Natural Killer Cell Dysfunction in Premenopausal BRCA1 Mutation Carriers: A Potential Mechanism for Ovarian Carcinogenesis.

BACKGROUND: Tissue-specificity for fimbrial fallopian tube ovarian carcinogenesis remains largely unknown in BRCA1 mutation carriers. We aimed to assess the cell autonomous and cell-nonautonomous implications of a germline BRCA1 mutation in the context of cancer immunosurveillance of CD3- CD56+ natural killer (NK) cells. METHODS: Premenopausal BRCA1 mutation carriers versus age-matched non-carriers were compared. Daily urinary 5ß-pregnanediol levels were used to determine progesterone metabolomics across an ovarian cycle. Using peripherally acquired NK cells the cell-mediated cytotoxicity of tumor targets (OVCAR-3, K-562) was determined using live cellular impedance (xCELLigence®) and multicolor flow cytometry. Hypoxia-inducible factor 1-alpha (HIF-1α) immunohistochemistry of cancer-free fallopian tube specimens allowed a comparison of proximal versus distal portions. Utilizing these findings the role of environmental factors relevant to the fimbrial fallopian tube (progesterone, hypoxia) on NK cell functional activity were studied in an ovarian phase-specific manner. RESULTS: BRCA1 mutation carriers demonstrate a differential progesterone metabolome with a phase-specific reduction of peripheral NK cell functional activity. Progesterone exposure further impairs NK cell-mediated cytotoxicity in a dose-dependent manner, which is reversed with the addition of mifepristone (1.25 µM). The fimbrial fallopian tube demonstrated significantly higher HIF-1α staining, particularly in BRCA1 mutation carriers, reflecting a site-specific 'hypoxic niche'. Exposure to hypoxic conditions (1% O2) can further impair tumor cytotoxicity in high-risk carriers. CONCLUSIONS: Phase-specific differential NK cell activity in BRCA1 mutation carriers, either systemically or locally, may favor site-specific pre-invasive carcinogenesis. These cumulative effects across a reproductive lifecycle in high-risk carriers can have a detrimental effect further supporting epidemiological evidence for ovulation inhibition.

Neurotropism of SARS-CoV-2: A Pathological Examination of Neurosurgical Specimens.

BACKGROUND AND OBJECTIVES: Neurological manifestations may occur in more than 80% of patients hospitalized with COVID-19 infection, including severe disruptions of the central nervous system (CNS), such as strokes, encephalitis, or seizures. Although the primary pathophysiological mechanism for the effects of COVID-19 in CNS remains unknown, evidence exists for both direct injury from neuroinvasion and indirect effects from disruptions in systemic inflammatory and coagulation pathways. In this study, we analyzed CNS tissue from living patients to better understand these processes. METHODS: With institutional review board approval and patient consent, samples that would be otherwise discarded from patients with active or recent (within 6 days of surgery) COVID-19 infection undergoing neurosurgical intervention were collected and tested for the presence of SARS-CoV-2 using immunohistochemistry, in situ hybridization, electron microscopy, and reverse transcription polymerase chain reaction. RESULTS: Five patients with perioperative mild-to-moderate COVID-19 infection met inclusion criteria (2 male, 3 female; mean age 38.8 ± 13.5 years). Neurosurgical diagnoses included a glioblastoma, a ruptured arteriovenous malformation, a ruptured posterior inferior cerebellar artery aneurysm, a middle cerebral artery occlusion, and a hemorrhagic pontine cavernous malformation. Samples analyzed included the frontal lobe cortex, olfactory nerve, arteriovenous malformation/temporal lobe parenchyma, middle cerebral artery, cerebellum, and cavernous malformation/brainstem parenchyma. Testing for the presence of SARS-CoV-2 was negative in all samples. CONCLUSION: The CNS is likely not a significant viral reservoir during mild-to-moderate COVID-19 infection, although direct neuroinvasion is not definitively excluded. Additional testing to help elucidate the relative contributions of direct and indirect pathways for CNS injury from COVID is warranted.

