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Alterations in Brca1 expression in mouse ovarian granulosa cells have short-term and long-term consequences on estrogen-responsive organs
Abstract and Figures
Incessant menstrual cycle activity, uninterrupted by either pregnancy or oral contraceptive use, is the most important risk factor for sporadic ovarian cancer. Menstrual cycle progression is partly controlled by steroid hormones such as estrogens and others that are secreted by the ovarian granulosa cells. We showed earlier that mice carrying a homozygous granulosa cell-specific knockout of Brca1, the homolog of BRCA1 that is associated with familial ovarian cancer predisposition in humans, develop benign epithelial tumors in their reproductive tract. These tumors are driven, at least in part, by a prolongation of the proestrus phase of the estrus cycle (equivalent to the follicular phase of the menstrual cycle) in Brca1 mutant mice, resulting in prolonged unopposed estrogen stimulation. Mutant mice synchronized in proestrus also showed increased circulating estradiol levels, but the possibility that this change also has a role in tumor predisposition was not investigated. We sought to determine whether these changes in hormonal stimulation result in measurable changes in tissues targeted by estrogen outside the ovary. Here we show that mice carrying a Brca1 mutation in their ovarian granulosa cells show increased endometrial proliferation during proestrus, implying that the effects of Brca1 inactivation on estrogen stimulation have short-term consequences, at least on this target organ. We further show that mutant mice develop increased femoral trabecular thickness and femoral length, which are well-known consequences of chronic estrogen stimulation. Estrogen biosynthesis by granulosa cells was increased not only in mice carrying a homozygous Brca1 mutation, but also in heterozygous mutants mimicking the mutational status in granulosa cells of human BRCA1 mutation carriers. The results suggest that human germline BRCA1 mutations, although associated with increased cancer risk, may also have beneficial consequences, such as increased bone strength, that may have contributed to the maintenance of mutated BRCA1 alleles in the human gene pool.
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... Specifically, estrogen levels were 33% higher while progesterone levels were 121% higher in women BRCA carriers relative to women with wild-type BRCA genes [21]. Studies in mice carrying a BRCA1 mutation in ovarian granulosa cells only confirmed these findings of greatly elevated circulating estrogen and progesterone levels, suggesting that a defect in ovarian steroid hormone biosynthesis contributes to breast and ovarian cancer risk in BRCA carriers [22][23][24]. Additionally, hormone-responsive tumors (more notably, breast and prostate cancer) readily generate significant local estrogen, androgen, progesterone and cortisol, from abundant circulating precursors, such as DHEA [25][26][27][28] and cholesterol [29][30][31][32]. ...
... While BRCA1/BRCA2 mutations clearly disrupt DNA repair signaling pathways (subsequently increasing the risk of cancer development), the presence of these mutations in all tissues does not explain the organ specific penetrance for breast and ovarian cancers among carriers. Notably, recent studies have demonstrated that BRCA1 or BRCA2 mutation carriers have higher serum (circulating) levels of estrogen and, in particular, progesterone [21][22][23][24]. This finding fully recapitulated in mice carrying a BRCA1 mutation expressed only in ovarian granulosa (i.e. ...
... This finding fully recapitulated in mice carrying a BRCA1 mutation expressed only in ovarian granulosa (i.e. hormone producing) cells, suggesting that BRCA1/ BRCA2 mutations confer heightened ovarian steroid hormone biosynthesis or decreased turnover [22][23][24]. The removal of both ovaries and fallopian tubes reduces the risk of ovarian and breast cancer, supporting a causal or contributing role for the dysregulation of ovarian hormone production in carriers. ...
Progesterone Receptors (PRs) are critical effectors of estrogen receptor (ER) signaling required for mammary gland development and reproductive proficiency. In breast and reproductive tract malignancies, PR expression is a clinical prognostic marker of ER action. While estrogens primarily regulate PR expression, other factors likely contribute to a dynamic range of receptor expression across diverse tissues. In this study, we identified estrogen-independent but progestin (R5020)-dependent regulation of ER target genes including PGR in ER+/PR+ cancer cell lines. R5020 (10nM-10μM range) induced dose-dependent PR mRNA and protein expression in the absence of estrogen but required both PR-B and ERα. Antagonists of either PR (RU486, onapristone) or ERα (ICI 182,780) attenuated R5020 induction of TFF1, CTSD, and PGR. Chromatin immunoprecipitation (ChIP) assays performed on ER+/PR+ cells demonstrated that both ERα and PR were recruited to the same ERE/Sp1 site-containing region of the PGR proximal promoter in response to high dose progestin (10μM). Recruitment of ERα and PR to chromatin and subsequent PR mRNA induction were dependent upon rapid activation of MAPK/ERK and AKT; inhibition of these kinase pathways via U0126 or LY294002 blocked these events. Overall, we have identified a novel mechanism of ERα activation initiated by rapid PR-dependent kinase pathway activation and associated with phosphorylation of ERα Ser118 for estrogen-independent but progestin-dependent ER/PR cross talk. These studies may provide insight into mechanisms of persistent ER-target gene expression during periods of hormone (i.e. estrogen) ablation and suggest caution following prolonged treatment with aromatase or CYP17 inhibitors (i.e. contexts when progesterone levels may be abnormally elevated).
