PVLSI (Pioneer Valley Life Sciences Institute) Posters - 2019

PVLSI (Pioneer Valley Life Sciences Institute) Posters - 2019https://scholarlycommons.libraryinfo.bhs.org/research_education/1014/thumbnail.jp

Discovery and replication of microRNAs for breast cancer risk using genome-wide profiling

BACKGROUND: Genome-wide miRNA expression may be useful for predicting breast cancer risk and/or for the early detection of breast cancer. RESULTS: A 41-miRNA model distinguished breast cancer risk in the discovery study (accuracy of 83.3%), which was replicated in the independent study (accuracy = 63.4%, P=0.09). Among the 41 miRNA, 20 miRNAs were detectable in serum, and predicted breast cancer occurrence within 18 months of blood draw (accuracy 53%, P=0.06). These risk-related miRNAs were enriched for HER-2 and estrogen-dependent breast cancer signaling. MATERIALS AND METHODS: MiRNAs were assessed in two cross-sectional studies of women without breast cancer and a nested case-control study of breast cancer. Using breast tissues, a multivariate analysis was used to model women with high and low breast cancer risk (based upon Gail risk model) in a discovery study of women without breast cancer (n=90), and applied to an independent replication study (n=71). The model was then assessed using serum samples from the nested case-control study (n=410). CONCLUSIONS: Studying breast tissues of women without breast cancer revealed miRNAs correlated with breast cancer risk, which were then found to be altered in the serum of women who later developed breast cancer. These results serve as proof-of-principle that miRNAs in women without breast cancer may be useful for predicting breast cancer risk and/or as an adjunct for breast cancer early detection. The miRNAs identified herein may be involved in breast carcinogenic pathways because they were first identified in the breast tissues of healthy women

Genetic Mutations Associated with Hormone-Positive Breast Cancer in a Small Cohort of Ethiopian Women

In Ethiopia, a breast cancer diagnosis is associated with a prognosis significantly worse than that of Europe and the US. Further, patients presenting with breast cancer in Ethiopia are far younger, on average, and patients are typically diagnosed at very late stages, relative to breast cancer patients of European descent. Emerging data suggest that a large proportion of Ethiopian patients have hormone-positive (ER+) breast cancer. This is surprising given (1) that patients have late-stage breast cancer at the time of diagnosis, (2) that African Americans with breast cancer frequently have triple negative breast cancer (TNBC), and (3) these patients typically receive chemotherapy, not hormone-targeting drugs. To further examine the similarity of Ethiopian breast tumors to those of African Americans or of those of European descent, we sequenced matched tumor and normal adjacent tissue from Ethiopian patients from a small pilot collection. We identified mutations in 615 genes across all three patients, unique to the tumor tissue. Across this analysis, we found far more mutations shared between Ethiopian patient tissue and that from white patients (103) than we did comparing to African Americans (3). Several mutations were found in extracellular matrix encoding genes with known roles in tumor cell growth and metastasis. We suggest future mechanistic studies on this disease focus on these genes first, toward finding new treatment strategies for breast cancer patients in Ethiopia. Keywords: BRCA; Extracellular matrix; MAPK; Metastasis; TCGA

Exposure to Low Doses of Oxybenzone During Perinatal Development Alters Mammary Gland Stroma in Female Mice

Mammary stroma is a prominent modulator of epithelial development, and a complex set of interactions between these tissue compartments is essential for normal development, which can be either permissive or restrictive in tumor initiation and progression. During perinatal development, exposures of mice to oxybenzone, a common UV filter, environmental pollutant and endocrine disruptor, induce alterations in mammary epithelium. Our prior research indicates that oxybenzone alters mammary epithelial structures at puberty and in adulthood. We had also previously observed changes in the expression of hormone receptors at puberty (e.g., oxybenzone induced a decrease in the number of epithelial cells positive for progesterone receptor) and in adulthood (e.g., oxybenzone induced a decrease in the number of estrogen receptor-positive epithelial cells), and increased body weight in adulthood. Here, we investigated mammary stromal changes in BALB/c animals exposed during gestation and perinatal development to 0, 30, or 3000 μg oxybenzone/kg/day. In mice exposed to 30 μg/kg/day, we observed morphological changes in adulthood (e.g., a thicker periductal stroma and adipocytes that were considerably larger). We also observed an increased number of mast cells in the mammary stroma at puberty which may represent a transient influence of oxybenzone exposure. These results provide additional evidence that even low doses of oxybenzone can disrupt hormone sensitive outcomes in the mammary gland when exposures occur during critical windows of development, and some of these effects manifest in later life. Keywords: 2-hydroxy-4methoxybenzophenone; collagen; endocrine disrupting chemical; terminal end bud; white fat; xenoestrogen

The influence of tumor necrosis factor-α on the tumorigenic Wnt-signaling pathway in human mammary tissue from obese women

