MicroRNA-367 Inhibits Breast Cancer and Promotes Apoptosis by Targeting AT-Rich Interactive Domain-Containing Protein 1B

Introduction: Breast cancer (BC) developed in the glandular epithelial tissue of breast. microRNA (miR)-367 is an important player in cancer progression, but has never been studied in BC. This experiment tries to probe the mechanism of miR-367 in BC treatment with downstream target gene. Materials and Methods: Human BC cell lines and healthy breast epithelium cells were applied in this study. After the transfection of miR-367 inhibitor or mimic into BC cells, functional assays were conducted to measure cell growth. Afterwards, flow cytometry was employed in apoptosis verification. Then, target relation between miR-367 and ARID1B was certified. Furthermore, ARID1B level was also measured. Results: miR-367 was underexpressed in human BC cells (p < 0.05). Besides, overexpressed miR-367 inhibited BC cell proliferation and encouraged apoptosis, while underexpressed miR-367 led to an opposite outcome (p < 0.05). This experiment then implied that miR-367 dramatically suppressed the activity of cell transfected with ARID1B-wild type. miR-367 overexpression quenched ARID1B level in BC cells; while silencing miR-367 upregulated ARID1B expression (p < 0.05). Conclusion: Our experiment discovered that miR-367 quenched BC cell growth and promoted apoptosis by targeting ARID1B. This investigation may provide novel insights in BC treatment.

Deep Proteome Profiling of Human Mammary Epithelia at Lineage and Age Resolution

SUMMARYAge is the major risk factor in most carcinomas, yet little is known about how proteomes change with age in any human epithelium. We present comprehensive proteomes comprised of >9,000 total proteins, and >15,000 phosphopeptides, from normal primary human mammary epithelia at lineage resolution from ten women ranging in age from 19 to 68. Data were quality controlled, and results were biologically validated with cell-based assays. Age-dependent protein signatures were identified using differential expression analyses and weighted protein co-expression network analyses. Up-regulation of basal markers in luminal cells, including KRT14 and AXL, were a prominent consequence of aging. PEAK1 was identified as an age-dependent signaling kinase in luminal cells, which revealed a potential age-dependent vulnerability for targeted ablation. Correlation analyses between transcriptome and proteome revealed age-associated loss of proteostasis regulation. Protein expression and phosphorylation changes in the aging breast epithelium identify potential therapeutic targets for reducing breast cancer susceptibility.

DNA Methylation and Breast Cancer Risk: An Epigenome-Wide Study of Normal Breast Tissue and Blood

Differential DNA methylation is a potential marker of breast cancer risk. Few studies have investigated DNA methylation changes in normal breast tissue and were largely confounded by cancer field effects. To detect methylation changes in normal breast epithelium that are causally associated with breast cancer occurrence, we used a nested case–control study design based on a prospective cohort of patients diagnosed with a primary invasive hormone receptor-positive breast cancer. Twenty patients diagnosed with a contralateral breast cancer (CBC) were matched (1:1) with 20 patients who did not develop a CBC on relevant risk factors. Differentially methylated Cytosine-phosphate-Guanines (CpGs) and regions in normal breast epithelium were identified using an epigenome-wide DNA methylation assay and robust linear regressions. Analyses were replicated in two independent sets of normal breast tissue and blood. We identified 7315 CpGs (FDR < 0.05), 52 passing strict Bonferroni correction (p < 1.22 × 10−7) and 43 mapping to known genes involved in metabolic diseases with significant enrichment (p < 0.01) of pathways involving fatty acids metabolic processes. Four differentially methylated genes were detected in both site-specific and regions analyses (LHX2, TFAP2B, JAKMIP1, SEPT9), and three genes overlapped all three datasets (POM121L2, KCNQ1, CLEC4C). Once validated, the seven differentially methylated genes distinguishing women who developed and who did not develop a sporadic breast cancer could be used to enhance breast cancer risk-stratification, and allow implementation of targeted screening and preventive strategies that would ultimately improve breast cancer prognosis.

Upregulation of lipid metabolism genes in the breast prior to cancer diagnosis

Histologically normal tissue adjacent to the tumor can provide insight of the microenvironmental alterations surrounding the cancerous lesion and affecting the progression of the disease. However, little is known about the molecular changes governing cancer initiation in cancer-free breast tissue. Here, we employed laser microdissection and whole-transcriptome profiling of the breast epithelium prior to and post tumor diagnosis to identify the earliest alterations in breast carcinogenesis. Furthermore, a comprehensive analysis of the three tissue compartments (microdissected epithelium, stroma, and adipose tissue) was performed on the breast donated by either healthy subjects or women prior to the clinical manifestation of cancer (labeled “susceptible normal tissue”). Although both susceptible and healthy breast tissues appeared histologically normal, the susceptible breast epithelium displayed a significant upregulation of genes involved in fatty acid uptake/transport (CD36 and AQP7), lipolysis (LIPE), and lipid peroxidation (AKR1C1). Upregulation of lipid metabolism- and fatty acid transport-related genes was observed also in the microdissected susceptible stromal and adipose tissue compartments, respectively, when compared with the matched healthy controls. Moreover, inter-compartmental co-expression analysis showed increased epithelium-adipose tissue crosstalk in the susceptible breasts as compared with healthy controls. Interestingly, reductions in natural killer (NK)-related gene signature and CD45+/CD20+ cell staining were also observed in the stromal compartment of susceptible breasts. Our study yields new insights into the cancer initiation process in the breast. The data suggest that in the early phase of cancer development, metabolic activation of the breast, together with increased epithelium-adipose tissue crosstalk may create a favorable environment for final cell transformation, proliferation, and survival.

