The uterine secretome initiates growth of gynecologic tissues in ectopic locations.

Endosalpingiosis (ES) and endometriosis (EM) refer to the growth of tubal and endometrial epithelium respectively, outside of their site of origin. We hypothesize that uterine secretome factors drive ectopic growth. To test this, we developed a mouse model of ES and EM using tdTomato (tdT) transgenic fluorescent mice as donors. To block implantation factors, progesterone knockout (PKO) tdT mice were created. Fluorescent lesions were present after oviduct implantation with and without WT endometrium. Implantation was increased (p<0.05) when tdt oviductal tissue was implanted with endometrium compared to oviductal tissue alone. Implantation was reduced (p<0.0005) in animals implanted with minced tdT oviductal tissue with PKO tdT endometrium compared to WT endometrium. Finally, oviductal tissues was incubated with and without a known implantation factor, leukemia inhibitory factor (LIF) prior to and during implantation. LIF promoted lesion implantation. In conclusion, endometrial derived implantation factors, such as LIF, are necessary to initiate ectopic tissue growth. We have developed an animal model of ectopic growth of gynecologic tissues in a WT mouse which will potentially allow for development of new prevention and treatment modalities.

Epigenome environment interactions accelerate epigenomic aging and unlock metabolically restricted epigenetic reprogramming in adulthood.

Our early-life environment has a profound influence on developing organs that impacts metabolic function and determines disease susceptibility across the life-course. Using a rat model for exposure to an endocrine disrupting chemical (EDC), we show that early-life chemical exposure causes metabolic dysfunction in adulthood and reprograms histone marks in the developing liver to accelerate acquisition of an adult epigenomic signature. This epigenomic reprogramming persists long after the initial exposure, but many reprogrammed genes remain transcriptionally silent with their impact on metabolism not revealed until a later life exposure to a Western-style diet. Diet-dependent metabolic disruption was largely driven by reprogramming of the Early Growth Response 1 (EGR1) transcriptome and production of metabolites in pathways linked to cholesterol, lipid and one-carbon metabolism. These findings demonstrate the importance of epigenome:environment interactions, which early in life accelerate epigenomic aging, and later in adulthood unlock metabolically restricted epigenetic reprogramming to drive metabolic dysfunction.

Prevalence of endosalpingiosis and other benign gynecologic lesions.

Endosalpingiosis, traditionally regarded as an incidental pathological finding, was recently reported to have an association with gynecologic malignancies. To determine the prevalence of endosalpingiosis, we evaluated all benign appearing adnexal lesions using the Sectioning and Extensively Examining-Fimbria (SEE-Fim) protocol, and queried the pathology database for the presence of endosalpingiosis, gynecologic malignancy, endometriosis, Walthard nests, and paratubal cysts. Using the SEE-Fim protocol, the prevalence of endosalpingiosis, endometriosis, Walthard nests, and paratubal cysts were 22%, 45%, 33%, and 42% respectively, substantially higher than previously reported. All lesions were observed to increase with age except endometriosis which increased until menopause then decreased dramatically. Among specimens including ovarian tissue, the prevalence of implantation of at least one lesion type was ubiquitous in patients age 51 and older (93%). The clinical significance of endosalpingiosis should be a continued area of research with larger trials assessing prevalence, factors affecting incidence, and association with malignancy. Our findings contribute to elucidating the origin of ectopic lesions and gynecologic disease risk.

Hepatic Tumor Formation in Adult Mice Developmentally Exposed to Organotin.