GRP78 Inhibitor YUM70 Suppresses SARS-CoV-2 Viral Entry, Spike Protein Production and Ameliorates Lung Damage.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, has given rise to many new variants with increased transmissibility and the ability to evade vaccine protection. The 78-kDa glucose-regulated protein (GRP78) is a major endoplasmic reticulum (ER) chaperone that has been recently implicated as an essential host factor for SARS-CoV-2 entry and infection. In this study, we investigated the efficacy of YUM70, a small molecule inhibitor of GRP78, to block SARS-CoV-2 viral entry and infection in vitro and in vivo. Using human lung epithelial cells and pseudoviral particles carrying spike proteins from different SARS-CoV-2 variants, we found that YUM70 was equally effective at blocking viral entry mediated by original and variant spike proteins. Furthermore, YUM70 reduced SARS-CoV-2 infection without impacting cell viability in vitro and suppressed viral protein production following SARS-CoV-2 infection. Additionally, YUM70 rescued the cell viability of multi-cellular human lung and liver 3D organoids transfected with a SARS-CoV-2 replicon. Importantly, YUM70 treatment ameliorated lung damage in transgenic mice infected with SARS-CoV-2, which correlated with reduced weight loss and longer survival. Thus, GRP78 inhibition may be a promising approach to augment existing therapies to block SARS-CoV-2, its variants, and other viruses that utilize GRP78 for entry and infection.

Embryological Insights into the Origin of Epithelial Cancers of the Female Reproductive Tract.

The embryology of the female reproductive organs is reviewed focusing on aspects relevant to the histogenesis of reproductive neoplasms. The evidence reviewed suggests that (1) there is no embryological link between the ovarian surface epithelium and the coelomic epithelium; (2) the ovarian surface epithelium is not composed of pluripotent cells that readily differentiate into various components of the reproductive tract before or after birth; (3) there is no embryological link between the ovarian surface epithelium and the Müllerian ducts, from which internal female reproductive organs other than the ovaries, including the endocervix, endometrium, and fallopian tubes, are derived; and (4) there is an embryological link between the Müllerian ducts and the renal collecting system, perhaps accounting for clear cell differentiation in some gynecological malignancies. Implications for our understanding of the origin of the tumors historically classified as ovarian epithelial neoplasms are discussed.

Antiprogestins reduce epigenetic field cancerization in breast tissue of young healthy women.

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.

Endoplasmic reticulum chaperone GRP78/BiP is critical for mutant Kras-driven lung tumorigenesis.

Lung cancer is the leading cause of cancer mortality worldwide and KRAS is the most commonly mutated gene in lung adenocarcinoma (LUAD). The 78-kDa glucose-regulated protein GRP78/BiP is a key endoplasmic reticulum chaperone protein and a major pro-survival effector of the unfolded protein response (UPR). Analysis of the Cancer Genome Atlas database and immunostain of patient tissues revealed that compared to normal lung, GRP78 expression is generally elevated in human lung cancers, including tumors bearing the KRASG12D mutation. To test the requirement of GRP78 in human lung oncogenesis, we generated mouse models containing floxed Grp78 and Kras Lox-Stop-Lox G12D (KrasLSL-G12D) alleles. Simultaneous activation of the KrasG12D allele and knockout of the Grp78 alleles were achieved in the whole lung or selectively in lung alveolar epithelial type 2 cells known to be precursors for adenomas that progress to LUAD. Here we report that GRP78 haploinsufficiency is sufficient to suppress KrasG12D-mediated lung tumor progression and prolong survival. Furthermore, GRP78 knockdown in human lung cancer cell line A427 (KrasG12D/+) leads to activation of UPR and apoptotic markers and loss of cell viability. Our studies provide evidence that targeting GRP78 represents a novel therapeutic approach to suppress mutant KRAS-mediated lung tumorigenesis.

A Phosphotyrosine Switch in Estrogen Receptor ß Is Required for Mouse Ovarian Function.

The two homologous estrogen receptors ERα and ERß exert distinct effects on their cognate tissues. Previous work from our laboratory identified an ERß-specific phosphotyrosine residue that regulates ERß transcriptional activity and antitumor function in breast cancer cells. To determine the physiological role of the ERß phosphotyrosine residue in normal tissue development and function, we investigated a mutant mouse model (Y55F) whereby this particular tyrosine residue in endogenous mouse ERß is mutated to phenylalanine. While grossly indistinguishable from their wild-type littermates, mutant female mice displayed reduced fertility, decreased ovarian follicular cell proliferation, and lower progesterone levels. Moreover, mutant ERß from female mice during superovulation is defective in activating promoters of its target genes in ovarian tissues. Thus, our findings provide compelling genetic and molecular evidence for a role of isotype-specific ERß phosphorylation in mouse ovarian development and function.

Non-Surgical Cancer Risk Reduction in BRCA1 Mutation Carriers: Disabling the Remote Control.

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.

Clinicopathological significance of endoplasmic reticulum stress proteins in ovarian carcinoma.

