Background
Breast cancer is one of the most common cancers among women, with more than one million cases and nearly 600,000 deaths annually worldwide [
1]. Breast cancer incidence rates vary markedly among countries. Breast cancer is the most frequently diagnosed cancer in women in the United States. Due to the high incidence rate along with social and cultural considerations, breast cancer ranks highest among women’s health concerns. Despite the advancement of new preventive strategies, the incidence of breast cancer has remained the same since 2005 [
2]. Approximately 70% of breast cancers are diagnosed in postmenopausal women [
3].
The steroid hormones estrogen and progesterone have long been thought to play a role in the etiology of breast cancer. Apart from breast cancer growth, these hormones also influence various physiological processes. After the cessation of ovarian function, a significant decrease in the ovarian hormones estrogen and progesterone leads to a variety of symptoms known as postmenopausal symptoms. The most common symptoms include hot flashes, night sweats, mood swings, and sleep disturbances. These symptoms negatively influence a woman’s quality of life. Additionally, estrogens have beneficial actions on bone and lipid metabolism and cardiovascular function [
4‐
7]. To alleviate postmenopausal symptoms, hormone replacement therapy (HRT) is used as a treatment. In particular, HRT has been shown to alleviate vasomotor symptoms, aid in the prevention of osteoporosis and improve serum lipid profiles [
8‐
11].
Despite positive effects of HRT, some exogenous hormones have been shown to increase the incidence of breast cancer. The Women’s Health Initiative (WHI) study, which utilized conjugated equine estrogen (0.625 mg per day) and medroxyprogesterone acetate (2.5 mg per day), revealed a 24% increased risk for invasive breast cancer [
12], with no major beneficial effects against cardiovascular disease, stroke, and thromboembolic diseases [
13]. These findings resulted in a 63% reduction of HRT use within 3 months after the WHI publication. However, recent analyses of the WHI data have shown that estrogen replacement therapy alone (without medroxyprogesterone acetate) actually decreased the risk of breast cancer [
12].
Aromatase inhibitors (AIs) are widely used for the adjuvant treatment of postmenopausal breast cancer, generally prescribed for five years at the conclusion of surgery, chemotherapy and/or radiation treatment. AIs target the aromatase enzyme, which converts adrenal androgens to estrogens. After the Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial showed AIs are equally effective to tamoxifen, the FDA approved AIs as a first-line endocrine therapy for preventing recurrence of hormone-positive postmenopausal breast cancer [
14‐
18]. However, several observational and meta-analyses revealed that AIs used for the prevention of postmenopausal breast cancer reduce cancer recurrence but also have serious side effects on bone and the cardiovascular system. AIs cause severe joint pain, hip fracture, increased osteoporosis risk, and musculoskeletal pain. Loss of learning and memory function is also an important adverse effect associated with AI treatment that can lead to dementia at later stages [
19,
20]. In a large cohort study using 8,769 breast cancer patients, approximately 51% of the patients discontinued their adjuvant hormonal therapies including tamoxifen and AIs due to the adverse side effects [
21]. Therefore, it is imperative to find alternative treatment regimens with fewer unfavorable side effects for postmenopausal breast cancer patients.
From the available literature and published data, it is clear that currently used treatments reduce breast cancer recurrence but also have serious undesirable side effects that limit their usefulness. In this study, we aimed to develop hormone treatments that will provide similar or improved survival rates compared with the drugs used currently, but without the harmful and undesirable side effects.
Discussion
Anti-estrogen therapies are currently the standard treatment for estrogen receptor-positive breast cancer recurrence. However, AIs are known to reduce bone mineral density, increasing risk of osteoporosis, and their side effect profile leads to a high discontinuation rate [
21].
The current study was performed to investigate hormonal therapeutic regimens that inhibit breast tumor growth without negative effects on cardiovascular and bone formation processes. The ultimate goal of the study was to develop a postmenopausal hormone treatment regimen that blocks the growth of breast cancer while enhancing overall health and quality of life.
The use of hormone replacement therapy for postmenopausal symptoms has been the subject of debate for the past two decades. Studies over that time have revealed a risk-benefit profile that varies by type of hormone, time since menopause, and organ/system in question [
24]. In addition, the dose, duration, and mode of administration of hormones are important factors in determining the efficiency and beneficial function of a particular treatment [
25,
26]. However, current standard of practice considers hormones of any type absolutely contraindicated after hormone-receptor-positive breast cancer, with the assumption being that hormones “throw fuel on the fire” of cancer. This assumption makes intuitive sense, since current treatment is to block remaining estrogens with aromatase inhibitors, the exact opposite.
