Navigating a plethora of progesterone receptors: Comments on the safety/risk of progesterone supplementation in women with a history of breast cancer or at high-risk for developing breast cancer.

The progesterone receptor gene ( PGR ) is well-studied as an estrogen-responsive gene, that is upregulated by the estrogen receptor (ER) in response to the estrogen stimulation of ER+ tissues, including the brain, uterus, and breast. For this reason, nPRs (i.e., both PR-A and PR-B) are used as a clinical biomarker of estrogen signaling during breast cancer diagnosis and signifies the presence of a functional ER in tumor cells. Breast cancers with high levels of both ER and nPRs (luminal A type) respond well to endocrine therapy. In contrast, luminal B breast cancers often exhibit lower levels of ER and have little to no nPR expression (ER+/PR-low/null). Such luminal B cancers are more likely to already be endocrine resistant at diagnosis or may rapidly become endocrine resistant during disease progression [ 5 ]. ER and nPR seldom function independently in breast cancer cells; nPR can inhibit ER actions (primarily PR-A) but can also be an important ER binding partner (primarily PR-B), helping to regulate numerous estrogen-regulated genes. In ER+ breast cancer models, engagement of nPRs with either progestin or anti-progestin ligands has been shown to inhibit cancer cell growth by reprogramming ER responses on genes that function to promote cell proliferation [ 6 , 7 ].

In sum, progesterone and related progestins may interact with a minimum of 7-8 different progesterone-binding proteins that are often co-expressed in the same tissues and cells. Discussions of cancer risk are primarily focused on nPRs relative to PGMRC1/2 or PAQRs, which have emerging roles in cancer. Historical epidemiological studies suggest that doctors and other healthcare professionals who advise women should consider the case-by-case benefits versus risks of hormonal interventions as they relate to a patient’s unique combination of individual risk factors such as family history, prior hormone exposure and reproductive history, genetics/BRCA status, breast density, and age/menopausal status. Due to the complexity of nPR signaling and cross talk with both ERα and other PR classes (mPRs, PGRMCs), progesterone remains a controversial hormone regarding the risks and benefits of exogenous supplementation. Further study is urgently needed to clearly define which PRs (nPRs, mPRs, PGMRC1/2 or combinations thereof) are most relevant to known biological outcomes and how these receptors and their signaling pathways interact so that improvements can be made towards the safe administration of progesterone or progestins. In addition, new ligands (both agonists and antagonists) or agents with mixed activity known as selective PR modulators (SPRMs) are needed that may be able to discriminate between receptor classes, aid research discovery, and ultimately enter the clinical arena to both improve health/wellbeing and longevity and prevent or treat cancer or other related disease states (fibroids, endometriosis) for women of all ages. As researchers gain new insights into the specificity and integration of PR rapid signaling and nuclear actions in the control of cell and tissue homeostasis and in concert with other sex hormones such as estrogen or androgen, we are hopeful that the benefits of progesterone therapy may be fully realized without the burden of increased women’s cancer risk.