Epithelial ovarian cancer (EOC) is a leading cause of cancer-related mortality in the United States due to the late-stage disease at diagnosis. Overexpression of GRP78 and PDI following endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) promote growth and invasion in cancer. To identify novel prognostic biomarkers in EOC, here we determined the expression of ER stress-associated proteins (GRP78, ATF6 and PERK) and correlated with clinical outcome in EOC. Tissue microarray (TMA) samples from 415 tissues collected from three cancer centers (UM, USC, and KCCRI) were used to assess the expression levels of ER-associated proteins using immunohistochemistry (IHC). We observed that the expression levels of GRP78 (p < 0.0001), ATF6 (p < 0.0001), and PERK (p < 0.0001) were significantly increased in specimens of EOC compared to normal tissues, including in the serous subtype (p < 0.0001). Previously we reported that high expression of PDI correlated with poor patient survival in EOC. Here we showed that overexpression of GRP78 and PDI protein expression correlated with poor patient survival (p = 0.03), while low expression of combined GRP78 and PDI correlated with better survival (p = 0.01) in high-grade serous. The increased expression of ER stress-associated proteins in EOC suggests a role for ER stress and the UPR in EOC. More importantly, our results demonstrate that GRP78 and PDI are potential biomarkers for EOC and could be used as dual prognostic markers.

Association between AXL promoter methylation and lung function growth during adolescence.

AXL is one of the TAM (TYRO3, AXL and MERTK) receptor tyrosine kinases and may be involved in airway inflammation. Little is known about how epigenetic changes in AXL may affect lung development during adolescence. We investigated the association between AXL DNA methylation at birth and lung function growth from 10 to 18 years of age in 923 subjects from the Children's Health Study (CHS). DNA methylation from newborn bloodspots was measured at multiple CpG loci across the regulatory regions of AXL using Pyrosequencing. Linear spline mixed-effects models were fitted to assess the association between DNA methylation and 8-year lung function growth. Findings were evaluated for replication in a separate population of 237 CHS subjects using methylation data from the Illumina HumanMethylation450 (HM450) array when possible. A 5% higher average methylation level of the AXL promoter region at birth was associated with a 48.4 ml decrease in mean FEV1 growth from 10 to 18 years of age in the primary study population (95% CI: -100.2, 3.4), and a 53.9 ml decrease in mean FEV1 growth from 11 to 15 years of age in the replication population (95% CI: -104.3, -3.5). One CpG locus in the promoter region, cg10564498, was significantly associated with decreased growth in FEV1, FVC and MMEF from 10 to 18 years of age and the negative associations were observed in a similar age range in the replication population. These findings suggest a potential association between AXL promoter methylation at birth and lower lung function growth during adolescence.

Mechanism of cytokinesis failure in ovarian cystadenomas with defective BRCA1 and P53 pathways.

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.

Self-reported prenatal tobacco smoke exposure, AXL gene-body methylation, and childhood asthma phenotypes.

BACKGROUND: Epigenetic modifications, including DNA methylation, act as one potential mechanism underlying the detrimental effects associated with prenatal tobacco smoke (PTS) exposure. Methylation in a gene called AXL was previously reported to differ in response to PTS. METHODS: We investigated the association between PTS and epigenetic changes in AXL and how this was related to childhood asthma phenotypes. We tested the association between PTS and DNA methylation at multiple CpG loci of AXL at birth using Pyrosequencing in two separate study populations, the Children's Health Study (CHS, n = 799) and the Newborn Epigenetic Study (NEST, n = 592). Plasma cotinine concentration was used to validate findings with self-reported smoking status. The inter-relationships among AXL mRNA and miR-199a1 expression, PTS, and AXL methylation were examined. Lastly, we evaluated the joint effects of AXL methylation and PTS on the risk of asthma and related symptoms at age 10 years old. RESULTS: PTS was associated with higher methylation level in the AXL gene body in both CHS and NEST subjects. In the pooled analysis, exposed subjects had a 0.51% higher methylation level in this region compared to unexposed subjects (95% CI 0.29, 0.74; p < 0.0001). PTS was also associated with 21.2% lower expression of miR-199a1 (95% CI - 37.9, - 0.1; p = 0.05), a microRNA known to regulate AXL expression. Furthermore, the combination of higher AXL methylation and PTS exposure at birth increased the risk of recent episodes of bronchitic symptoms in childhood. CONCLUSIONS: PTS was associated with methylation level of AXL and the combination altered the risk of childhood bronchitic symptoms.

Expression of protein disulfide isomerase family members correlates with tumor progression and patient survival in ovarian cancer.