... The incidence of these tumors correlates with the magnitude of consequences of such mutations on estrous cycle homeostasis, which include prolongation of the estrogen-dominant pre-ovulatory phase and increased circulating levels of estradiol [10]. The physiological significance of these observations is underscored by evidence of increased estrogen stimulation in organs targeted by this hormone in mice harboring Brca1-deficient ovaries including increased proliferative index in the endometrium during the pre-ovulatory phase of the estrous cycle and increased length of long bones [10,11]. Such multi-systemic effects, although more severe in mice that carry a homozygous Brca1 mutation in their ovarian granulosa cells, are also significant in animals that carry a heterozygous mutation such as present in human BRCA1 mutation carriers [11,12]. ...
... The physiological significance of these observations is underscored by evidence of increased estrogen stimulation in organs targeted by this hormone in mice harboring Brca1-deficient ovaries including increased proliferative index in the endometrium during the pre-ovulatory phase of the estrous cycle and increased length of long bones [10,11]. Such multi-systemic effects, although more severe in mice that carry a homozygous Brca1 mutation in their ovarian granulosa cells, are also significant in animals that carry a heterozygous mutation such as present in human BRCA1 mutation carriers [11,12]. The relevance of these observations in experimental animals to human BRCA1 mutation carriers is supported by findings of increased endometrial thickness during the proliferative phase of the menstrual cycle in such carriers, as well as of higher levels of circulating sex steroid hormones [12]. ...
Women-specific cancers are a major health issue, particularly those associated with the BRCA1 germline mutation carrier state, which include triple-negative basal breast carcinomas and high-grade serous ovarian carcinomas (referred to as extra-uterine Müllerian carcinomas). Whereas many chronic diseases can currently be prevented (e.g., cardiovascular diseases), no recent tangible progress was made in cancer prevention of BRCA1 mutation carriers apart from surgical resections of at-risk organs. This lack of progress is largely due to (1) poor understanding of the initiating events triggered by known risk factors in the development of these cancers, (2) the fact that current preventive measures rely on evidence obtained from adjuvant breast cancer treatment that fail to protect against poor prognostic cancers, and (3) problems with using cancer incidence in high-risk women as an ethically justifiable endpoint in cancer prevention trials. Here, we propose that cancer predisposition in BRCA1 mutation carriers is driven, at least in part, by cell-nonautonomous mechanisms (i.e., driven by consequences of this carrier state on hormonal and other systemic factors controlled in organs other than those that are cancer-prone) and that biomarkers of epigenomic reprogramming, hypothesized to be a direct consequence of such cell-nonautonomous mechanisms, are attractive as intermediate surrogate endpoints to assess the efficacy of cancer risk-reducing strategies targeting these mechanisms.
... Indeed, our data suggest that these consequences of reduced BRCA1 expression on premature progression to anaphase resulting in cytokinesis failure are only significant in cells subjected to accelerated replicative aging, a condition that can be triggered in mammary and in serous extra-uterine Müllerian epithelia due to cell-nonautonomous consequences of the BRCA1 mutation carrier state. 18,[25][26][27] Evidence from observations with animal models as well as with human studies 18,[25][26][27] suggest that the BRCA1 mutation carrier state leads to changes in the hormonal fluctuations associated with the menstrual cycle, which are the most important risk factor for the sporadic (non-familial) forms of high-grade serous extra-uterine Müllerian and breast carcinomas. 28,29 Such hormonal fluctuations can influence mitogenic pathways such as, for example, the RANK pathway in targeted tissues, 17,30 resulting in premature replicative aging due to increased cellular proliferation. ...
... Indeed, our data suggest that these consequences of reduced BRCA1 expression on premature progression to anaphase resulting in cytokinesis failure are only significant in cells subjected to accelerated replicative aging, a condition that can be triggered in mammary and in serous extra-uterine Müllerian epithelia due to cell-nonautonomous consequences of the BRCA1 mutation carrier state. 18,[25][26][27] Evidence from observations with animal models as well as with human studies 18,[25][26][27] suggest that the BRCA1 mutation carrier state leads to changes in the hormonal fluctuations associated with the menstrual cycle, which are the most important risk factor for the sporadic (non-familial) forms of high-grade serous extra-uterine Müllerian and breast carcinomas. 28,29 Such hormonal fluctuations can influence mitogenic pathways such as, for example, the RANK pathway in targeted tissues, 17,30 resulting in premature replicative aging due to increased cellular proliferation. ...