Specific conditions confer increased breast cancer risk, including a family history of breast cancer (in one or more first-degree relatives) and/or alterations in sex hormone levels. Lifetime elevated levels of estrogen and progesterone (e.g. early onset of menarche, late onset of menopause, late pregnancy) are associated with increased breast cancer risk, while early pregnancy (before age 20) is protective [ 12 ]. Post-menopausal hormone replacement therapy (HRT) that includes both estrogen and a synthetic progestin (namely, medroxyprogesterone acetate (MPA)) is associated with increased breast cancer risk; women taking combined HRT had larger tumors that were of higher grade [ 13 ]. Breast cancer incidence returned to normal levels upon cessation of combined HRT, indicative of tumor promoting (i.e., reversible) effects of this treatment. The mechanism of tumor promoting effects of MPA when combined with estrogen are unclear but may in part be due to off-target effects of MPA on other steroid hormone receptors, for example by modulation (i.e., inhibition) of androgen receptor (AR) signaling [ 14 ]. Notably, natural progesterone, when used as part of combined HRT, was not associated with increased breast cancer risk [ 15 ]. However, small sample sizes across multiple studies may limit the ability to detect small increases in risk. Heritable genetic factors also contribute to elevated breast cancer risk and include germline mutations, the most notable being BRCA1/2 gene mutations. Deleterious mutations in the BRCA1/2 genes results in a deficiency in a critical DNA damage repair pathway known as homologous recombination (HR), due to the loss of function of either or both of these tumor suppressor proteins. Additionally, germline BRCA1/2 carriers exhibit increased ovarian steroid hormone biosynthesis due to the tissue-specific loss of function of BRCA1/2 in the ovaries, resulting in lifelong elevated estrogen and progesterone levels [ 16 ]. Elevated hormone levels in BRCA1/2 carriers may contribute to breast or ovarian cancer initiation because BRCA proteins participate in transcriptional complexes with steroid hormone receptors (ER and nPR), and normally function to repair single-strand DNA breaks created during the process of transcription elongation [ 17 ]. Thus, loss of this essential repair step during repeated cycles of sex hormone-induced transcription is predicted to result in the accumulation of genetic damage. BRCA proteins have also been shown to inhibit PR by unclear mechanisms [ 18 ]. Additional conditions of hormonal imbalance may contribute to increased breast cancer risk. For example, some women experience abnormal or excessive uterine bleeding, termed menorrhagia, due to high levels of estrogen and low levels of progesterone. Intra-uterine progestin (levonorgestrel) treatment of women with menorrhagia was associated with decreased ovarian, lung, and colon cancers, but dose-dependently increased breast cancer [ 19 ]. This finding of protective effects in diverse tissues outside the breast suggests that in addition to nPRs expressed in breast tissues, PGMRC1/2 and/or selected mPRs may have been impacted by systemic leakage of intra-uterine applied levonorgestrel. Finally, high mammographic breast density is associated with increased breast cancer risk, in part because small tumors in dense breast tissue are hard to detect using state-of-the-art mammography screens [ 20 ]. However, the tissue microenvironment (TME) is clearly altered in regions of high breast density relative to low breast density, and oncogenic signaling pathways are elevated [ 21 ]. Breast tumors tend to form in regions of high breast density, and hormones (namely, progesterone) can reversibly modulate (i.e., increase) breast density [ 22 ]. In sum, women considered to be at high risk for breast cancer development, due to known family history, BRCA status, or hormonal condition, or who have mammographic dense breast tissue should use caution or avoid hormone-based drugs, including progesterone.

Progesterone and progestins (synthetic progesterone receptor agonists) are powerful steroid hormones with complex actions. Numerous receptors or receptor families exist that bind to and are modulated by progesterone or by a growing array of progestin ligands, including classical or nuclear progesterone receptors (nPR) encoded by the PGR gene, whose ligand-dependent activation leads to activation or repression of transcriptional activity at PR-target genes. The nPRs include two major co-expressed isoforms (PR-A and PR-B) that may reside in the cytoplasm and/or are associated with signaling molecules at the cell membrane as well as in the nucleus, thereby functioning as both signaling molecules and ligand-activated transcription factors. Liganded PRs are capable of direct binding and activation of signaling molecules positioned at or near the cell membrane. Historically, Migliaccio and colleagues documented the direct involvement of