BACKGROUND: Tributyltin (TBT) is a persistent and bioaccumulative environmental toxicant. Developmental exposure to TBT has been shown to cause fatty liver disease (steatosis), as well as increased adiposity in many species, leading to its characterization as an obesogen. OBJECTIVE: We aimed to determine the long-term effects of developmental TBT exposure on the liver. METHODS: C57BL/6J mice were exposed to a dose of TBT (0.5mg/kg body weight per day; 3.07µM) below the current developmental no observed adverse effect level (NOAEL) via drinking water, or drinking water alone, provided to the dam from preconception through lactation. Sires were exposed during breeding and lactation. Pups from two parity cycles were included in this study. Animals were followed longitudinally, and livers of offspring were analyzed by pathological evaluation, immunohistochemistry, immunoblotting, and RNA sequencing. RESULTS: Developmental exposure to TBT led to increased adiposity and hepatic steatosis at 14 and 20 weeks of age and increased liver adenomas at 45 weeks of age in male offspring. Female offspring displayed increased adiposity as compared with males, but TBT did not lead to an increase in fatty liver or tumor development in female offspring. Liver tumors in male mice were enriched in pathways and gene signatures associated with human and rodent nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC). This includes down-regulation of growth hormone receptor (GHR) and of STAT5 signaling, which occurred in response to TBT exposure and preceded liver tumor development. CONCLUSIONS: These data reveal a previously unappreciated ability of TBT to increase risk for liver tumorigenesis in mice in a sex-specific manner. Taken together, these findings provide new insights into how early life environmental exposures contribute to liver disease in adulthood. https://doi.org/10.1289/EHP5414.

Loss of E-cadherin Enhances IGF1-IGF1R Pathway Activation and Sensitizes Breast Cancers to Anti-IGF1R/InsR Inhibitors.

Purpose: Insulin-like growth factor 1 (IGF1) signaling regulates breast cancer initiation and progression and associated cancer phenotypes. We previously identified E-cadherin (CDH1) as a repressor of IGF1 signaling and in this study examined how loss of E-cadherin affects IGF1R signaling and response to anti-IGF1R/insulin receptor (InsR) therapies in breast cancer.Experimental Design: Breast cancer cell lines were used to assess how altered E-cadherin levels regulate IGF1R signaling and response to two anti-IGF1R/InsR therapies. In situ proximity ligation assay (PLA) was used to define interaction between IGF1R and E-cadherin. TCGA RNA-seq and RPPA data were used to compare IGF1R/InsR activation in estrogen receptor-positive (ER+) invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC) tumors. ER+ ILC cell lines and xenograft tumor explant cultures were used to evaluate efficacy to IGF1R pathway inhibition in combination with endocrine therapy.Results: Diminished functional E-cadherin increased both activation of IGF1R signaling and efficacy to anti-IGF1R/InsR therapies. PLA demonstrated a direct endogenous interaction between IGF1R and E-cadherin at points of cell-cell contact. Increased expression of IGF1 ligand and levels of IGF1R/InsR phosphorylation were observed in E-cadherin-deficient ER+ ILC compared with IDC tumors. IGF1R pathway inhibitors were effective in inhibiting growth in ER+ ILC cell lines and synergized with endocrine therapy and similarly IGF1R/InsR inhibition reduced proliferation in ILC tumor explant culture.Conclusions: We provide evidence that loss of E-cadherin hyperactivates the IGF1R pathway and increases sensitivity to IGF1R/InsR targeted therapy, thus identifying the IGF1R pathway as a potential novel target in E-cadherin-deficient breast cancers. Clin Cancer Res; 24(20); 5165-77. ©2018 AACR.

Determinants of prolactin in postmenopausal Chinese women in Singapore.

PURPOSE: Mechanistic and observational data together support a role for prolactin in breast cancer development. Determinants of prolactin in Asian populations have not been meaningfully explored, despite the lower risk of breast cancer in Asian populations. METHODS: Determinants of plasma prolactin were evaluated in 442 postmenopausal women enrolled in the Singapore Chinese Health Study, a population-based prospective cohort study. At baseline all cohort members completed an in-person interview that elicited information on diet, menstrual and reproductive history, and lifestyle factors. One year after cohort initiation we began collecting blood samples. Quantified were plasma concentrations of prolactin, estrone, estradiol, testosterone, androstenedione, and sex hormone-binding globulin (SHBG). Analysis of covariance method was used for statistical analyses with age at blood draw, time since last meal, and time at blood draw as covariates. RESULTS: Mean prolactin levels were 25.1% lower with older age at menarche (p value = 0.001), and 27.6% higher with greater years between menarche and menopause (p value = 0.009). Prolactin levels were also positively associated with increased sleep duration (p value = 0.005). The independent determinants of prolactin were years from menarche to menopause, hours of sleep, and the plasma hormones estrone and SHBG (all p values < 0.01). CONCLUSION: The role of prolactin in breast cancer development may involve reproductive and lifestyle factors, such as a longer duration of menstrual cycling and sleep patterns.