We previously described an in vitro model in which serous ovarian cystadenomas were transfected with SV40 large T antigen, resulting in loss of RB and P53 functions and thus mimicking genetic defects present in early high‐grade serous extra‐uterine Müllerian (traditionally called high‐grade serous ovarian) carcinomas including those associated with the BRCA1 mutation carrier state. We showed that replicative aging in this cell culture model leads to a mitotic arrest at the spindle assembly checkpoint. Here we show that this arrest is due to a reduction in microtubule anchoring that coincides with decreased expression of the BUB1 kinase and of the phosphorylated form of its substrate, BUB3. The ensuing prolonged mitotic arrest leads to cohesion fatigue resulting in cell death or, in cells that recover from this arrest, in cytokinesis failure and polyploidy. Down‐regulation of BRCA1 to levels similar to those present in BRCA1 mutation carriers leads to increased and uncontrolled microtubule anchoring to the kinetochore resulting in overcoming the spindle assembly checkpoint. Progression to anaphase under those conditions is associated with formation of chromatin bridges between chromosomal plates due to abnormal attachments to the kinetochore, significantly increasing the risk of cytokinesis failure. The dependence of this scenario on accelerated replicative aging can, at least in part, account for the site specificity of the cancers associated with the BRCA1 mutation carrier state, as epithelia of the mammary gland and of the reproductive tract are targets of cell‐nonautonomous consequences of this carrier state on cellular proliferation associated with menstrual cycle progressions. This article is protected by copyright. All rights reserved.
... They concluded that mice carrying a Brca1 mutation had both increased and prolonged estrogen stimulation unopposed by progesterone (64). The physiological relevance of these findings is underscored by a follow up study showing evidence of increased steroid hormone stimulation in end-organs targeted by estrogens such as the endometrium and long bones in Brca1-deficient mice (65). Mutant mice showed increased endometrial thickness and increased bone length and density, providing strong support for the idea that the presence of a Brca1 mutation in ovarian granulosa cells leads to increased estrogen stimulation. ...
... Mutant mice showed increased endometrial thickness and increased bone length and density, providing strong support for the idea that the presence of a Brca1 mutation in ovarian granulosa cells leads to increased estrogen stimulation. Although the authors used mice carrying homozygous Brca1 mutations in these studies in order to magnify the measurable consequences of such mutations, they also showed that mice carrying heterozygous mutations, such as present in human BRCA1 mutation carriers, showed effects similar to those present in homozygous mutants on sex steroid hormone biosynthesis, albeit of lesser magnitude (65). ...
Serous extra-uterine Müllerian tumors include lesions previously classified either as serous ovarian, fimbrial, or primary peritoneal tumors. They should be distinguished from intra-uterine (endometrial) serous tumors in spite of their common Müllerian differentiation lineage due to distinctive clinical-pathological parameters. Increased risk for these cancers is driven primarily by cell non-autonomous hormonal factors associated with menstrual cycle progression, which can be accentuated by mutations in BRCA1/2. Additional factors, such as inflammatory conditions and others, may also contribute to elevated cancer risk in some individuals. The most consistent molecular alterations associated with serous extra-uterine Müllerian carcinomas, whether they arise in the context of familial predisposition or BRCA-independent hormonal factors, include severe aneuploidy superimposed on P53 mutations. Their near polyploid nature strongly suggests a role for mitotic errors associated with cytokinesis failure in their pathogenesis. A molecular mechanism is proposed involving a combination of both cell non-autonomous and cell autonomous factors in the development and progression of serous extra-uterine Müllerian tumors. Cell non-autonomous factors lead to increased cellular proliferation in extra-uterine serous Müllerian epithelium and contribute to the site-specificity of cancers associated with germline BRCA mutations. Cell autonomous factors in individuals with BRCA1 mutations lead to failure of cytokinesis following recovery from a cell cycle arrest at the mitotic spindle assembly checkpoint. In individuals with intact BRCA1/2 function, recovery from such arrest might be facilitated by CCNE1 amplification and ensuing centrosome duplication leading to increased microtubule anchoring, accounting for frequent amplification of this cyclin observed in tumors not associated with BRCA1/2 mutations. The cell non-autonomous factors provide an opportunity for using pharmacological means to control cancer incidence in individuals at elevated risk for these cancers such as BRCA1/2 mutation carriers, underscoring the importance of better understanding their determinants and downstream targets. The cell autonomous factors discussed here account for the molecular features of high-grade serous extra-uterine Müllerian carcinomas, which are regarded as a disease of chromosomes, and underscore the potential merit of targeting components of the spindle assembly checkpoint in their clinical management.
... The idea that germline mutations in the ubiquitously expressed BRCA1 gene drive cancer development primarily via a classical tumour-suppressor mechanism triggered by a reduced "chromosome custodian" function [14] does not account for the organspecificity of the cancers associated with this carrier state [15]. We previously reported evidence for cellnonautonomous mechanisms of cancer predisposition in humans and experimental animals carrying germline BRCA1 or Brca1 mutations [16][17][18][19][20][21]. By definition, such mechanisms are driven by consequences of this carrier state on organs that are different than (and are upstream of ) those that are cancer-prone. ...