90 YEARS OF PROGESTERONE: Progesterone receptor signaling in the normal breast and its implications for cancer

Progesterone is considered as the pregnancy hormone and acts on many different target tissues. Progesterone receptor (PR) signaling is important for normal development and the physiologic function of the breast and impinges on breast carcinogenesis. Both systemically and locally, in the breast epithelium, there are multiple layers of complexity to progesterone action, many of which have been revealed through experiments in mice. The hormone acts via its receptor expressed in a subset of cells, the sensor cells, in the breast epithelium with different signaling outcomes in individual cells eliciting distinct cell-intrinsic and paracrine signaling involving different mediators for different intercellular interactions. PR expression itself is developmentally regulated and the biological outcome of PR signaling depends on the developmental stage of the mammary gland and the endocrine context. During both puberty and adulthood PR activates stem and progenitor cells through Wnt4-driven activation of the myoepithelium with downstream Adamts18-induced changes in extracellualr matrix (ECM) / basal membrane (BM). During estrous cycling and pregnancy, the hormone drives a major cell expansion through Rankl. At all stages, PR signaling is closely tied to estrogen receptor α (ER) signaling. As the PR itself is a target gene of ER, the complex interactions are experimentally difficult to dissect and still poorly understood. Ex vivo models of the human breast and studies on biopsy samples show that major signaling axes are conserved across species. New intraductal xenograft models hold promise to provide a better understanding of PR signaling in the normal breast epithelium and in breast cancer development in the near future.

Mutation Mechanisms of Breast Cancer among the Female Population in China

Background: Cancer is a genetic disease caused by the accumulation of gene mutations. It is important to derive the number of driver mutations that are needed for the development of human breast cancer, which may provide insights into the tumor diagnosis and therapy. Objective: This work is designed to investigate whether there is any difference for the mutation mechanism of breast cancer between the patients in the USA and those in China. We study the mechanisms of breast cancer development in China, and then compare these mechanisms with those in the USA. Methods: This work designed a multistage model including both gene mutation and clonal expansion of intermediate cells to fit the dataset of breast cancer in China from 2004 to 2009. Results: Our simulation results show that the maximum number of driver mutations for breast epithelium stem cells of females in China is 13 which is less than the 14 driver mutations of females in the USA. In addition, the two-hit model is the optimal one for the tumorigenesis of females in China, which is also different from the three-hit model that was predicted as the optimal model for the tumorigenesis of females in the USA. Conclusion: The differences of the mutation mechanisms between China and the USA reflect a variety of lifestyle, genetic influences, environmental exposure, and the availability of mammography screening.

SUN-126 Distinct Molecular Phenotypes of Non-Diseased Breast Adipose Tissue of Pre-Menopausal Obese and Non-Obese Women May Underlie Differing Breast Cancer Risks

Obesity is a major risk factor for many chronic diseases including postmenopausal breast cancer. Paradoxically, breast cancer susceptibility is inversely linked to obesity in pre-menopausal women. Adipose tissues are active endocrine organs that play major roles in tumor development and progression; however, fat depots at different anatomical sites are biologically and functionally distinct and their singular influence on breast epithelial biology remains unclear. To study the early events by which breast adiposity may provide a microenvironment predisposing normal breast epithelial cells to tumorigenesis, we collected breast tissue from pre-menopausal (n=10/group) non-obese (NO, BMI=27.6±0.8) and obese (O, BMI=44.5±2.8) women of comparable ages (NO: 36.1± 3.3; O: 40.0±2.0) with no breast cancer and undergoing elective breast reduction surgery. Breast adipose tissue and corresponding glandular cells were analyzed histologically and evaluated for expression of genes (adipokines, cytokines, steroid hormone signaling) by QPCR and proteins (proliferation, apoptosis, inflammation) by IHC. Adipocyte size distributions from NO and O breasts did not differ (P=0.9). However, adipose mRNA levels for pro-inflammatory cytokines (IL-6, IL-8, CSF-1, MCP-1) and adipokines (LEP, CFD) were higher for O than NO (P&lt;0.05). AdipoQ, ER-α, and ER-β transcript levels were lower for O than NO (P&lt;0.05), while those for CYP19 and PTGS2 showed reverse trends (O&gt;NO, P&lt;0.05). In the corresponding glandular cells, NO had higher mRNA levels for IL-6, IL-8, ER-α, and ER-β than O (P&lt;0.05). Immunostaining with anti-Ki67 antibodies indicated that O glandular cells were 3-fold less proliferative than those for NO, consistent with their lower Cyclin D1 mRNA levels (P&lt;0.05). Galectin-1, a pro-fibrotic protein, showed predominant myo- vs. luminal epithelial localization, with staining intensities for O tending to be higher (P=0.07) than for NO. Perilipin immunostaining was specific for adipocytes and did not differ for O and NO. A non-targeted approach using a Human Cytokine Array (R&D Systems) was employed to further evaluate the inflammation status of O vs. NO adipose. The analyses confirmed the higher expression of IL-8, Leptin and CFD (by QPCR) in O vs. NO and identified C-reactive protein, EMMPRIN, Trefoil Factor-3, Cystatin-3 and Macrophage Migration Inhibitory Factor-1 as greater in O than NO (~2-fold). Our findings demonstrate marked differences in gene and protein expression patterns of O and NO breast adipose tissue, which were accompanied by a suppression of proliferation of O relative to NO breast epithelium. We speculate that early exposure of the breast epithelium to a highly inflammatory environment fueled by breast adiposity may promote a senescent state that confers protection from pre-menopausal breast cancer.