Estrogen biosynthesis and action in ovarian cancer.

Steroid sulfatase is a member of the arylsulfatase family, which is known for the hydrolysis of sulfate ester bonds in a wide range of substrates. The corresponding gene is located on the X-chromosome and contains 10 exons. The group of Hernandez-Guzman et al. ( 64 ) was the first to isolate STS from the human placenta, to purify and crystallize it. This enzyme consists of two membrane-spanning hydrophobic alpha helices that are arranged anti-parallel. The amino acid proline on position 212 acts on the cytosolic side of the endoplasmatic reticulum membrane as turning point. STS is present in the ER of many tissues and is especially high in ovarian granulose cells ( 65 , 66 ). It plays an essential role in the synthesis of E2, by converting E1-S into active E1 (Figure 2 ). Estrogen sulfotransferase belongs to the family of SULTs, phase II detoxification enzymes. Ten isoforms of SULT are known to be expressed in humans. These enzymes have a wide range of substrates including hydroxysteroids, thyroid hormones, phenols, arylamines, and primary alcohols. Because SULT1E1 has the highest affinity of all SULTs for E1 and E2, it is also called estrogen specific sulfotransferase. It catalyzes the transfer of a sulfuryl group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to nucleophilic groups of SULT1E1 substrates. PAPS are synthesized by PAPS synthesizing enzymes (PAPS synthetases). Estrogen sulfotransferase acts in an antagonistic way to STS by converting E1 to E1-S. Therefore, it is inactivating the estrogen. The corresponding gene is located on chromosome 4q3.12, has eight exons and a length of 20 kB. SULT1E1 is present in various tissues, including liver, testis, mammary epithelium cells, and endometrium ( 67 – 69 ). 17β-HSDs form a family of enzymes (14 isoforms in vertebrates) that catalyze the conversion between highly active 17β-hydroxy steroid hormones and 17-keto steroid hormones with lower or even missing activity. 17β-HSD isoenzymes have different enzymatic properties and a characteristic cell-specific expression pattern according to their different physiological functions. They are multifunctional and modulate other steroid structures as well. Among their substrates are bile acids, retinoids, fatty acids, and others. The reductive 17β2-HSD isoenzyme activates E1 to E2 and 4-Dione to testosterone. Also the isoforms 7 and 12 are reductive estrogenic enzymes. In contrast, isoforms 4, 8, 10, and 14 are oxidative enzymes responsible for E2 inactivation ( 63 ). In the normal ovary, 17β-HSDs are detected in granulosa cells of developing follicles, but not in the normal OSE. However, a variety of EOC have been reported to be positive for different 17β-HSD isoenzymes. For example, a recent study showed that reducing 17β12-HSD is overexpressed in many human carcinomas including ovarian carcinoma, while it is not present in the normal OSE. Importantly, patients with EOC tumors with a weak or moderate expression of 17β12-HSD had a better overall survival than those with strongly 17β12-HSD positive tumors ( 55 ).

Currently, ovarian cancer is the fifth most common cancer in women in industrialized countries. It is mainly a disease of postmenopausal women, because more than 80% of all cases are being diagnosed in women older than 50 years. Although a falling rate of new ovarian cancer cases of 1.1% each year and an increase in the relative 5-year survival time from 33.6% in 1975 to 45.2% in 2010 was observed in industrialized countries, ovarian cancer is still the deadliest of all gynecologic malignancies worldwide. The alarming data are greatly attributed to the generally late diagnosis of the disease. More than 80% of all newly detected cases are advanced epithelial ovarian cancers (EOC) with peritoneal metastases and/or metastases in distant organs [Féderation Internationale de Gynécologie et d’Obstétrique (FIGO) stage III–IV]. The FIGO staging system was established by the FIGO and is most commonly used in EOC diagnosis ( 1 ). Effective preventative measures and reliable screening tools for an early detection are not yet available. Although the majority of women experience a variety of non-specific symptoms in the year before diagnosis, the disease is not commonly recognized until the tumor reaches an advanced stage. Another problem is the early development of resistance to the standard chemotherapy regimens with cisplatin/oxaliplatin in combination with paclitaxel. This leads to an early relapse and tumor progression. Considering these problems, it is clear that reliable diagnostic tools for an early detection of these cancers and more treatment options are urgently needed ( 2 – 4 ). Epidemiological data show that induction and biology of ovarian cancer is related to estrogen exposure and metabolism. Experimental data demonstrated that ovarian cancer cells share a number of estrogen regulated pathways with other hormone-dependent cancers such as breast and endometrial cancer. Such pathways were studied in more details already in these tumors ( 5 – 7 ). This review gives an overview on ovarian cancer heterogeneity and estrogen-related mechanisms in ovarian cancer biology. Thereby, data on enzymes active to synthesize and inactivate estrogens as well as on estrogen receptors (ER) are shown and strategies to target these pathways are discussed.