Endocrine Disruptors and Developmental Origins of Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a growing epidemic worldwide, particularly in countries that consume a Western diet, and can lead to life-threatening conditions such as cirrhosis and hepatocellular carcinoma. With increasing prevalence of NAFLD in both children and adults, an understanding of the factors that promote NAFLD development and progression is crucial. Environmental agents, including endocrine-disrupting chemicals (EDCs), which have been linked to other diseases, may play a role in NAFLD development. Increasing evidence supports a developmental origin of liver disease, and early-life exposure to EDCs could represent one risk factor for the development of NAFLD later in life. Rodent studies provide the strongest evidence for this link, but further studies are needed to define whether there is a causal link between early-life EDC exposure and NAFLD development in humans. Elucidating the molecular mechanisms underlying development of NAFLD in the context of developmental EDC exposures may identify biomarkers for people at risk, as well as potential intervention and/or therapeutic opportunities for the disease.

Functional characterization of lysine-specific demethylase 2 (LSD2/KDM1B) in breast cancer progression.

Flavin-dependent histone demethylases govern histone H3K4 methylation and act as important chromatin modulators that are extensively involved in regulation of DNA replication, gene transcription, DNA repair, and heterochromatin gene silencing. While the activities of lysine-specific demethylase 1 (LSD1/KDM1A) in facilitating breast cancer progression have been well characterized, the roles of its homolog LSD2 (KDM1B) in breast oncogenesis are relatively less understood. In this study, we showed that LSD2 protein level was significantly elevated in malignant breast cell lines compared with normal breast epithelial cell line. TCGA- Oncomine database showed that LSD2 expression is significantly higher in basal-like breast tumors compared to other breast cancer subtypes or normal breast tissue. Overexpression of LSD2 in MDA-MB-231 cells significantly altered the expression of key important epigenetic modifiers such as LSD1, HDAC1/2, and DNMT3B; promoted cellular proliferation; and augmented colony formation in soft agar; while attenuating motility and invasion. Conversely, siRNA-mediated depletion of endogenous LSD2 hindered growth of multiple breast cancer cell lines while shRNA-mediated LSD2 depletion augmented motility and invasion. Moreover, LSD2 overexpression in MDA-MB-231 cells facilitated mammosphere formation, enriched the subpopulation of CD49f+/EpCAM- and ALDHhigh, and induced the expression of pluripotent stem cell markers, NANOG and SOX2. In xenograft studies using immune-compromised mice, LSD2-overexpressing MDA-MB-231 cells displayed accelerated tumor growth but significantly fewer lung metastases than controls. Taken together, our findings provide novel insights into the critical and multifaceted roles of LSD2 in the regulation of breast cancer progression and cancer stem cell enrichment.

Potential Mechanisms underlying the Protective Effect of Pregnancy against Breast Cancer: A Focus on the IGF Pathway.

A first full-term birth at an early age protects women against breast cancer by reducing lifetime risk by up to 50%. The underlying mechanism resulting in this protective effect remains unclear, but many avenues have been investigated, including lobular differentiation, cell fate, and stromal composition. A single pregnancy at an early age protects women for 30-40 years, and this long-term protection is likely regulated by a relatively stable yet still modifiable method, such as epigenetic reprograming. Long-lasting epigenetic modifications have been shown to be induced by pregnancy and to target the IGF pathway. Understanding how an early first full-term pregnancy protects against breast cancer and the role of epigenetic reprograming of the IGF system may aid in developing new preventative strategies for young healthy women in the future.

Endocrine-disrupting chemicals and uterine fibroids.