OBJECTIVE: Protein disulfide isomerase (PDI) is an oxidoreductase that is overexpressed in several cancers. PDI family members (PDIs) play a role in various diseases including cancer. Select PDIs were reported as useful markers in other cancers but their expression in ovarian cancer has not been thoroughly assessed. We sought to evaluate the expression of PDI, PDIA6, PDIR, ERp57, ERp72 and AGR3 in ovarian cancer patient samples and examine their prognostic significance. METHODS: TMA samples from 415 tissues collected from three cancer centers (UM, USC, and KCCRI) were used to assess the expression levels of PDI family proteins using IHC. RESULTS: We observed significant increases in PDI (p = 9.16E-36), PDIA6 (p = 5.51E-33), PDIR (p = 1.81E-12), ERp57 (p = 9.13E-07), ERp72 (p = 3.65E-22), and AGR3 (p = 4.56E-24) expression in ovarian cancers compared to normal tissues. Expression of PDI family members also increases during disease progression (p <0.001). All PDI family members are overexpressed in serous ovarian cancer (p<0.001). However, PDI, PDIA6, PDIR, ERp72 and AGR3 are more significantly overexpressed (p<0.001) than ERp57 (p<0.05) in clear cell ovarian carcinoma. Importantly, overexpression of PDI family members is associated with poor survival in ovarian cancer (p = 0.045 for PDI, p = 0.047 for PDIR, p = 0.037 for ERp57, p = 0.046 for ERp72, p = 0.040 for AGR3) with the exception of PDIA6 (p = 0.381). CONCLUSIONS: Our findings demonstrate that select PDI family members (PDI, PDIR, ERp72, ERp57 and AGR3) are potential prognostic markers for ovarian cancer.

Epigenetic regulation of AXL and risk of childhood asthma symptoms.

Background: AXL is one of the TAM (TYRO3, AXL and MERTK) receptor tyrosine kinases and may affect numerous immune-related health conditions. However, the role for AXL in asthma, including its epigenetic regulation, has not been extensively studied. Methods: We investigated the association between AXL DNA methylation at birth and risk of childhood asthma symptoms at age 6 years. DNA methylation of multiple CpG loci across the regulatory regions of AXL was measured in newborn bloodspots using the Illumina HumanMethylation450 array on a subset of 246 children from the Children's Health Study (CHS). Logistic regression models were fitted to assess the association between asthma symptoms and DNA methylation. Findings were evaluated for replication in a separate population of 1038 CHS subjects using Pyrosequencing on newborn bloodspot samples. AXL genotypes were extracted from genome-wide data. Results: Higher average methylation of CpGs in the AXL gene at birth was associated with higher risk of parent-reported wheezing, and the association was stronger in girls than in boys. This relationship reflected the methylation status of the gene-body region near the 5' end, for which a 1% higher methylation level was significantly associated with a 72% increased risk of ever having wheezed by 6 years. The association of one CpG locus, cg00360107 was replicated using Pyrosequencing. Increased AXL methylation was also associated with lower mRNA expression level of this gene in lung tissue from the Cancer Genome Atlas (TCGA) dataset. Furthermore, AXL DNA methylation was strongly linked to underlying genetic polymorphisms. Conclusions: AXL DNA methylation at birth was associated with higher risk for asthma-related symptoms in early childhood.

GRP78 haploinsufficiency suppresses acinar-to-ductal metaplasia, signaling, and mutant Kras-driven pancreatic tumorigenesis in mice.

Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal disease in critical need of new therapeutic strategies. Here, we report that the stress-inducible 78-kDa glucose-regulated protein (GRP78/HSPA5), a key regulator of endoplasmic reticulum homeostasis and PI3K/AKT signaling, is overexpressed in the acini and PDAC of Pdx1-Cre;KrasG12D/+;p53f/+ (PKC) mice as early as 2 mo, suggesting that GRP78 could exert a protective effect on acinar cells under stress, as during PDAC development. The PKC pancreata bearing wild-type Grp78 showed detectable PDAC by 3 mo and rapid subsequent tumor growth. In contrast, the PKC pancreata bearing a Grp78f/+ allele (PKC78f/+ mice) expressing about 50% of GRP78 maintained normal sizes during the early months, with reduced proliferation and suppression of AKT, S6, ERK, and STAT3 activation. Acinar-to-ductal metaplasia (ADM) has been identified as a key tumor initiation mechanism of PDAC. Compared with PKC, the PKC78f/+ pancreata showed substantial reduction of ADM as well as pancreatic intraepithelial neoplasia-1 (PanIN-1), PanIN-2, and PanIN-3 and delayed onset of PDAC. ADM in response to transforming growth factor α was also suppressed in ex vivo cultures of acinar cell clusters isolated from mouse pancreas bearing targeted heterozygous knockout of Grp78 (c78f/+ ) and subjected to 3D culture in collagen. We further discovered that GRP78 haploinsufficiency in both the PKC78f/+ and c78f/+ pancreata leads to reduction of epidermal growth factor receptor, which is critical for ADM initiation. Collectively, our studies establish a role for GRP78 in ADM and PDAC development.