There is strong epidemiological evidence that etiology, pathogenesis, and progression of ovarian cancers are greatly dependent on the activity of estrogens. Furthermore, the balance between estrogen and progesterone is critical for the formation of ovarian cancers ( 22 ). Statistical analyses show that the incidence of ovarian cancer is much higher in industrial countries than in developing countries. The birth rates in industrial countries are low compared to developing countries ( 23 ). There is strong evidence that reproductive factors including multiple pregnancies, breastfeeding, and use of oral conceptive pill (OCP) protect against ovarian cancer. With each pregnancy, the risk of developing ovarian cancer decreases by 10–16% and a pregnancy at the age of 35 years is twice as protective as at the age of 25 years ( 24 , 25 ). Also, a significant protective effect is seen in women that do breastfeeding for more than 18 months ( 26 , 27 ). Similarly, application of OCP for more than 3 years causes a 30–50% reduced risk of developing ovarian cancer ( 28 ). In contrast to these protective factors, women with an early first period and a late menopause as well as women that receive drugs for the treatment of infertility (gonadotropin releasing-hormone antagonists or clomiphene) have an increased risk of developing ovarian cancers. The latter is thought to be caused by high concentrations of estrogen after stimulation of the sex-steroid hormone synthesis in the ovary ( 29 ). Also, application of hormone replacement therapy (HRT) was found to be a risk factor for ovarian cancer. An approximately 22% increased risk of ovarian cancer over 5 years was seen in postmenopausal women using unopposed estrogen as HRT. The risk was still significantly increased (by approximately 10%) by the application of a combination of estrogen and progestin ( 30 , 31 ). Data from a study in a large patient cohort in England revealed that the incidence of ovarian cancer increased with longer duration of HRT therapy, especially if HRT was taken for 10 or more years. There was a higher relative risk for developing EOCs rather than MCs, ECs, or CCCs. But the composition of the HRT did not influence the risk ( 32 ). A more recent study revealed that women taking unopposed oral estrogen therapy have an increased risk of HGSCs, LGSCs, and ECs. Only the risk for MCs was decreased. Similar, an increased risk for serous carcinomas and ECs was found in women receiving an estrogen/progestin combination as HRT ( 33 ). On the other hand, women with previous HRT have a better prognosis when diagnosed with ovarian cancer. They are more likely to be diagnosed at younger age and lower tumor stage allowing a complete surgical removal of the tumor mass (complete debulking without any signs of a residual tumor mass). An increased overall survival, specifically in the subgroup of patients, which had a complete debulking, was found in these studies. No correlation was seen with the progression free survival ( 34 ). Data from another study, however, showed that if HRT was administered following tumor debulking, the prognosis remained unchanged. Furthermore, the survival time was independent on the expression of estrogen and progesterone receptors (PGR) in the cancer tissues. This study was done with a small number of patients only, and therefore, data may not be representative for a larger cohort of ovarian cancer patients ( 35 ). After tumor operation, especially younger ovarian cancer patients may suffer from estrogen withdrawal symptoms, and therefore, they will consider HRT treatment. Since data are not clear yet, studies with more patients suffering from different types of ovarian cancer are urgently needed. To explain the effects of estrogens in the etiology of ovarian