Yet hormones have myriad effects throughout the body, effects which influence survival and quality of life as much as breast cancer recurrence does or more. We explored a radical hypothesis: Could an optimal choice of hormones lead to improved survival factors and quality of life enough to outweigh any negative effect on tumor recurrence?
In our experiments, we used steroids in their bioidentical form, as these hormones have been shown to possess a more positive risk-benefit profile than synthetic hormones which have been molecularly altered for patentability or oral bioavailability [
27‐
30]. The first question we sought to address was influence on overall health of an optimal hormone regimen. In the landmark Women’s Health Initiative study, a negative risk-benefit profile was seen with oral equine estrogens and oral synthetic medroxyprogesterone acetate (PremPro), a drug combination based on an estrogen formulation first approved in 1942 and which continues to dominate the market in English-speaking countries. We therefore chose estradiol and progesterone delivered non-orally, as is commonly used in southern European countries and increasingly in English-speaking countries as well, based on an extensive literature indicating more favorable global risk-benefit profile [
31].Our results show that the right combination of hormone treatments is essential to achieving the desired effect on postmenopausal symptoms and the risks associated with osteoporosis and cardiovascular disease (CVD). E plus P plus T treatment was associated with increased cognition, physical activity, and cardiovascular and bone health in the mouse model, and demonstrates the potential significance of hormone treatment in postmenopausal women. Testosterone is critical to both physical activity and mental health. Testosterone has been shown to be beneficial to cognitive function and memory. It also functions like vasodilator and enhances endothelial functions to improve cardiac health [
32,
33]. In our study we found that addition of testosterone along with estradiol and progesterone improves the cognitive function, physical activity and cardiac health. In agreement with our study, testosterone therapy has also been shown to reduce breast cancer incidence in postmenopausal women and breast tumor growth in animal models [
34‐
37].
Because estrogen-blocking aromatase inhibitors are the current adjuvant treatment after hormone-sensitive breast cancer, common sense would lead to the assumption that any treatment containing estrogen itself would lead to opposite, highly negative impact on tumor growth. However, this turned out not to be the case. As was the case for general health markers, maximal reduction in tumor growth was achieved by E plus P plus T treatment. In only one group, the lowest-dose E plus P group, did addition of estrogen result in tumor volumes slightly worse than control. Our results thus did not confirm the “throwing fuel on the fire” conception prevalent among clinicians.
Furthermore, the antitumor effect of AI treatment, though notable when compared to control, did not excel when compared to hormone treatment. Treatment with AI had initial antitumor activity, consistent with the results of preclinical studies leading to the approval of AIs. However, three of five hormone treatment regimens provided similar suppression of tumor volume to the AI regimen. And with cessation of the AI treatment phase (chosen to be equivalent to the current clinical standard of care, 5 years), the antitumor effect of AIs diminished, leading to a steepened rise in tumor volume, while the most effective hormone regimens, including E plus P plus T, continued to more effectively suppress tumor volume.
A frequent criticism of studies in a mouse model is that they may have limited utility in predicting eventual clinical outcomes. Anticipating this objection, we designed our study to exactly mimic conditions such as dose and length of treatment used in pre-approval studies of anastrozole (Arimidex®), a leading aromatase inhibitor currently on the market. These murine-model results were seen to accurately predict outcomes later seen in large clinical trials such as the ATAC (Arimidex, Tamoxifen, Alone or in Combination) trial and were part of the basis for approval of anastrozole [
38,
39].
Although an E plus P plus T regimen performed better in our study than AIs (the current standard of care) on measures of both tumor growth and general health, considerable momentum, as well as market forces, works against a reversal in treatment practice from hormone inhibitors to hormones. We therefore sought to determine whether addition of optimal hormones could improve quality of life and general health indicators when added to—instead of substituting for—AI treatment, without worsening tumor outcomes. Our results indicated the viability of this approach. When added to AIs, estradiol and progesterone significantly improved the general health of the animals as measured by cardiac and bone health markers (although positive impact of hormones on cardiac and bone health markers was not as marked when added to AIs as when used alone), without promoting breast tumor growth. We discuss possible explanations for this seeming paradox—improved general health but lack of tumor stimulation—below.