Uterine fibroids are the most frequent gynecologic tumor, affecting 70% to 80% of women over their lifetime. Although these tumors are benign, they can cause significant morbidity and may require invasive treatments such as myomectomy and hysterectomy. Many risk factors for these tumors have been identified, including environmental exposures to endocrine-disrupting chemicals (EDCs) such as genistein and diethylstilbestrol. Uterine development may be a particularly sensitive window to environmental exposures, as some perinatal EDC exposures have been shown to increase tumorigenesis in both rodent models and human epidemiologic studies. The mechanisms by which EDC exposures may increase tumorigenesis are still being elucidated, but epigenetic reprogramming of the developing uterus is an emerging hypothesis. Given the remarkably high incidence of uterine fibroids and their significant impact on women's health, understanding more about how prenatal exposures to EDCs (and other environmental agents) may increase fibroid risk could be key to developing prevention and treatment strategies in the future.

Tumor Phenotype and Gene Expression During Early Mammary Tumor Development in Offspring Exposed to Alcohol In Utero.

BACKGROUND: Alcohol exposure in utero increases susceptibility to carcinogen-induced mammary tumorigenesis in adult offspring and causes tumors with a more malignant phenotype. This study was conducted to identify changes early in tumor development that might lead to this outcome. METHODS: Pregnant Sprague-Dawley rats were fed a liquid diet containing 6.7% ethanol (alcohol), an isocaloric liquid diet without alcohol (pair-fed), or rat chow ad libitum (ad lib) from gestation day 7 until parturition. At birth, female progeny were cross-fostered to control dams. Pups were weaned at postnatal day (PND) 21 and fed rat chow ad libitum for the remainder of the experiment. Female offspring were administered N-nitroso-N-methylurea (NMU; 50 mg/kg body weight) on PND 50. Mammary glands were palpated weekly, and offspring were euthanized at 16 weeks post-NMU injection. RESULTS: At 16 weeks post-NMU, tumor multiplicity was greater in alcohol-exposed offspring compared with control groups. Estrogen receptor-α (ER) mRNA expression was decreased in tumors from alcohol-exposed offspring, and these animals developed more ER-negative tumors relative to the pair-fed group. Alcohol-exposed offspring also tended to develop more progesterone receptor (PR)-positive tumors. All tumors were HER2-negative. PR positivity was associated with higher Ki67 expression, suggesting that PR-positive tumors were more proliferative. Tumors from alcohol-exposed animals exhibited increased mRNA expression of the insulin-like growth factor (IGF) family members IGF-II and IGFBP-5. IGF-II and DNA methyltransferase mRNA tended to be greater in the normal contralateral mammary glands of these animals. CONCLUSIONS: These data indicate that alcohol exposure in utero may shift NMU-induced tumor development toward a more aggressive phenotype and that alterations in IGF-II expression may contribute to these changes. Additional studies should be aimed at epigenetic mechanisms that underlie IGF-II expression to further delineate how this gene is altered in mammary glands of adults exposed to alcohol in utero.

Targeted DNA Methylation Screen in the Mouse Mammary Genome Reveals a Parity-Induced Hypermethylation of Igf1r That Persists Long after Parturition.

The most effective natural prevention against breast cancer is an early first full-term pregnancy. Understanding how the protective effect is elicited will inform the development of new prevention strategies. To better understand the role of epigenetics in long-term protection, we investigated parity-induced DNA methylation in the mammary gland. FVB mice were bred or remained nulliparous and mammary glands harvested immediately after involution (early) or 6.5 months following involution (late), allowing identification of both transient and persistent changes. Targeted DNA methylation (109 Mb of Ensemble regulatory features) analysis was performed using the SureSelectXT Mouse Methyl-seq assay and massively parallel sequencing. Two hundred sixty-nine genes were hypermethylated and 128 hypomethylated persistently at both the early and late time points. Pathway analysis of the persistently differentially methylated genes revealed Igf1r to be central to one of the top identified signaling networks, and Igf1r itself was one of the most significantly hypermethylated genes. Hypermethylation of Igf1r in the parous mammary gland was associated with a reduction of Igf1r mRNA expression. These data suggest that the IGF pathway is regulated at multiple levels during pregnancy and that its modification might be critical in the protective role of pregnancy. This supports the approach of lowering IGF action for prevention of breast cancer, a concept that is currently being tested clinically.

