HRT.46 These data are consistent with the requirement that, discovered in the laboratory, breast cancer cells need at least 5 years of an estrogen-free environment to create clones that undergo apoptosis with estrogen. Current estrogen users alone never had a lower relative risk of breast cancer compared with never-users, which contrast dramatically with the WHI.46
THE CREATION OF CELLULAR MODELS OF LTED ER-POSITIVE BREAST CANCER TO SCREEN STRUCTURE FUNCTION RELATIONSHIPS OF MOLECULES TO TREAT AROMATASE-RESISTANT BREAST CANCER
Cheap and effective treatments for breast cancer are essential to prevent the fracture of the family following death from breast cancer. To this end, investigators are advancing novel agents to clinical trial.
Clearly, the target for estrogen-induced apoptosis is the ER in LTED breast cancer cells. Early structure function studies using estrogen-induced prolactin synthesis in primary cultures of immature mouse pituitary cells47–52 and cultured breast cancer cells53,54 mapped out the ligand-induced functional changes that occur with synthetics molecules that bind to the ER. With the discovery of estrogen-induced apoptosis in LTED breast cancer cells27,29,30,55 renewed efforts in medicinal chemistry focused on new ligands for clinical applications. One such effort resulted in the synthesis and identification of candidates56–59 to be validated in clinical trials. The compound TTC-352 has completed a phase 1 trial.60 In addition, estetrol, produced by the fetal liver during pregnancy, is of interest for the treatment of advanced breast cancer.61–64
Interestingly enough, a recent report65 of a nonsteroidal compound called ErSO demonstrated the strong and cytotoxic activation of the unfolded protein response in wild-type and ER mutant–positive breast cancer cells. Clearly, this compound creates a unique conformation in the ErSO receptor complex that kills breast cancer through inappropriate triggering of the unfolded protein response. There is clearly much that remains to be discovered in this novel area of therapeutic research.
The clinical description8 and discovery of estrogen-induced apoptosis with further clinical application7 in 2 Karnofsky lectures, separated by 38 years, have now provided a mechanistic insight into the adjuvant treatment of breast cancer,66 an insight into the “unexpected” results of the WHI investigation of estrogen and estrogen/progestin given to women as hormone replacement at the age of 60 years40,55 versus the MWS of HRTs in the general population. The results of 2 clinical and epidemiological studies were not comparable but instructive about mechanisms of hormone action in the real world if long-term estrogen deprivation occurs at menopause before HRT administration of estrogen alone produces a sustained decrease in breast cancer, and the addition of MPA not only reverses but increases breast carcinogenesis. Mechanisms are documented in the laboratory.40
The enormous advances made in the understanding and development of estrogen-induced apoptosis lead to the idea that if the key triggers of ER-regulated estrogen-induced apoptosis are deciphered, then new approaches to tumor cell killing could be found by the discovery of novel agents to switch on cancer cell apoptosis without the need for an ER trigger.
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