Effects of Prolonged GRP78 Haploinsufficiency on Organ Homeostasis, Behavior, Cancer and Chemotoxic Resistance in Aged Mice.

GRP78, a multifunctional protein with potent cytoprotective properties, is an emerging therapeutic target to combat cancer development, progression and drug resistance. The biological consequences of prolonged reduction in expression of this essential chaperone which so far has been studied primarily in young mice, was investigated in older mice, as older individuals are likely to be important recipients of anti-GRP78 therapy. We followed cohorts of Grp78+/+ and Grp78+/- male and female mice up to 2 years of age in three different genetic backgrounds and characterized them with respect to body weight, organ integrity, behavioral and memory performance, cancer, inflammation and chemotoxic response. Our results reveal that body weight, organ development and integrity were not impaired in aged Grp78+/- mice. No significant effect on cancer incidence and inflammation was observed in aging mice. Interestingly, our studies detected some subtle differential trends between the WT and Grp78+/- mice in some test parameters dependent on gender and genetic background. Our studies provide the first evidence that GRP78 haploinsufficiency for up to 2 years of age has no major deleterious effect in rodents of different genetic background, supporting the merit of anti-GRP78 drugs in treatment of cancer and other diseases affecting the elderly.

Aberrant regulation of RANKL/OPG in women at high risk of developing breast cancer.

Breast cancer is the most common female cancer, affecting approximately one in eight women during their lifetime in North America and Europe. Receptor Activator of NF-kB Ligand (RANKL), its receptor RANK and the natural antagonist osteoprotegerin (OPG) are essential regulators of bone resorption. We have initially shown that RANKL/RANK are essential for hormone-driven mammary epithelial proliferation in pregnancy and RANKL/RANK have been implicated in mammary stem cell biology. Using genetic mouse-models, we and others identified the RANKL/RANK system as a key regulator of sex hormone, BRCA1-mutation, and oncogene-driven breast cancer and we proposed that RANKL/RANK might be involved in the initiation of breast tumors. We now report that in postmenopausal women without known genetic predisposition, high RANKL and progesterone serum levels stratify a subpopulation of women at high risk of developing breast cancer 12-24 months before diagnosis (5.33-fold risk, 95%CI 1.5-25.4; P=0.02). In women with established breast cancer, we demonstrate that RANKL/OPG ratios change dependent on the presence of circulating tumor cells (CTCs). Finally, we show in a prospective human breast cancer cohort that alterations in RANKL/OPG ratios are significantly associated with breast cancer manifestation. These data indicate that the RANKL/RANK/OPG system is deregulated in post-menopausal women at high risk for breast cancer and in women with circulating tumor cells. Thus, serum levels of RANKL/OPG are potentially indicative of predisposition and progression of breast cancer in humans. Advancement of our findings towards clinical application awaits prior validation in independent patient cohorts.

Epigenetic reprogramming of fallopian tube fimbriae in BRCA mutation carriers defines early ovarian cancer evolution.

The exact timing and contribution of epigenetic reprogramming to carcinogenesis are unclear. Women harbouring BRCA1/2 mutations demonstrate a 30-40-fold increased risk of high-grade serous extra-uterine Müllerian cancers (HGSEMC), otherwise referred to as 'ovarian carcinomas', which frequently develop from fimbrial cells but not from the proximal portion of the fallopian tube. Here we compare the DNA methylome of the fimbrial and proximal ends of the fallopian tube in BRCA1/2 mutation carriers and non-carriers. We show that the number of CpGs displaying significant differences in methylation levels between fimbrial and proximal fallopian tube segments are threefold higher in BRCA mutation carriers than in controls, correlating with overexpression of activation-induced deaminase in their fimbrial epithelium. The differentially methylated CpGs accurately discriminate HGSEMCs from non-serous subtypes. Epigenetic reprogramming is an early pre-malignant event integral to BRCA1/2 mutation-driven carcinogenesis. Our findings may provide a basis for cancer-preventative strategies.