Transcriptome analysis reveals disruption of circadian rhythms in late gestation dairy cows may increase risk for fatty liver and reduced mammary remodeling

Circadian disruption increased insulin resistance and decreased mammary development in late gestation, non-lactating (dry) cows. The objective was to measure the effect of circadian disruption on transcriptomes of the liver and mammary gland. At 35 d before expected calving (BEC) multiparous dry cows were assigned to either control (CON) or phase-shifted treatments (PS). CON was exposed to 16 h light and 8 h of dark. PS was exposed to 16 h light to 8 h dark, but phase of the light-dark cycle was shifted 6 h every 3 d. On d 21 BEC, liver and mammary were biopsied. RNA was isolated (n=6 CON, n=6 PS per tissue), libraries prepared and sequenced using paired end reads. Reads mapping to bovine genome averaged 27 M ± 2 M, and aligned to 14,222 protein coding genes in liver and 15,480 in mammary analysis. In the liver, 834 genes, and in the mammary gland, 862 genes were different (nominal P < 0.05) between PS and CON. In the liver, genes upregulated in PS functioned in cholesterol biosynthesis, endoplasmic reticulum stress, wound healing, and inflammation. Genes downregulated in liver function in cholesterol efflux. In the mammary gland, genes upregulated functioned in mRNA processing and transcription, downregulated genes encoded extracellular matrix proteins and proteases, cathepsins and lysosomal proteases, lipid transporters and regulated oxidative phosphorylation. Increased cholesterol synthesis and decreased efflux suggests circadian disruption potentially increases the risk of fatty liver in cows. Decreased remodeling and lipid transport in mammary may decrease milk production capacity during lactation.

Mammary Development in Gilts at One Week Postnatal Is Related to Plasma Lysine Concentration at 24 h after Birth, but Not Colostrum Dose

Perinatal nutrition affects future milk production. The number of mammary epithelial cells affect milk production capacity. Therefore, it was hypothesized that the level of colostrum intake affects the proliferation rate and the total number of mammary epithelial cells in the gland. The ratio of newly synthesized protein to newly synthesized DNA reflects the relative amount of cellular differentiation to cell division. The study objective was to determine the relationship between the level of colostrum intake and 24 h-level of circulating amino acid, glucose and insulin with mammary parenchyma histological features, cell division and protein synthesis over the first week postnatal. One of two standardized doses of a homogenate colostrum sample, 10% (n = 8) and 20% (n = 8) of birth bodyweight, was fed to gilts over the first 24 h postnatal. Gilts were administered deuterium oxide immediately after birth and daily to label newly synthesized DNA and proteins. Gilts were euthanized on postnatal day seven, and DNA and protein were isolated from mammary parenchyma. DNA and protein fractional synthesis (f) and fractional synthetic rate (FSR) were calculated using mass isotopomer distribution analysis. The ratio of protein f and FSR to DNA f and FSR were calculated and used to indicate the relative amounts of differentiation to cell division. Mammary morphological development was also analyzed by measuring the parenchymal epithelial area and the stromal and epithelial proliferation index on postnatal day seven. Colostrum dose was not related to any of the variables used to evaluate mammary development. However, plasma lysine levels at 24 h postnatal were positively related to average daily gain (ADG; r = 0.54, p = 0.05), DNA f (r = 0.57; p = 0.03) and DNA FSR (r = 0.57; p = 0.03) in mammary parenchyma. Plasma lysine was inversely related to the ratio of protein to DNA f and FSR (r = −0.56; p = 0.04). ADG was related to the parenchymal epithelial area and DNA and protein f and FSR (p < 0.05). These relationships support the idea that the nutritional environment affects early mammary development and that higher lysine levels in the perinatal period favored a greater degree of cell division versus differentiation in mammary of neonatal pigs and thus, warrant further investigations.

Insight into mammary gland development and tumor prevention in a newly developed metastatic mouse model of breast cancer

The development of breast cancer has been observed due to altered regulation of mammary gland developmental processes. Thus, a better understand of the normal mammary gland development can reveal possible mechanism in how normal cells are re-programmed to become malignant cells. E2F1-4 are part of the E2F transcription factor family with varied roles in mammary development. However, little is known about the role of E2F5 in mammary gland development. A combination of scRNAseq and predictive signature tools demonstrate the presence of E2F5 in the mammary gland and showed altered activity during the various phases of mammary gland development and function. Testing the hypothesis that E2F5 regulates mammary function, we generated a mammary-specific E2F5 knockout mouse model, resulting in modest mammary gland development changes. However, after a prolonged latency the E2F5 conditional knockout mice developed highly metastatic mammary tumors with metastases in both the lung and liver. Transplantation of the tumors revealed metastases to lymph nodes that was enriched through serial transplantation. Through whole genome sequencing and RNAseq analysis we identified, and then confirmed in vivo, that Cyclin D1 was dysregulated in E2F5 conditional knockout mammary glands and tumors. Based on these findings, we propose that loss of E2F5 leads altered regulation of Cyclin D1, which facilitates the development of mammary tumors.