cancer, different hypothesis are available. The “incessant ovulation hypothesis” was developed already in 1971, but the more recent “incessant menstruation hypothesis” is now favored ( 36 – 38 ). This hypothesis attributes ovarian cancer formation to repetitive wounding during ovulation and the subsequent activation of repair mechanisms. These processes are associated with an increased number of mutations accumulating in epithelial cells. This finally drives tumor formation and progression ( 39 ). The association between sex steroids and cancer can be explained by processes that take place during the menstrual cycles, in which the ovarian surface epithelium (OSE) plays pivotal roles during ovulation and postovulatory wound repair. Indeed, most of the total proliferative activity of the OSE is related to ovulation repair and formation of the corpus luteum . In the menstrual cycle, the OSE covering growing follicles enters into the proliferative phase during pro-estrus/estrus transition. After the ovulation, the proliferation rate of OSE cells covering the newly formed corpus luteum decreases. Also, the exposure of the OSE to high doses of the gonadotropins luteinizing hormone (LH) and follicle stimulating hormone (FSH) during the menstrual cycle promotes cell proliferation and tumor growth ( 40 ). As a positive effect, progesterone, which is increased during pregnancy and during OCP application, promotes clearing of transformed cells from the ovarian surface epithelial layers ( 37 , 41 ). High-grade serous ovarian cancer are sought to derive from cells in the fimbriae of the fallopian tubes, which are floating in bloody peritoneal fluid. Thereby, they are exposed to iron-induced oxidative stress derived from retrograde menstruation. The genotoxic effect of reactive oxygen species, generated from hemolysis of erythrocytes by pelvic macrophages would explain the distal site of tubal intraepithelial neoplasia ( 37 , 38 , 41 ). At the cellular level, tumor promoting effects of estrogen are conferred in a (i) receptor-dependent and (ii) -independent way. Receptor-dependent ways: binding of estrogen to the nuclear estrogen receptor-α (ER-α) leads to the transcriptional activation of estrogen-responsive genes, which provide signaling systems for cell division and differentiation. Among these genes are proto-oncogenes, such as c-fos, c-myc, and HER2/neu; cell cycle regulating cyclins, growth factors, and others ( 42 ). Binding to membrane-bound G-protein-coupled estrogen receptor (GPER, formerly known as GPR30) activates second messenger systems. Thereby, GPER confers rapid non-genomic effects of estrogens ( 43 ). In a receptor independent way, formation of reactive metabolites via cytochrome P450 enzymes (CYPs) may lead to the generation of mutagenic DNA adducts. Free radicals generated by the metabolic activation of estrogens cause mutations. Accumulation of mutations in various genes in cells in the fallopian tubes and in the ovary will lead to the neoplastic transformation of cells (Figure 1 ) ( 10 , 44 , 45 ). Pathways for estrogen to convert tumor promoting effects in cells in the fallopian tubes and the ovaries . (a.) Activation of the nuclear estrogen receptor-α (ER-α) leads to the transcriptional activation of estrogen-responsive genes, which stimulate cell proliferation. (b.) Binding to membrane-bound G-protein-coupled estrogen receptor (GPER) activates second messenger systems. In cancer cells, estrogen induces extracellular-signal regulated kinase (ERK), phosphoinositide 3-kinase (PI3K), and epidermal growth factor receptor (EGFR) leading to enhanced cell proliferation. (c.) The formation of reactive metabolites leads to the generation of mutagenic DNA adducts. Free radicals from the metabolic activation of estrogens will cause