Epidemiological studies have convincingly suggested that obesity is an important risk factor for postmenopausal breast cancer, but the mechanisms responsible for this relationship are still not fully understood. We hypothesize that obesity creates a low-grade inflammatory microenvironment, which stimulates Wnt-signaling and thereby promotes the development of breast cancer. To test this hypothesis, we evaluated the correlations between expression of multiple inflammatory cytokines and Wnt pathway downstream genes in mammary tissues from women (age ≥ 50) undergoing reduction mammoplasty. Moreover, we specifically examined the role of tumor necrosis factor-α (TNF-α), an important proinflammatory cytokine associated with obesity and a possible modulator of the Wnt pathway. The regulatory effects of TNF-α on Wnt pathway targets were measured in an ex vivo culture of breast tissue treated with anti-TNF-α antibody or TNF-α recombinant protein. We found that BMI was positively associated with the secretion of inflammatory cytokines IL-1β, IL-6 and TNF-α, all of which were negatively correlated with the expression of SFRP1. The transcriptional expression of Wnt-signaling targets, AXIN2 and CYCLIN D1, were higher in mammary tissue from women with BMI ≥ 30 compared to those with BMI \u3c 30. Our ex vivo work confirmed that TNF-α is causally linked to the up-regulation of active β-CATENIN, a key component in the Wnt pathway, and several Wnt-signaling target genes (i.e. CYCLIN D1, AXIN2, P53 and COX-2). Collectively, these findings indicate that obesity-driven inflammation elevates Wnt-signaling in mammary tissue and thereby creates a microenvironment conducive to the development of breast cancer

Investigation of the adolescent female breast transcriptome and the impact of obesity

BACKGROUND: Early life environmental exposures affect breast development and breast cancer risk in adulthood. The breast is particularly vulnerable during puberty when mammary epithelial cells proliferate exponentially. In overweight/obese (OB) women, inflammation increases breast aromatase expression and estrogen synthesis and promotes estrogen-receptor (ER)-positive breast cancer. In contrast, recent epidemiological studies suggest that obesity during childhood decreases future breast cancer risk. Studies on environmental exposures and breast cancer risk have thus far been limited to animal models. Here, we present the first interrogation of the human adolescent breast at the molecular level and investigate how obesity affects the immature breast. METHODS: We performed RNA-seq in 62 breast tissue samples from adolescent girls/young women (ADOL; mean age 17.8 years) who underwent reduction mammoplasty. Thirty-one subjects were non-overweight/obese (NOB; mean BMI 23.4 kg/m2) and 31 were overweight/obese (OB; BMI 32.1 kg/m2). We also compared our data to published mammary transcriptome datasets from women (mean age 39 years) and young adult mice, rats, and macaques. RESULTS: The ADOL breast transcriptome showed limited (30%) overlap with other species, but 88% overlap with adult women for the 500 most highly expressed genes in each dataset; only 43 genes were shared by all groups. In ADOL, there were 120 differentially expressed genes (DEG) in OB compared with NOB samples (padj \u3c 0.05). Based on these DEG, Ingenuity Pathway Analysis (IPA) identified the cytokines CSF1 and IL-10 and the chemokine receptor CCR2 as among the most highly activated upstream regulators, suggesting increased inflammation in the OB breast. Classical ER targets (e.g., PR, AREG) were not differentially expressed, yet IPA identified the ER and PR and growth factors/receptors (VEGF, HGF, HER3) and kinases (AKT1) involved in hormone-independent ER activation as activated upstream regulators in OB breast tissue. CONCLUSIONS: These studies represent the first investigation of the human breast transcriptome during late puberty/young adulthood and demonstrate that obesity is associated with a transcriptional signature of inflammation which may augment estrogen action in the immature breast microenvironment. We anticipate that these studies will prompt more comprehensive cellular and molecular investigations of obesity and its effect on the breast during this critical developmental window

Durable antibody and effector memory T cell responses in breastmilk from women with SARS-CoV-2

Background: Given that only 25% of pregnant women elect to receive a COVID-19 vaccine, maternal SARS-CoV-2 infection remains an important route of conferring protective passive immunity to breastfed infants of mothers who are not vaccinated. Methods: We enrolled 30 lactating participants between December 2020 and March 2021 who had a positive PCR-test and their first COVID-19 symptoms within the previous 21 days. Participants were asked to provide serial bilateral milk samples at 12 timepoints (~ every 3 days) over a period of 35 days. A second set of samples was collected at least four months after the beginning of the first set. Participants also were asked to provide their dried blood spots and infant stool samples. All samples were tested for receptor-binding domain (RBD)-specific immunoglobulin (Ig)A, IgG, and IgM. Milk samples were assessed for neutralizing ability against the spike protein and four SARS-CoV-2 variants: D614G, Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P.1). Permeability of the breast epithelium was assessed by measuring the sodium to potassium ions (Na:K) in milk. Using flow cytometry, memory CD4 and CD8 T cells (CD45RO+ and CCR7+/-) and mucosal-homing CD4 and CD8 T cells (CD103+) were determined in cells from milk expressed at 35 days and at least 4 months after their first milk donation. Results: Milk antibodies from SARS-CoV-2 positive participants neutralized the spike complex. Milk from 73, 90, and 53% of participants had binding reactivities to RBD-specific IgA, IgG, and IgM, respectively. In contrast to blood spots, which showed increased levels of IgG, but not IgA or IgM, the COVID-19 response in milk was associated with a robust IgA response. Twenty-seven percent of participants had increased breast-epithelium permeability, as indicated by Na:K ≥ 0.6. The percentage of CD45RO+CCR7- effector-memory T cells in the day ≥120 milk samples was significantly higher than day 35 samples (P\u3c 0.05). Conclusions: Antibodies in milk from participants with recent SARS-CoV-2 infection and those who recovered can neutralize the spike complex. For the first time we show that breastmilk T cells are enriched for mucosal memory T cells, further emphasizing the passive protection against SARS-CoV-2 conferred to infants via breastmilk. Keywords: COVID-19; SARS-CoV-2; breast milk; breast permeability; flow cytometry; memory T cells; mucosal immunity