PR with membrane-associated or cytoplasmic signaling complexes. Seminal studies highlighted the presence of membrane-associated PR-B/ERα/c-Src kinase ternary complexes [ 1 ]. Boonyaratanakornkit and colleagues [ 2 ] later showed that PRs can directly engage with and activate c-Src through its conventional SH2 (phosphotyrosine-binding) and SH3 (proline-rich motif-binding) domains. Specifically, in the N-terminal region of PR-B, there is a proline-rich segment that directly interacts with c-Src’s SH3 domain, and this interaction is unique to PR-B, but not PR-A. Additionally, PR-B directly interacts with ERα via two ER interacting domains (ERID1 and ERID2) that flank the N-terminal poly-Pro region [ 3 ]. When membrane-associated PR interacts with its ligand, it triggers swift activation of c-Src and downstream kinases within seconds to minutes (3-5 min). This activation extends to both the Ras/Raf/MEK/MAPK and PI3K/AKT/mTOR/S6 kinase cascades. Other families of progesterone receptors with emerging significance in cancer biology include heme-associated progesterone receptor membrane components one and two (PGRMC1 and PGRMC2), and the ubiquitous progestin-binding progestin/ a dipo Q receptor (PAQR) family members (also called mPRs). The PGRMC1/2 receptors are highly expressed in reproductive tissues, can be either membrane-localized or nuclear, and are involved in cholesterol and steroid biosynthesis. The membrane PRs resemble seven-transmembrane G-protein coupled receptors capable of modulating the abundance of intracellular cAMP and other second messengers that impact cellular decisions, including pro-survival and proliferation ( Fig. 1 ). Notably, mPR and nPR cross-talk is extensive and reviewed in [ 4 ]. The complexity of progesterone action via a plethora of diverse receptors that are often co-expressed in the same tissues or cells warrants careful consideration of the clinical applications of progesterone or progestin agonists, which primarily include ovarian suppression during birth control (typically mediated by progestins) and hormonal supplementation following menopause (typically via oral micronized progesterone for women with an intact uterus who also supplement estrogens, wherein progesterone is used to block estrogen-driven proliferation). Herein, we will focus on the benefits and risks of progesterone supplementation, drawing from the current literature. As nPR is a clinically used biomarker of ER action in breast tumor tissues, we will focus primarily on the role of estrogen-induced PGR gene products, PR-A and PR-B (collectively, nPR) in breast cancer, with some comments on ovarian cancer. The two other major classes of progesterone-binding proteins (PGRMCs and mPRs) have been recently reviewed [ 4 ]. We conclude that progesterone-only supplementation is considered safe for most reproductive-age women. Women who currently have ER+ breast cancer or have had such cancer in the past should not take sex hormones, including progesterone. Women who are at high-risk for developing breast cancer, due to family history or known genetic or hormonal conditions, should proceed with caution when considering using sex hormones to mitigate menopausal symptoms and/or improve quality of life after menopause. These individuals are urged to consult with a qualified OB-GYN physician over the risks and benefits of sex hormone supplementation that includes progesterone or commonly used synthetic progestin agonists, such as levonorgestrel. Considering the recent FDA approval of over the counter norgestrel-containing contraceptives in the US, these considerations are especially timely.

In women of reproductive age, numerous progestin-only contraceptives have been evaluated for association with breast cancer risk, with variable outcomes [ 23 ]. Progesterone-only pills (POPs), including norethindrone, levonorgestrel, and desogestrel are the most well-studied. For example, in the Norwegian-Swedish Women’s Lifestyle and Health Cohort study of 103,027, exclusive past (ever) use of POPs was not associated with increased breast cancer risk at the time the study was performed. However, the risk of developing breast cancer was elevated among those who were currenly using or had recently used POPs at the start of the study’s follow-up [ 24 ]. This finding suggests that progestins are not carcinogens, but largely act as tumor promoters that promote cell division or survival of pre-initiated cells during continuous use; these effects are reversible and thus, once one stops taking progestin, the risk returns to baseline. Similarly, a temporary elevation of breast cancer risk occurs after pregnancy [ 25 – 27 ] and in women who took equine estrogen and a synthetic progestin (MPA) as part of combined HRT; the increased risk associated with HRT returned to baseline upon cessation of hormone exposure [ 28 , 29 ]. Study size remains a significant challenge for detection of small increases in risk. The above findings were only partially