mutations. Accumulation of mutations will lead to neoplastic transformation of proliferating cells ( 10 , 42 , 44 – 46 ) Transcriptional effects of estrogens on target genes are mediated by activation of nuclear receptors, the estrogen receptor-α (ER-α), and estrogen receptor-β (ER-β). Upon binding of estrogens to ER-α, transcription of a battery of genes, which stimulate cell proliferation, is induced. Enhanced proliferation is associated with an increased risk of mutations that accumulate in cells finally leading to malignant transformation. ER-β was found to counteract the growth stimulating effects of ER-α in ovarian cancer cells ( 47 ). Another member of the nuclear receptor superfamily, which confers estrogenic effects in ovarian cancer is the estrogen-related receptor-α (ERR-α). It is known to regulate metabolic homeostasis under conditions of high energy demand, e.g., in brown adipocytes. The increased expression and activity of ERR-α was associated with a less favorable clinical outcome of ovarian cancer ( 42 ). Estrogens also promote tumor progression by influencing signaling pathways. Via the seven-transmembrane spanning G-protein-coupled receptor, named GPER, estrogen rapidly activates the extracellular signal-regulated kinases (ERK)-1 and ERK-2, and confers estrogenic effects to cells devote of the classical nuclear ER ( 48 ). A recent study in endometrial cancer cells showed that GPER mediates the estrogen stimulated induction of the kinases ERK-1 and -2 and the phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K) via activation of matrix metalloproteinase. This is followed by the subsequent transactivation of the epidermal growth factor receptor (EGFR) ( 46 ). Metabolism of estrogen may cause DNA damage by the formation of mutagenic purinergic DNA adducts and by generation of free radicals from the metabolic activation to reactive catechol estrogens. Catechol estrogens are formed by aromatic hydroxylation of primary estrogens at either the C-2 or C-4 position. The catechol metabolites are inactivated by their conjugation with active sulfate or uridine-diphosphate (UDP)-glucuronic acid by steroid sulfotransferases (SULTs) and UDP-glucuronosyltransferases, respectively. Before their conjugation to more water-soluble metabolites, hydroxylation of the steroid moiety via specific CYP isoenzymes, namely CYP1A1 and CYP1A2, which catalyze hydroxylation in position 2, and CYP1B1, which is an estrogen 4-hydroxylase, occurs ( 44 ). Especially, 4-hydroxyestrogens can be oxidized to quinone intermediates, which react with purine bases of the DNA. This results in depurinating adducts, which generate highly mutagenic, apurinic sites. 2-hydroxyestrogens produce less genotoxic DNA adducts. Studies in rodents demonstrated that E2 and E1, as well as their catechol metabolites, in particular 4-hydroxy E2/E1 (4-OH E2/E1), have carcinogenic effects. In a redox cycle, 4-OH E2/E1 is converted to the quinone derivatives. The conversion back to 4-OH E2/E1 is associated with the formation of oxygen radicals. DNA mutations caused by free radicals will lead to the neoplastic transformation of cells ( 45 ). Breast and ovarian cancer mostly arise sporadically, but a small number of cases (approximately 10%) of these cancers are associated with mutations in BRCA1/2 genes. Defects in the DNA damage response or in the DNA repair pathways in patients with the BRCA1/2 mutation are responsible for the high penetrance of these cancers in the breast and/or ovary. It was shown that carriers of BRCA1/2 mutations have also increased levels of estrogen, which may trigger breast and ovarian cancers ( 49 ). Indeed, in premenopausal patients with BRCA1/2 mutations, removal of both ovaries and of the fallopian tubes reduces the risk of these cancers ( 50 ).