Aromatase classically converts C19 steroids (androgens) to C18 steroids (estrogens) with the addition of a hydroxyl group. Because it has a wide range of substrate specificity, it accepts DHEA as a substrate and converts it to estrogens [
40,
41]. Estrogen, which is predominantly produced in postmenopausal women by the aromatization of DHEA, selectively activates ERα [
42,
43]. Aromatase has high expression in breast tumor cells and the surrounding stroma in postmenopausal women [
44,
45]. AIs inhibit aromatase and reduce the conversion of androgens to estrogens in postmenopausal breast cancer patients.
However, this inhibition is not reliably effective long-term because many patients develop resistance to AI treatment [
46‐
48]. Furthermore, inhibiting aromatase in tissues outside the breast is associated with a variety of negative sequelae in joints, bone, and other tissues [
49‐
53]. Our data indicate that an optimal dose of estradiol and progesterone can overcome effects on bone, cardiovascular, and cognitive health. Furthermore, the addition of testosterone along with estradiol and progesterone enhances the beneficial effects.
Large observational studies suggest estrogens have a cardioprotective effect [
13,
54,
55]. Abnormal serum lipid levels have been associated with an increased risk for CVDs [
13,
54]. Low HDL and high LDL levels in serum are mainly attributed to an increased risk of CVDs. Several clinical and experimental studies indicate that estradiol treatment is beneficial to the heart by reducing LDL levels and increasing HDL levels in circulation [
56‐
58]. Based on epidemiological studies, CVDs are prevalent in postmenopausal women, and serum concentrations of estrogen are inversely associated with CVD risk [
59]. Estrogen replacement therapy initiated within 5 years after menopause has a beneficial effect on cardiovascular risk factors, but not if the therapy is begun later [
60]. Natural hormone 17-b estradiol was more effective in reducing CVD risk factors than conjugated equine estrogens, and it is also affected by an oral or transdermal route of delivery [
61]. Taking all of these factors into account, the cellular mechanism of estradiol-induced cardioprotection involves the contribution of several factors including time of administration, type of hormone administered, and mode of administration. Our data demonstrate that administration of hormones immediately after ovariectomy results in improved cardiac health, in agreement with data from clinical studies.
The role of progesterone versus synthetic progestins in cardiovascular health is the subject of much debate. The WHI study demonstrated increased atherosclerosis upon the use of synthetic progestins [
13,
62,
63]. In a long-term randomized study that accessed and compared the effects of synthetic progestins and progesterone on serum lipids, synthetic progestins negatively influenced the beneficial effect of estrogens by lowering serum HDL levels [
55]. Progesterone, in contrast, has been shown to support the cardioprotective actions of estrogen in several other studies [
64‐
70]. Our results indicate that estrogen and progesterone improve the serum lipid profile and reduce the risk of CVDs in a postmenopausal breast cancer mouse model.
Osteoporosis is a major concern in postmenopausal women. Several studies have shown that estradiol increases bone formation and prevents osteoporosis [
71,
72]. Similarly, depletion of estrogen resulted in osteoporosis, supporting the notion that estrogens are important for bone formation [
73,
74]. Clinical studies have indicated that progesterone treatment helps maintain bone mass [
75‐
77]. Progesterone supports bone formation by preventing glucocorticoid-induced bone loss [
54]. Several animal and human studies have demonstrated progesterone’s positive effect on bone formation as well as inhibition of bone resorption [
76‐
78]. Studies evaluating estrogen and progesterone supplementation suggest estrogen and progesterone have distinct but complementary roles in bone maintenance [
75‐
77,
79]. The addition of testosterone positively influences bone mass by preventing urinary calcium loss. Our findings demonstrate that the addition of hormones along with AI treatment is beneficial for bone health in postmenopausal women.
Our data on physical activity, cognition, and spatial learning clearly demonstrate the importance of hormones in addition to AIs for breast cancer treatment. Cyclical administration of hormones appears to have a slightly better effect versus administration of steady levels of hormones. It is interesting that the addition of testosterone has a significant positive impact on all aspects that were studied in this investigation.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
RL designed the study, carried out the experiments, supervised the project and prepared the manuscript. AA carried out the experiments, collected and analyzed the data. EL conceived of the study, participated in study design, and was involved in manuscript preparation. All authors read and approved the final manuscript.