Genetic variation in sensitivity to estrogens and breast cancer risk

Breast cancer risk is intimately intertwined with exposure to estrogens. While more than 160 breast cancer risk loci have been identified in humans, genetic interactions with estrogen exposure remain to be established. Strains of rodents exhibit striking differences in their responses to endogenous ovarian estrogens (primarily 17β-estradiol). Similar genetic variation has been observed for synthetic estrogen agonists (ethinyl estradiol) and environmental chemicals that mimic the actions of estrogens (xenoestrogens). This review of literature highlights the extent of variation in responses to estrogens among strains of rodents and compiles the genetic loci underlying pathogenic effects of excessive estrogen signaling. Genetic linkage studies have identified a total of the 35 quantitative trait loci (QTL) affecting responses to 17β-estradiol or diethylstilbestrol in five different tissues. However, the QTL appear to act in a tissue-specific manner with 9 QTL affecting the incidence or latency of mammary tumors induced by 17β-estradiol or diethylstilbestrol. Mammary gland development during puberty is also exquisitely sensitive to the actions of endogenous estrogens. Analysis of mammary ductal growth and branching in 43 strains of inbred mice identified 20 QTL. Regions in the human genome orthologous to the mammary development QTL harbor loci associated with breast cancer risk or mammographic density. The data demonstrate extensive genetic variation in regulation of estrogen signaling in rodent mammary tissues that alters susceptibility to tumors. Genetic variants in these pathways may identify a subset of women who are especially sensitive to either endogenous estrogens or environmental xenoestrogens and render them at increased risk of breast cancer

The influence of obesity on folate status, DNA methylation and cancer-related gene expression in normal breast tissues from premenopausal women

Epidemiological studies have established obesity as a critical risk factor for postmenopausal breast cancer (post-BC), whereas a reverse association holds prior to menopause. A significant scientific gap exists in understanding the mechanism(s) underpinning this epidemiological phenomenon, particularly the reverse association between obesity and premenopausal breast cancer (pre-BC). This study aimed to understand how folate metabolism and DNA methylation informs the association between obesity and pre-BC. Fifty normal breast tissue samples were collected from premenopausal women who underwent reduction mammoplasty. We modified the Lactobacillus Casei microbiological folate assay and measured folate levels in our breast tissue samples. The DNA methylation of LINE-1, a biomarker of genome-wide methylation, and the expression of a panel of breast cancer-related genes was measured by pyrosequencing and real-time PCR. We found that a high BMI is associated with an increase of folate levels in mammary tissue, with an increase of 2.65 ng/g of folate per every 5-unit increase of BMI (p \u3c 0.05). LINE-1 DNA methylation was significantly associated with BMI (p \u3c 0.05), and marginally associated with folate concentration (p = 0.087). A high expression of SFRP1 was observed in subjects with high BMI or high folate status (p \u3c 0.05). This study demonstrated that, in premenopausal women, obesity is associated with increased mammary folate status, genome-wide DNA methylation and SFRP1 gene expression. Our findings indicated that the improved folate and epigenetic status represents a novel mechanism responsible for the reverse association between obesity and pre-BC. Keywords: DNA methylation; Obesity; folate; premenopausal breast cancer

Using Breast Milk to Assess Breast Cancer Risk: The Role of Mass Spectrometry-Based Proteomics

Although mammography and treatment advances have led to declines in breast cancer mortality in the United States, breast cancer remains a major cause of morbidity and mortality. Breast cancer in young women is associated with increased mortality and current methods of detecting breast cancers in this group of women have known limitations. Tools for accurately assessing personal breast cancer risk in young women are needed to identify those women who would benefit the most from earlier intervention. Proteomic analysis of breast milk could identify biomarkers of breast cancer risk and provide a tool for identifying women at increased risk. A preliminary analysis of milk from four women provides a proof of concept for using breast milk to assess breast cancer risk