Background
Breast cancer is a leading cause of death in premenopausal women. Progesterone drives expansion of luminal progenitor cells, leading to the development of poor-prognostic breast cancers. However, it is not known if antagonising progesterone can prevent breast cancers in humans. We suggest that targeting progesterone signalling could be a means of reducing features which are known to promote breast cancer formation.
Methods
In healthy premenopausal women with and without a BRCA mutation we studied (i) estrogen and progesterone levels in saliva over an entire menstrual cycle ( n = 20); (ii) cancer-free normal breast-tissue from a control population who had no family or personal history of breast cancer and equivalently from BRCA1/2 mutation carriers ( n = 28); triple negative breast cancer (TNBC) biopsies and healthy breast tissue taken from sites surrounding the TNBC in the same individuals ( n = 14); and biopsies of ER+ve/PR+ve stage T1–T2 cancers and healthy breast tissue taken from sites surrounding the cancer in the same individuals ( n = 31); and (iii) DNA methylation and DNA mutations in normal breast tissue (before and after treatment) from clinical trials that assessed the potential preventative effects of vitamins and antiprogestins (mifepristone and ulipristal acetate; n = 44).
Results
Daily levels of progesterone were higher throughout the menstrual cycle of BRCA1/2 mutation carriers, raising the prospect of targeting progesterone signalling as a means of cancer risk reduction in this population. Furthermore, breast field cancerization DNA methylation signatures reflective of (i) the mitotic age of normal breast epithelium and (ii) the proportion of luminal progenitor cells were increased in breast cancers, indicating that luminal progenitor cells with elevated replicative age are more prone to malignant transformation. The progesterone receptor antagonist mifepristone reduced both the mitotic age and the proportion of luminal progenitor cells in normal breast tissue of all control women and in 64% of BRCA1/2 mutation carriers. These findings were validated by an alternate progesterone receptor antagonist, ulipristal acetate, which yielded similar results. Importantly, mifepristone reduced both the TP53 mutation frequency as well as the number of TP53 mutations in mitotic-age-responders.
Conclusions
These data support the potential usage of antiprogestins for primary prevention of poor-prognostic breast cancers.
Trial registration
Clinical trial 1 Mifepristone treatment prior to insertion of a levonorgestrel releasing intrauterine system for improved bleeding control – a randomized controlled trial , clinicaltrialsregister.eu, 2009-009014-40 ; registered on 20 July 2009.
Clinical trial 2 The effect of a progesterone receptor modulator on breast tissue in women with BRCA1 and 2 mutations , clinicaltrials.gov, NCT01898312 ; registered on 07 May 2013.
Clinical trial 3 A pilot prevention study of the effects of the anti- progestin Ulipristal Acetate (UA) on surrogate markers of breast cancer risk , clinicaltrialsregister.eu, 2015-001587-19 ; registered on 15 July 2015.
... The demultiplexed sequencing reads were quality checked, trimmed and filtered (Sickle v1.33 [25]) and adapters and primers removed (Cutadapt v1.10 [26]). Overlapping paired-end reads were merged for full 12 length V1-V3 16S amplicons (FLASh v1.2.11) [27], clustered (CD-HIT v4.6) [28], and chimeric sequences removed (UCHIME v4.2.40) [29]. ...
Background
The composition of the microbiome plays an important role in human health and disease. Whether there is a direct association between the cervicovaginal microbiome and the host’s epigenome is largely unexplored.
Results
Here we analyzed a total of 448 cervicovaginal smear samples and studied both the DNA methylome of the host and the microbiome using the Illumina EPIC array and next-generation sequencing, respectively. We found that those CpGs that are hypo-methylated in samples with non-lactobacilli (O-type) dominating communities are strongly associated with gastrointestinal differentiation and that a signature consisting of 819 CpGs was able to discriminate lactobacilli-dominating (L-type) from O-type samples with an area under the receiver operator characteristic curve (AUC) of 0.84 (95% CI = 0.77–0.90) in an independent validation set. The performance found in samples with more than 50% epithelial cells was further improved (AUC 0.87) and in women younger than 50 years of age was even higher (AUC 0.91). In a subset of 96 women, the buccal but not the blood cell DNA showed the same trend as the cervicovaginal samples in discriminating women with L- from O-type cervicovaginal communities.
Conclusions
These findings strongly support the view that the epithelial epigenome plays an essential role in hosting specific microbial communities.
... Studies have shown a negative relationship between BRCA1 expression and oestradiol synthesis through aromatase P450 (CYP19A1), suggesting an additional role for BRCA1 in GCs, besides DNA repair. Consistent with this, mutation of BRCA1 in human and mice have shown increased levels of oestradiol and increased endometrial stromal cell proliferation (Chand, Simpson and Clyne, 2009;Yen et al., 2012). Hong et al. showed that adult mice that do not express BRCA1 in their GCs have a more extended proestrus phase, increasing the time of exposition to high levels of oestrogen . ...
Altered maternal nutrition around conception can affect oocyte and embryo development which influences later-life health outcomes. Low folate is related to poor reproductive outcomes. Folic acid (FA) supplementation and fortification have been effective strategies to avoid NTD. On the other hand, the FA levels in women of reproductive age are increased more than recommended, with unknown consequences. This project aims to determine the effect of high FA diet on the ovary and embryo development.