confirmed in a larger Danish study of 1.8 million women who were current and previous hormonal contraceptive users (relative to never users), in which levonorgestrel-containing but not norethindrone- or desogestrel-containing POPs were associated with increased breast cancer risk [ 30 ]. Recent analyses from the Nurses’ Health Study II [ 31 ] reported that current combined oral contraceptive (e.g. estrogen plus a progestin) users had a higher risk of invasive breast cancer relative to never users, but that risk in former users was comparable to that of never users after 5 years since cessation of use. Interestingly, only formulations of ethinyl estradiol that contained levonorgestrel or norgestrel were associated with higher breast cancer risk. No significant association was observed for monophasic levonorgestrel, or combined formulations of norethindrone, norethindrone acetate, and ethynodiol diacetate, desogrestrel and norgestimate, and drospirenone, although sample sizes were small for these newer progestins [ 31 ]. The limitations of studies of less popular or newer non-oral routes of hormone administration that reported no risk or increased risk include the small number of users and confounding factors such as prior use of combined oral contraceptives [ 23 ]. In sum, increased risk is often associated with formulations of oral contraceptives that combine an estrogen with a progestin. Importantly, the effects of progestins on breast cancer risk appear to be reversible, with elevated risk experienced during current use returning to baseline levels following cessation. Research findings on breast cancer risk and the use of progestin-only contraceptives remain inconsistent; more large cohort studies are needed as well as accurate tracking of the various PRs by tissue. Epidemiological studies have also implicated reproductive status and hormone exposure in the pathogenesis of ovarian cancers [ 32 ]. Progesterone is widely believed to be antiproliferative and thus protective for uterine and ovarian cancers in young women, via exposure to progestins as components of oral or non-oral contraceptives or during the pregnancy-associated elevation in endogenous progesterone. Full-term pregnancies are associated with reduced epithelial ovarian cancer risk, reducing the incidence of early-stage cancers [ 33 ]. Numerous studies conducted between 1970-1991 consistently found that ever-use of oral contraceptives reduced risk by 35% [ 34 ]. In contrast to pre-menopausal women, combined HRT in post-menopausal women has recently been associated with increased ovarian cancer risk [ 35 ]. Thus, like findings related to breast cancer risk (discussed above), the type of progestin used (progesterone, synthetic progestins), past/current use, and age of exposure appear to be important factors that modify women’s ovarian cancer risk. The impact of progesterone or progestin supplementation on ovarian cancer has recently been reviewed by Mauro et. al [ 36 ].

Most breast cancer treatments target proliferating cells. In ER+ breast cancer, tumor cells are dependent on the transcriptional activity of ER to promote proliferation. Thus, endocrine therapies that target ER function (antiestrogens such as Selective ER Modulators (SERMs, e.g. tamoxifen) and Selective ER Degraders (SERDs, e.g. fulvestrant), and aromatase inhibitors) are very effective initial treatments for ER+ breast cancer. However, these treatments are not durable and at least 10%-41% patients experience late (>5 years) recurrence [ 8 ]. Non-proliferative (G0 phase of the cell cycle) cancer cell states such as quiescence or tumor cell dormancy are poorly targeted by endocrine therapies that primarily interfere with proliferation, but do not kill cancer cells (i.e., are cytostatic instead of cytotoxic). Cancer stem cells (CSCs) are non-proliferating cells that readily evade these therapies and expand slowly over time, repopulating tumors at distant metastatic sites [ 9 , 10 ]. Recent studies in mouse models of breast cancer suggest that antiestrogen therapies such as tamoxifen inhibit breast cancer proliferation, but can simultaneously promote CSC expansion and activity, potentially driving metastasis [ 11 ]. Progesterone and progestins are well known to be proliferative in the normal breast. More recently, in ER+ breast cancer models, nPRs (bound to either progestins or antiprogestins) have been shown to block estrogen-driven proliferation [ 6 , 7 ]. However, only nPR agonists (progesterone or progestins) potently promote non-proliferative (G0) cell phenotypes associated with cancer metastasis such as increased cancer cell survival, endocrine resistance, changes in cancer metabolism, dissemination (migration/invasion), and most notably CSC self-renewal/expansion [ 5 ]. This progesterone-dependent support of circulating dormant breast CSCs demonstrates the danger of prescribing progesterone or progestins to women with a current or prior diagnosis of ER+ breast cancer.