Ovarian cancer is the deadliest of all gynecological malignancies in women and these tumors are usually seen in women after the age of 50 years. The still poor prognosis for ovarian cancer is partly attributed to the fact that the diagnosis is usually made at a late stage, when the cancer has already spread to other organs. There are only limited options for a successful chemotherapeutic treatment so far and novel strategies are needed. Several epidemiological and experimental data revealed that ovarian cancer shares many estrogen regulated pathways with other hormone-dependent cancers, e.g., breast cancer. Therefore, local estrogen synthesis from circulating steroid hormone precursors by steroid-forming and steroid-inactivating enzymes may be important to drive ovarian cancer progression in women after the menopause. Indeed, these enzymes and receptors were identified in ovarian cancer cells and their expression was shown to be related to clinical parameters. So far, such studies were mostly done in a small group of patients, which were not selected based on their age, ovarian cancer subtype, hormone-receptor status, and resistance to standard chemotherapy. Because ovarian cancer is a heterogeneous disease and tumors vary with respect to their origin, behavior, and prognosis, they may also differ in their sensitivity to hormonal therapy. At least in subgroups of patients, who express enzymes for estrogen biosynthesis and receptors for estrogen signaling in their tumors, hormonal therapy might offer an additional possibility to prevent recurrence of ovarian cancer. The review explains the role of estrogen in ovarian cancer and it gives an overview on ovarian cancer subtypes. Furthermore, enzymes active to synthesize and metabolize estrogens as well as estrogen signaling pathways are described. Strategies to target these pathways are discussed.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

There is evidence that estrogens play a role in the progression of ovarian cancer. An overexpression of STS will lead to an increased level of E2 and this may contribute to cancer progression. In postmenopausal women, the local conversion from circulating steroid hormone precursors, e.g., E1-S and DHEA-S to active E2 could promote ovarian cancer progression. Indeed, the expression levels of key enzymes vary between normal tissues and different subtypes of EOC. For example, STS was detected in 30% of serous carcinomas, in 70% of CCCs, and in 8% of MCs ( 98 ). Ovarian cancer studies further showed that a longer progression free survival is significantly associated with lower STS levels. That can be explained by the fact that through a high expression of STS, more E2 is synthesized ( 99 ). Similar effects of STS were also reported from breast cancer studies, where STS activity correlated with the E2 serum levels. On the opposite, high estrogen inactivating SULT1E1 levels were associated with smaller tumors, a better prognosis, and a negative lymph node status in ovarian cancer patients ( 100 – 103 ). In the same way, in breast cancer a high expression of SULT1E1 together with a decreased level of STS correlates with a better prognosis, smaller tumor size, and a negative lymph status at the time of diagnosis. Another evidence that SULT1E1 acts as a tumor suppressor is based on a study with xenograft models carried out by Xu et al. ( 69 ) showing that an overexpression of SULT1E1 inhibited estrogen-dependent cell growth and induced tumor cell apoptosis. High E2 levels were often observed in ovarian cancer patients ( 102 ). E2 was shown to increase the mobility of ovarian cancer cells via the inhibition of cell–cell adhesion. This promotes metastasis ( 104 ), and a similar effect of E2 was also observed in breast cancer ( 105 ). Mostly earlier studies in ovarian cancer patients reported that high levels of ER-α and low levels ER-β are associated with a worse prognosis ( 106 – 110 ). However, in other studies, high expression of ER-α was found to be associated with a better prognosis ( 111 , 112 ). Also higher ER-β levels were significantly associated with longer disease-free survival and a longer overall survival in one study. The reduction of ER-β significantly correlated with the hyper-methylation of the ER-β promoter, causing an inhibition of gene translation ( 113 ). A proof for the protective effect of ER-β is that this isoform is the dominant ER isoform in healthy ovaries. But in all serous tumors and also in metastasis, ER-α is usually dominant, and ER-β expression is rather weak. This leads to the conclusion that the gradual reduction of ER-β during tumor progression (from normal to borderline to malign tissue to metastasis) is a continuous process and important for malignant transformation and cancer cell proliferation ( 114 , 115 ). Progesterone has been proved to decrease the proliferative effect of estrogens and inhibit inflammation and cancer penetration by suppressing ovulation. It also initiates apoptosis in tumor cells. PGR is a biomarker for a better prognosis and longer overall survival in ovarian cancer ( 116 ). This is in line with findings that PGR levels are significantly lower in benign, borderline, and malignant ovarian tumors than in