C57BL/6 female mice at PND74 were fed with control (1mg FA/kg food) or high (5mg FA/kg food) FA diet for four weeks and culled at diestrus stage (PND102). In parallel, a group of animals were maintained on the control diet for another four weeks (PND130) or either mated and culled at 3.5 days post coitum (dpc). Ovaries and embryos were collected, RNA extracted and analysed by qPCR. Morphological and immunostaining analyses were also performed to determine the effect of FA in the ovary and blastocyst.
High FA diet reduced expression of follicle developmental control genes at PND102 such as Fshr and Oct4, but also epigenetic writers like Ezh2 and Bmi1. In contrast, four weeks after FA diet release, the same genes were upregulated in the ovary. Females with a preconceptional high FA diet showed an increased mating period compared to the control group. The embryos of these mice showed reduced TE cells and lower expression of CDX2. In parallel, embryos exposed to high FA diet exclusively during preimplantation showed delayed development with decreased total cell number and lower expression of lineage markers (Oct4, NANOG and Gata6).
High FA diet not only altered follicle growth factors during and after supplementation in a different pattern but also affected blastocyst biogenesis. This could impact on later-life health outcomes of the offspring.
... Cell-nonautonomous effects. High-penetrance germline mutations (such as mutations in BRCA1/2) are able to modulate the influence of various endocrine factors [54][55][56][57][58][59] (such as higher levels of oestrogen and progesterone production in the ovaries). These mutations might then have specific effects on the epigenomes of cells that are receptive to these signals. ...
The incidence of cancer is continuing to rise and risk-tailored early diagnostic and/or primary prevention strategies are urgently required. The ideal risk-predictive test should: integrate the effects of both genetic and nongenetic factors and aim to capture these effects using an approach that is both biologically stable and technically reproducible; derive a score from easily accessible biological samples that acts as a surrogate for the organ in question; and enable the effectiveness of risk-reducing measures to be monitored. Substantial evidence has accumulated suggesting that the epigenome and, in particular, DNA methylation-based tests meet all of these requirements. However, the development and implementation of DNA methylation-based risk-prediction tests poses considerable challenges. In particular, the cell type specificity of DNA methylation and the extensive cellular heterogeneity of the easily accessible surrogate cells that might contain information relevant to less accessible tissues necessitates the use of novel methods in order to account for these confounding issues. Furthermore, the engagement of the scientific community with health-care professionals, policymakers and the public is required in order to identify and address the organizational, ethical, legal, social and economic challenges associated with the routine use of epigenetic testing.
... Although cell nonautonomous alterations such as hormonal alterations leading to aberrant growth of hormone-sensitive target cells may be particularly relevant to sporadic cancers ( Veronesi et al., 2005), recent evidence linking BRCA-associated risk and hormonal factors (Domchek et al., 2010;Dubeau, 2015) suggests an interplay between cell-autonomous and cell nonautonomous factors in BRCAmutation carriers. Preclinical studies (Chodankar et al., 2005;Hong et al., 2010;Yen et al., 2012) show mice carrying a BRCA1 mutation in the steroid-hormone-producing granulosa cells have a longer prooestrous phase, corresponding with the oestrogen-dominant follicular phase of the human menstrual cycle, as well as elevated basal E2 levels and evidence of increased oestrogen exposure in target organs such as bones. Recently, we demonstrated altered endometrial thickness and higher E2 and P levels in well-defined parts of the luteal phase in BRCA-mutation carriers vs wild-type controls (Widschwendter et al., 2013). ...
Breast cancer development in BRCA1/2 mutation carriers is a net consequence of cell-autonomous and cell nonautonomous factors which may serve as excellent targets for cancer prevention. In light of our previous data we sought to investigate the consequences of the BRCA-mutation carrier state on RANKL/osteoprotegerin (OPG) signalling. We analysed serum levels of RANKL, OPG, RANKL/OPG complex, oestradiol (E2), and progesterone (P) during menstrual cycle progression in 391 BRCA1/2-mutation carriers and 782 noncarriers. These studies were complemented by analyses of RANKL and OPG in the serum and mammary tissues of female cynomolgus macaques (n = 88) and serum RANKL and OPG in postmenopausal women (n = 150). BRCA-mutation carriers had lower mean values of free serum OPG in particular in BRCA1-mutation carriers (p = 0.018) compared with controls. Among BRCA1/2 mutation carriers, lower OPG levels were associated with germline mutation locations known to confer an increased breast cancer risk (p = 0.003). P is associated with low OPG levels in serum and tissue, particularly in BRCA-mutation carriers (rho = - 0.216; p = 0.002). Serum OPG levels were inversely correlated (rho = - 0.545, p < 0.001) with mammary epithelial proliferation measured by Ki67 expression and increased (p = 0.01) in postmenopause. The P-RANKL/OPG system is dysregulated in BRCA-mutation carriers. These and previously published data provide a strong rationale for further investigation of antiprogestogens or an anti-RANKL antibody such as denosumab for breast cancer prevention.