Scaffold attachment factor B2 (SAFB2)-null mice reveal non-redundant functions of SAFB2 compared with its paralog, SAFB1.

Scaffold attachment factors SAFB1 and SAFB2 are multifunctional proteins that share >70% sequence similarity. SAFB1-knockout (SAFB1(-/-)) mice display a high degree of lethality, severe growth retardation, and infertility in male mice. To assess the in vivo role of SAFB2, and to identify unique functions of the two paralogs, we generated SAFB2(-/-) mice. In stark contrast to SAFB1(-/-), SAFB2(-/-) offspring were born at expected Mendelian ratios and did not show any obvious defects in growth or fertility. Generation of paralog-specific antibodies allowed extensive expression analysis of SAFB1 and SAFB2 in mouse tissues, showing high expression of both SAFB1 and SAFB2 in the immune system, and in hormonally controlled tissues, with especially high expression of SAFB2 in the male reproductive tract. Further analysis showed a significantly increased testis weight in SAFB2(-/-) mice, which was associated with an increased number of Sertoli cells. Our data suggest that this is at least in part caused by alterations in androgen-receptor function and expression upon deletion of SAFB2. Thus, despite a high degree of sequence similarity, SAFB1(-/-) and SAFB2(-/-) mice do not totally phenocopy each other. SAFB2(-/-) mice are viable, and do not show any major defects, and our data suggest a role for SAFB2 in the differentiation and activity of Sertoli cells that deserves further study.

Fetal alcohol exposure and mammary tumorigenesis in offspring: role of the estrogen and insulin-like growth factor systems.

Fetal alcohol spectrum disorders affect a significant number of live births each year, indicating that alcohol consumption during pregnancy is an important public health issue. Environmental exposures and lifestyle choices during pregnancy may affect the offspring's risk of disease in adulthood, leading to the idea that a woman's risk of breast cancer may be pre-programmed prior to birth. Exposure of pregnant rats to alcohol increases tumorigenesis in the adult offspring in response to mammary carcinogens. The estrogen and insulin-like growth factor (IGF-I) axes occupy central roles in normal mammary gland development and breast cancer. 17-ß estradiol (E2) and IGF-I synergize to regulate formation of terminal end buds and ductal elongation during pubertal development. The intracellular signaling pathways mediated by the estrogen and IGF-I receptors cross-talk at multiple levels through both genomic and non-genomic mechanisms. Several components of the E2 and IGF-I systems are altered in early development in rat offspring exposed to alcohol in utero, therefore, these changes may play a role in the enhanced susceptibility to mammary carcinogens observed in adulthood. Alcohol exposure in utero induces a number of epigenetic alterations in non-mammary tissues in the offspring and other adverse in utero exposures induce epigenetic modifications in the mammary gland. Future studies will determine if fetal alcohol exposure can induce epigenetic modifications in genes that regulate E2/IGF action at key phases of mammary development, ultimately leading to changes in susceptibility to carcinogens.

Epigenetic reprogramming in breast cancer: from new targets to new therapies.

Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer death among women in the United States. Recently, interest has grown in the role of epigenetics in breast cancer development and progression. Epigenetic changes such as DNA methylation, histone modifications, and abnormal expression of non-coding RNAs emerged as novel biomarkers in breast cancer diagnosis, therapy, and prevention. This review focuses on the most recent mechanistic findings underlying epigenetic changes in breast cancer development and their role as predictors of breast cancer risk. The rapid progress in our understanding of epigenetic findings in breast cancer has opened new avenues for potential therapeutic approaches via identification of epigenetic targets. We highlight the development of novel epigenetically targeted drugs, relevant clinical trials in breast cancer patients, and recent approaches combining epigenetic agents with chemotherapy and/or endocrine therapy that may incrementally improve long-term outcomes in appropriately selected breast cancer patients. Biomarkers of response are needed, however, to identify patient subsets that are most likely to benefit from epigenetic treatment strategies.