The miR-200 family in normal mammary gland development

The miR-200 family of microRNAs plays a significant role in inhibiting mammary tumor growth and progression, and its members are being investigated as therapeutic targets. Additionally, if future studies can prove that miR-200s prevent mammary tumor initiation, the microRNA family could also offer a preventative strategy. Before utilizing miR-200s in a therapeutic setting, understanding how they regulate normal mammary development is necessary. No studies investigating the role of miR-200s in embryonic ductal development could be found, and only two studies examined the impact of miR-200s on pubertal ductal morphogenesis. These studies showed that miR-200s are expressed at low levels in virgin mammary glands, and elevated expression of miR-200s have the potential to impair ductal morphogenesis. In contrast to virgin mammary glands, miR-200s are expressed at high levels in mammary glands during late pregnancy and lactation. miR-200s are also found in the milk of several mammalian species, including humans. However, the relevance of miR-200s in milk remains unclear. The increase in miR-200 expression in late pregnancy and lactation suggests a role for miR-200s in the development of alveoli and/or regulating milk production. Therefore, studies investigating the consequence of miR-200 overexpression or knockdown are needed to identify the function of miR-200s in alveolar development and lactation.

Core circadian clock transcription factor BMAL1 regulates mammary epithelial cell growth, differentiation, and milk component synthesis

The role the mammary epithelial circadian clock plays in gland development and lactation is unknown. We hypothesized that mammary epithelial clocks function to regulate mammogenesis and lactogenesis, and propose the core clock transcription factor BMAL1:CLOCK regulates genes that control mammary epithelial development and milk synthesis. Our objective was to identify transcriptional targets of BMAL1 in undifferentiated (UNDIFF) and lactogen differentiated (DIFF) mammary epithelial cells (HC11) using ChIP-seq. Ensembl gene IDs with the nearest transcriptional start site to ChIP-seq peaks were explored as potential targets, and represented 846 protein coding genes common to UNDIFF and DIFF cells and 2773 unique to DIFF samples. Genes with overlapping peaks between samples (1343) enriched cell-cell adhesion, membrane transporters and lipid metabolism categories. To functionally verify targets, an HC11 line with Bmal1 gene knocked out (BMAL1-KO) using CRISPR-CAS was created. BMAL1-KO cultures had lower cell densities over an eight-day growth curve, which was associated with increased (p<0.05) levels of reactive oxygen species and lower expression of superoxide dismutase 3 (Sod3). RT-qPCR analysis also found lower expression of the putative targets, prolactin receptor (Prlr), Ppara, and beta-casein (Csn2). Findings support our hypothesis and highlight potential importance of clock in mammary development and substrate transport.

Mammary Development and Breast Cancer: a Notch Perspective

Mammary gland development primarily occurs postnatally, and this unique process is complex and regulated by systemic hormones and local growth factors. The mammary gland is also a highly dynamic organ that undergoes profound changes at puberty and during the reproductive cycle. These changes are driven by mammary stem cells (MaSCs). Breast cancer is one of the most common causes of cancer-related death in women. Cancer stem cells (CSCs) play prominent roles in tumor initiation, drug resistance, tumor recurrence, and metastasis. The highly conserved Notch signaling pathway functions as a key regulator of the niche mediating mammary organogenesis and breast neoplasia. In this review, we discuss mechanisms by which Notch contributes to breast carcinoma pathology and suggest potentials for therapeutic targeting of Notch in breast cancer. In summary, we provide a comprehensive overview of Notch functions in regulating MaSCs, mammary development, and breast cancer.

Baicalin Promotes Mammary Gland Development via Steroid-Like Activities

Baicalin, the main flavonoid component extracted from Scutellaria roots, has a variety of biological activities and is therefore used in the treatment of many kinds of diseases. However, whether baicalin affects the normal development of tissues and organs is still unclear. Here, using a mouse mammary gland model, we investigated the effects of baicalin on the expansion of mammary stem cells (MaSCs) and mammary development, as well as breast cancer progression. Interestingly, we found that baicalin administration significantly accelerates duct elongation at puberty, and promotes alveolar development and facilitates milk secretion during pregnancy. Furthermore, self-renewal of MaSCs was significantly promoted in the presence of baicalin. Moreover, in a tumor xenograft model, baicalin promoted tumor growth of the MDA-MB-231 cell line, but suppressed tumor growth of the ZR-751 cell line. Mechanistically, baicalin can induce expression of the protein C receptor, while inhibiting the expression of the estrogen receptor. Transcriptome analysis revealed that baicalin is involved in signaling pathways related to mammary gland development, immune response, and cell cycle control. Taken together, our results from comprehensive investigation of the biological activity of baicalin provide a theoretical basis for its rational clinical application.

Estrogen receptor-α signaling in post-natal mammary development and breast cancers

Abstract17β-estradiol controls post-natal mammary gland development and exerts its effects through Estrogen Receptor ERα, a member of the nuclear receptor family. ERα is also critical for breast cancer progression and remains a central therapeutic target for hormone-dependent breast cancers. In this review, we summarize the current understanding of the complex ERα signaling pathways that involve either classical nuclear “genomic” or membrane “non-genomic” actions and regulate in concert with other hormones the different stages of mammary development. We describe the cellular and molecular features of the luminal cell lineage expressing ERα and provide an overview of the transgenic mouse models impacting ERα signaling, highlighting the pivotal role of ERα in mammary gland morphogenesis and function and its implication in the tumorigenic processes. Finally, we describe the main features of the ERα-positive luminal breast cancers and their modeling in mice.