Germline mutations present in inherited cancer syndromes are thought to have a local effect in an organ that is predisposed to the development of cancer. This may explain the organ-specific cancer penetrance seen for example with breast and ovarian tumors in BRCA1 and BRCA2 mutation carriers. In fact, evidence from preclinical models suggest that both hormone production and sensitivity of end organs is altered in carriers of BRCA1 mutations [1,2]. In this article the authors postulate that both endometrial thickness, as a surrogate of hormonal activity, and serum ovarian steroid hormone titers at particular stages of menstrual cycle, differ between carriers of the BRCA1/BRCA2 mutations and women without these mutations. Participants, women aged above 35 years with no previous or subsequent history of cancer, were recruited from the UK Familial Ovarian Cancer Screening Study between June 2002 and September 2010. Endometrial thickness and date of the last menstrual period were routinely recorded during sonography performed in the context of ovarian cancer screening, germline mutations in the coding sequence of BRCA1 and BRCA2 were assessed, and estradiol and progesterone were measured with automated immunoassays. Results showed clear differences in cyclical endometrial thickness and sex hormone titers between carries of BRCA1/BRCA2 mutations and wild-type controls. These results support the hypothesis that the organ-specific cancer penetrance in carriers is, in part, due to hormonal dysregulation and might be the basis for novel chemoprevention trials using strategies (e.g., anti-RANKL monoclonal antibody or progesterone antagonist/modulator) that exploit them.
Epidemiological studies have shown that taller people are at increased risk of cancer, but it is unclear if height-associated risks vary by cancer site, or by other factors such as smoking and socioeconomic status. Our aim was to investigate these associations in a large UK prospective cohort with sufficient information on incident cancer to allow direct comparison of height-associated risk across cancer sites and in relation to major potential confounding and modifying factors.
Information on height and other factors relevant for cancer was obtained in 1996-2001 for middle-aged women without previous cancer who were followed up for cancer incidence. We used Cox regression models to calculate adjusted relative risks (RRs) per 10 cm increase in measured height for total incident cancer and for 17 specific cancer sites, taking attained age as the underlying time variable. We also did a meta-analysis of published results from prospective studies of total cancer risk in relation to height.
1 297 124 women included in our analysis were followed up for a total of 11·7 million person-years (median 9·4 years per woman, IQR 8·4-10·2), during which time 97 376 incident cancers occurred. The RR for total cancer was of 1·16 (95% CI 1·14-1·17; p<0·0001) for every 10 cm increase in height. Risk increased for 15 of the 17 cancer sites we assessed, and was statistically significant for ten sites: colon (RR per 10 cm increase in height 1·25, 95% CI 1·19-1·30), rectum (1·14, 1·07-1·22), malignant melanoma (1·32, 1·24-1·40), breast (1·17, 1·15-1·19), endometrium (1·19, 1·13-1·24), ovary (1·17, 1·11-1·23), kidney (1·29, 1·19-1·41), CNS (1·20, 1·12-1·29), non-Hodgkin lymphoma (1·21, 1·14-1·29), and leukaemia (1·26, 1·15-1·38). The increase in total cancer RR per 10 cm increase in height did not vary significantly by socioeconomic status or by ten other personal characteristics we assessed, but was significantly lower in current than in never smokers (p<0·0001). In current smokers, smoking-related cancers were not as strongly related to height as were other cancers (RR per 10 cm increase in height 1·05, 95% CI 1·01-1·09, and 1·17, 1·13-1·22, respectively; p=0·0004). In a meta-analysis of our study and ten other prospective studies, height-associated RRs for total cancer showed little variation across Europe, North America, Australasia, and Asia.
Cancer incidence increases with increasing adult height for most cancer sites. The relation between height and total cancer RR is similar in different populations.
Cancer Research UK and the UK Medical Research Council.
Menstrual cycle activity is the most important risk factor for sporadic serous ovarian carcinoma, whereas a germ-line mutation in BRCA1 is the most important risk factor for the familial form. Given the rarity of BRCA1 mutations in sporadic ovarian cancers, we hypothesized that BRCA1 influences the menstrual cycle in a way that mimics the factors underlying sporadic ovarian cancer predisposition, making BRCA1 mutations redundant in such cancers. We compared the length of each phase of the estrus cycle (equivalent to the human menstrual cycle) and of circulating levels of estradiol in control mice and in mice carrying a Brca1 mutation in their ovarian granulosa cells, two thirds of which develop ovarian or uterine epithelial tumors. We also compared the length of the different phases of the cycle in mutants that subsequently developed tumors with those in mutants that remained tumor-free. Mutant mice as well as oophorectomized wild-type mice harboring mutant ovarian grafts showed a relative increase in the average length of the proestrus phase of the estrus cycle, which corresponds to the estrogen-dominated follicular phase of the human menstrual cycle. Total circulating levels of estradiol were also increased in mutant mice injected with pregnant mare serum gonadotropins. The relative increase in proestrus length was highest in mutant mice that subsequently developed reproductive epithelial tumors. We conclude that loss of a functional Brca1 increases murine ovarian epithelial tumor predisposition by increasing estrogen stimulation in the absence of progesterone, recapitulating conditions associated with sporadic ovarian cancer predisposition in humans.