Inhibition of histone demethylase, LSD2 (KDM1B), attenuates DNA methylation and increases sensitivity to DNMT inhibitor-induced apoptosis in breast cancer cells.

Increasing evidence suggests that dysfunction of histone lysine demethylase is associated with abnormal chromatin remodeling and gene silencing, contributing to breast tumorigenesis. In silico analysis shows that the newly identified histone demethylase lysine-specific demethylase 2 is highly expressed in breast cancer, especially in invasive tumors. However, it is currently unknown how LSD2 regulates chromatin remodeling and gene expression regulation in breast cancer. Using short hairpin RNA, we stably knocked down LSD2 (LSD2-KD) in MDA-MB-231 breast cancer cells. LSD2-KD led to accumulation of H3K4me1/2 without changing methylation levels of other key histone lysine residues, suggesting that LSD2 acts as a bona fide H3K4 demethylase in breast cancer cells. LSD2-KD resulted in decreased colony formation and attenuated global DNA methylation in MDA-MB-231 cells. Additionally, treatment with the DNMT inhibitor, 5-aza-deoxycytidine (DAC), synergistically increased mRNA expression of aberrantly silenced genes important in breast cancer development, including PR, RARß, ERα, SFRP1, SFRP2, and E-cadherin in LSD2-KD cells. Furthermore, LSD2-KD cells are more susceptible to cell death than scramble controls, and combined treatment with tranylcypromine, an LSD2 inhibitor, and DAC resulted in synergistic growth inhibition of breast cancer cells. DNMT inhibition by DAC in LSD2-KD cells led to internucleosomal DNA fragmentation, enhanced PARP cleavage and increased sub-G1 apoptotic cell population. These results demonstrate an important role for LSD2 in regulation of DNA methylation and gene silencing in breast cancer, and suggest that inhibition of LSD2 in combination with DNA methyltransferase inhibition represents a novel approach for epigenetic therapy of breast cancer.

Crosstalk between lysine-specific demethylase 1 (LSD1) and histone deacetylases mediates antineoplastic efficacy of HDAC inhibitors in human breast cancer cells.

Our previous studies demonstrated that lysine-specific demethylase 1 (LSD1) and histone deacetylases (HDACs) closely interact in controlling growth of breast cancer cells. However, the underlying mechanisms are largely unknown. In this study, we showed that knockdown of LSD1 expression (LSD1-KD) by RNAi decreased mRNA levels of HDAC isozymes in triple-negative breast cancer (TNBC) cells. Small interfering RNA (siRNA)-mediated depletion of HDAC5 expression induced the most significant accumulation of H3K4me2, a specific substrate of LSD1. Combined treatment with LSD1 inhibitor, pargyline, and HDAC inhibitor, SAHA (Vorinostat), led to superior growth inhibition and apoptotic death in TNBC cells, but exhibited additive or antagonistic effect on growth inhibition in non-TNBC counterparts or non-tumorigenic breast cells. Additionally, LSD1-KD enhanced SAHA-induced reexpression of a subset of aberrantly silenced genes, such as NR4A1, PCDH1, RGS16, BIK, and E-cadherin whose reexpression may be tumor suppressive. Genome-wide microarray study in MDA-MB-231 cells identified a group of tumor suppressor genes whose expression was induced by SAHA and significantly enhanced by LSD1-KD. We also showed that concurrent depletion of RGS16 by siRNA reduced overall cytotoxicity of SAHA and blocked the reexpression of E-cadherin, CDKN1C and ING1 in LSD1-deficient MDA-MB-231 cells. Furthermore, cotreatment with RGS16 siRNA reversed the downregulation of nuclear factor-kappaB expression induced by combined inhibition of LSD1 and HDACs, suggesting a crucial role of RGS16 in controlling key pathways of cell death in response to combination therapy. Taken together, these results provide novel mechanistic insight into the breast cancer subtype-dependent role of LSD1 in mediating HDAC activity and therapeutic efficacy of HDAC inhibitor.