healthy tissue ( 108 , 117 ). Recently, a multi-center investigation in 2933 women with invasive EOCs showed that PGR and ER are positive prognostic biomarkers for ECs and HGSCs ( 118 ). There is evidence that estrogen has an influence on the progression of the EOCs at least in some subgroups of patients ( 119 ). To combat estrogen stimulated tumor cell growth, selective estrogen receptor modulators (SERMs) that function as agonists or antagonists for ERs, have been developed. However, modulation of the tissue-specific expression of ER subtypes, expression of co-regulatory proteins, and varying ER conformational changes induced by ligand binding may change the activity of the hormonal therapy. The best studied SERM is tamoxifen that is highly effective to block the ER-signaling pathways. It prevents breast cancer recurrence in many patients with positive ER status. In postmenopausal patients failing tamoxifen therapy, the synthesis of estrogen can be blocked by aromatase inhibitors anastrozole, letrozole, and exemestane ( 120 ). In ovarian cancer, the therapeutic value for SERMs and aromatase inhibitors to block tumor progression and recurrence is not thoroughly established, yet. Only small-scale studies were done so far. Additionally, in the few studies, the patients were not selected based on their hormone-receptor status (ER positive or negative) or age (pre- vs. postmenopausal). Moreover, in some studies, patients were only selected after resistance to standard chemotherapy. Later, the group of Tropé et al. ( 121 ) compared data from different studies with tamoxifen (in total a collective of 647 patients) and found a response rate ranging from 0 to 56%, with a mean response rate of 11%. The treatment with aromatase inhibitor letrozole showed a response rate ranging from 0 to 35.7%, with stable disease rates ranging from 20 to 42% ( 122 – 124 ). For anastrozole, response rates of 1.9–4.3% and stable disease rates of 42–61% were reported ( 125 , 126 ). Whether ovarian cancer patients, who express ERs and estrogen synthesizing enzymes such as STS and 17β-HSDs in their tumors, may have a better response rate to hormonal therapy, should be investigated. Nevertheless, inhibition of estrogen activating STS would offer a novel approach to combat ovarian cancer. Among already available STS inhibitors, the cyclopentane carboxylate derivate STX64 (irosustat) is currently undergoing clinical trials for therapy of prostate, endometrial and breast cancer. With this drug, serum levels of E1, E2, 4-Dione and DHEA were decreased and stable disease for up to 7 months was even seen in breast cancer patients with advanced disease. However, for a more efficient depletion of tumor estrogen, application of STS inhibitors together with an aromatase inhibitor could also be of benefit in postmenopausal women in order to block both, E2 formation via the aromatase and sulfatase pathway ( 57 ). Also, subgroups of ovarian cancers should be studied in different therapeutic settings. This would help to identify patients, for which hormonal therapy might offer an additional possibility to prevent recurrence of ovarian cancer.

Progesterone is synthesized from cholesterol in the corpus luteum , follicles, placenta, and in other organs, e.g., the adrenal gland. During transport through the blood plasma, it is bound to cortisol binding globulin, because of its lipophilic nature and also to avoid degradation. In the ovary, progesterone plays an important role in the follicle maturation and moreover, it is responsible for preparing the female genital tract for pregnancy. It also maintains the pregnancy after fertilization. Progesterone promotes the growth of the uterus musculature and changes the endometrium from a proliferative to a secretory tissue. It also decreases the myometrium activity during pregnancy and changes the quality and quantity of the cervix mucus, thus preventing the entrance of sperms into the uteri and the tubes. In the ovary, it works in concert with estrogen to promote follicle maturation, ovulation, and formation of the corpus luteum . The effects of progesterone are mediated by the two members of nuclear PGR isoforms. Like other receptors of this family, PGR consists of four domains, namely the ligand-binding domain, the Hinge-Region as the flexible link between the ligand-binding domain and the DNA-binding domain, and the N-terminal domain ( 91 ). The two isoforms PR-A and PR-B are encoded by one gene but the transcription is carried out by two different promoters. Apart from the lack of a 164 amino-sequence at the N-terminal end in PR-A, the two PGRs are identical. PR-B is responsible for the transcriptional activation of progesterone responsive genes, but it can be inhibited by PR-A ( 92 – 95 ). PR-A and PR-B act either as homo- or heterodimers and both are synthesized in equal amounts in normal epithelial cells ( 96 ). Progesterone as an antagonist of estrogen has an antiproliferative effect on specific cells. It acts in part by decreasing the production of ERs, and through activation of 17β-HSD and SULT1E1. Indeed, elevated PGR levels were associated with a significantly better survival rate in EOC patients as data from a recent meta-analysis showed ( 97 ).