Although the role of the osteoclast in bone resorption is becoming better understood, much remains to be learned about osteoclastogenesis and the exact mechanism of action of anti-resorbing agents such as 17β-estradiol. This study investigated bone and morphologic osteoclast alterations following long-term estrogen administration to the B6D2F1 mouse. B6D2F1 mice aged 4-5 weeks were exposed to high levels of estrogen via implanted silastic tubing for at least 12 weeks; controls received empty tubing. Femurs of control and treated mice were assessed with radiology, quantitative histomorphometry and transmission electron microscopy.
After 8 weeks of treatment, there was radiologic evidence of severe osteosclerosis and 86% of femoral marrow space was replaced with bone. After 12 weeks histologic studies of treated animals revealed that osteoclasts were positive for tartrate-resistant acid phosphatase but showed markedly abnormal ultrastructure which prevented successful bone resorption.
Findings extend our understanding of osteoclast structure and function in the mouse exposed in vivo to high doses of estrogen. Ultrastructural examination showed that osteoclasts from estrogen-treated mice were unable to seal against the bone surface and were unable to form ruffled borders.
Adult height and body mass index (BMI) influence the risk of breast cancer in women. Whether these associations reflect growth patterns of the fetus or growth during childhood and adolescence is unknown. We investigated the association between growth during childhood and the risk of breast cancer in a cohort of 117,415 Danish women. Birth weight, age at menarche, and annual measurements of height and weight were obtained from school health records. We used the data to model individual growth curves. Information on vital status, age at first childbirth, parity, and diagnosis of breast cancer was obtained through linkages to national registries. During 3,333,359 person-years of follow-up, 3340 cases of breast cancer were diagnosed. High birth weight, high stature at 14 years of age, low BMI at 14 years of age, and peak growth at an early age were independent risk factors for breast cancer. Height at 8 years of age and the increase in height during puberty (8–14 years of age) were also associated with breast cancer. The attributable risks of birth weight, height at 14 years of age, BMI at 14 years of age, and age at peak growth were 7%, 15%, 15%, and 9%, respectively. No effect of adjusting for age at menarche, age at first childbirth, and parity was observed. Birth weight and growth during childhood and adolescence influence the risk of breast cancer.
Cancers that develop in BRCA1 mutation carriers are usually near tetraploid/polyploid. This led us to hypothesize that BRCA1 controls the mitotic checkpoint complex, as loss of such control could lead to mitotic errors resulting in tetraploidy/polyploidy and subsequent aneuploidy. We used an in vitro system mimicking premalignant conditions, consisting of cell strains derived from the benign counterparts of serous ovarian carcinomas (cystadenomas) and expressing SV40 large T antigen, conferring the equivalent of a p53 mutation. We previously showed that such cells undergo one or several doublings of their DNA content, as they age in culture and approach the phenomenon of in vitro crisis. Here, we show that such increase in DNA content reflects a cell cycle arrest possibly at the anaphase promoting complex, as evidenced by decreased BrdU incorporation and increased expression of the mitotic checkpoint complex. Down-regulation of BRCA1 in cells undergoing crisis leads to activation of the anaphase promoting complex and resumption of growth kinetics similar to those seen in cells before they reach crisis. Cells recovering from crisis after BRCA1 down-regulation become multinucleated, suggesting that reduced BRCA1 expression may lead to initiation of a new cell cycle without completion of cytokinesis. This is the first demonstration that BRCA1 controls a physiological arrest at the M phase apart from its established role in DNA damage response, a role that could represent an important mechanism for acquisition of aneuploidy during tumor development. This may be particularly relevant to cancers that have a near tetraploid/polyploid number of chromosomes.
Synthetic glucocorticoids (GCs) like dexamethasone (DEX) are effective immunosuppressants indicated for autoimmune and inflammatory diseases. However, they often promote osteoporosis and bone fractures. Here, we investigated the anti-mitogenic effect of GCs in primary osteoblast cultures. DEX did not affect cell cycle progression in confluent (day 2), or early post-confluent cultures. Starting on day 5, however, DEX strongly inhibited the G1/S cell cycle transition. Day 5 also marked the beginning of a ∼2-day commitment stage, during which cultures acquired resistance to the inhibitory effect of DEX on mineralization. Considering the importance of Wnt signaling in osteoblast development, we analyzed the effect of DEX on the Wnt pathway. DEX treatment did not inhibit the TOPGAL Wnt reporter before, but only during and after the commitment stage. However, this inhibition was not associated with repression of neither cyclin D1 nor c-Myc mRNA, well-established Wnt targets regulating cell cycle progression. On the other hand, acute (24-h) and chronic (7 days) DEX treatment significantly reduced both the mRNA and protein levels of cyclin A, another cell cycle regulator. Moreover, cyclin A repression by DEX was not observed before, but only during and after the commitment stage. Using gel shift and reporter assays, we identified an ATF/CREB-binding site critical for the DEX-mediated repression of cyclin A transcription. Furthermore, and similar to cyclin A, Atf4 expression was repressed by DEX only during and after commitment. Our data suggest that GCs attenuate cell cycle progression in osteoblasts in a developmental stage-specific manner by repressing Atf4-dependent cyclin A gene expression.
To add to the existing evidence that comes mostly from White populations, we conducted a nested case-control study to examine the association between sex hormones and breast cancer risk within the Multiethnic Cohort that includes Japanese American, White, Native Hawaiian, African American, and Latina women. Of the postmenopausal women for whom we had a plasma sample, 132 developed breast cancer during follow-up. Two controls per case, matched on study area (Hawaii, Los Angeles), ethnicity/race, birth year, date and time of blood draw and time fasting, were randomly selected from the women who had not developed breast cancer. Levels of estradiol (E(2)), estrone (E(1)), androstenedione, dehydroepiandrosterone (DHEA), and testosterone were quantified by RIA after organic extraction and Celite column partition chromatography. E(1) sulfate, DHEA sulfate (DHEAS), and sex hormone-binding globulin (SHBG) were quantified by direct immunoassays. Based on conditional logistic regression, the sex hormones were positively associated and SHBG was negatively associated with breast cancer risk. All associations, except those with DHEAS and testosterone showed a significant linear trend. The odds ratio (OR) associated with a doubling of E(2) levels was 2.26 (95% confidence interval (CI) 1.58-3.25), and the OR associated with a doubling of testosterone levels was 1.34 (95% CI 0.98-1.82). The associations in Japanese American women, who constituted 54% of our sample, were similar to or nonsignificantly stronger than in the overall group. This study provides the best evidence to date that the association between sex hormones and breast cancer risk is generalizable to an ethnically diverse population.
Inherited mutations of the breast cancer susceptibility gene BRCA1 confer a high risk for breast cancer development. The (300)RXKK and (266)KXK motifs have been identified previously as sites for acetylation of the estrogen receptor-alpha (ER-alpha), and (302)K was also found to be a site for BRCA1-mediated mono-ubiquitination of ER-alpha in vitro. Here we show that ER-alpha proteins with single or double lysine mutations of these motifs (including K303R, a cancer-associated mutant) are resistant to inhibition by BRCA1, even though the mutant ER-alpha proteins retain the ability to bind to BRCA1. We also found that BRCA1 overexpression reduced and knockdown increased the level of acetylated wild-type ER-alpha, without changing the total ER-alpha protein level. Increased acetylation of ER-alpha due to BRCA1 small interfering RNA was dependent upon phosphatidylinositol 3-kinase/Akt signaling and on up-regulation of the coactivator p300. In addition, using an in vitro acetylation assay, we found that in vitro-translated wild-type BRCA1 but not a cancer-associated point mutant (C61G) inhibited p300-mediated acetylation of ER-alpha. Furthermore, BRCA1 overexpression increased the levels of mono-ubiquitinated ER-alpha protein, and a BRCA1 mutant that is defective for ubiquitin ligase activity but retains other BRCA1 functions (I26A) did not ubiquitinate ER-alpha or repress its activity in vivo. Finally, ER-alpha proteins with mutations of the (300)RXKK or (266)KXK motifs showed modest or no BRCA1-induced ubiquitination. We propose a model in which BRCA1 represses ER-alpha activity, in part, by regulating the relative degree of acetylation vs. ubiquitination of ER-alpha.
It has become increasingly clear that use of menopausal hormone therapy (HT) is associated with an increased risk of ovarian cancer; however, the effects by type of formulation and duration of use are less clear. A systematic review of the HT and ovarian cancer literature was conducted to identify population-based case-control studies, cohort studies, and randomized trials that examined effects by formulation of HT (estrogen-alone [ET] and estrogen plus progestin [EPT]) and duration of use.
Pub-Med (www.pubmed.gov) was used to identify relevant publications through December 2007; 14 studies were identified. The authors abstracted relative risks (RRs) and 95% confidence intervals (CIs) in relation to duration of HT use (ET and EPT separately). The authors used the risk estimates per year of HT use if these were provided; otherwise, they calculated a duration-response for a log-linear model of the duration of HT use against risk.
Ovarian cancer risk was increased among ET users (RR per 5 years of use, RR(5) = 1.22; 95% CI, 1.18-1.27; P < .0001), and a lower but still statistically significant increased risk was seen with EPT use (RR(5) = 1.10; 95% CI, 1.04-1.16; P = .001). The increased risk in ET users was statistically significantly higher than the increased risk in EPT users (P = .004).
ET use increases risk of ovarian cancer in a duration-dependent manner, and it appears that the addition of progestins blocks this effect, at least to some extent. Whether the effect of estrogens would be completely blocked if progestins were given every day is unclear.