BRCA2 Haploinsufficiency in Telomere Maintenance

Our previous studies showed an association between monoallelic BRCA2 germline mutations and dysfunctional telomeres in epithelial mammary cell lines and increased risk of breast cancer diagnosis for women with BRCA2 999del5 germline mutation and short telomeres in blood cells. In the current study, we analyzed telomere dysfunction in lymphoid cell lines from five BRCA2 999del5 mutation carriers and three Fanconi Anemia D1 patients by fluorescence in situ hybridization (FISH). Metaphase chromosomes were harvested from ten lymphoid cell lines of different BRCA2 genotype origin and analyzed for telomere loss (TL), multitelomeric signals (MTS), interstitial telomere signals (ITS) and extra chromosomal telomere signals (ECTS). TL, ITS and ECTS were separately found to be significantly increased gradually between the BRCA2+/+, BRCA2+/- and BRCA2-/- lymphoid cell lines. MTS were found to be significantly increased between the BRCA2+/+ and the BRCA2+/- heterozygous (p < 0.0001) and the BRCA2-/- lymphoid cell lines (p < 0.0001) but not between the BRCA2 mutated genotypes. Dysfunctional telomeres were found to be significantly increased in a stepwise manner between the BRCA2 genotypes indicating an effect of BRCA2 haploinsufficiency on telomere maintenance.

MYC regulates a pan-cancer network of co-expressed oncogenic splicing factors

MYC is dysregulated in >50% of cancers, but direct targeting of MYC has been clinically unsuccessful. Targeting downstream MYC effector pathways represents an attractive alternative. MYC regulates alternative mRNA splicing, a hallmark of cancer, but the mechanistic links between MYC and the splicing machinery remain underexplored. Here, we identify a network of splicing factors (SFs) co-expressed as SF-modules in MYC-active breast tumors. Of these, one is a pan-cancer SF-module, correlating with MYC-activity across 33 tumor types. In mammary cell models, MYC activation leads to co-upregulation of pan-cancer module SFs and to changes in >4,000 splicing events. In breast cancer organoids, co-overexpression of the pan-cancer SF-module is sufficient to induce splicing events that are also MYC-regulated in patient tumors and to increase organoid size and invasiveness, while its knockdown decreases organoid size. Finally, we uncover a pan-cancer splicing signature of MYC activity which correlates with survival in multiple tumor types. Our findings provide insight into the mechanisms and function of MYC-regulated splicing and for the development of therapeutics for MYC-driven tumors.

Mammary cell gene expression atlas links epithelial cell remodeling events to breast carcinogenesis

The female mammary epithelium undergoes reorganization during development, pregnancy, and menopause, linking higher risk with breast cancer development. To characterize these periods of complex remodeling, here we report integrated 50 K mouse and 24 K human mammary epithelial cell atlases obtained by single-cell RNA sequencing, which covers most lifetime stages. Our results indicate a putative trajectory that originates from embryonic mammary stem cells which differentiates into three epithelial lineages (basal, luminal hormone-sensing, and luminal alveolar), presumably arising from unipotent progenitors in postnatal glands. The lineage-specific genes infer cells of origin of breast cancer using The Cancer Genome Atlas data and single-cell RNA sequencing of human breast cancer, as well as the association of gland reorganization to different breast cancer subtypes. This comprehensive mammary cell gene expression atlas (https://mouse-mammary-epithelium-integrated.cells.ucsc.edu) presents insights into the impact of the internal and external stimuli on the mammary epithelium at an advanced resolution.

Epigenetics: New Insights into Mammary Gland Biology

The mammary gland undergoes important anatomical and physiological changes from embryogenesis through puberty, pregnancy, lactation and involution. These steps are under the control of a complex network of molecular factors, in which epigenetic mechanisms play a role that is increasingly well described. Recently, studies investigating epigenetic modifications and their impacts on gene expression in the mammary gland have been performed at different physiological stages and in different mammary cell types. This has led to the establishment of a role for epigenetic marks in milk component biosynthesis. This review aims to summarize the available knowledge regarding the involvement of the four main molecular mechanisms in epigenetics: DNA methylation, histone modifications, polycomb protein activity and non-coding RNA functions.

Long Non-Coding RNA MPNCR is a Negative Regulator of Bovine Mammary Epithelial Cell Proliferation

BackgroundNumerous studies have reported that many long non-coding RNAs (lncRNAs) may play multiple essential roles during mammary gland development; however, little is known about their functions and regulatory mechanism.ResultsIn this study, we confirmed a novel lncRNA, MPNCR, that was highly expressed during lactation compared to the dry period in dairy cattle. The potential biological and regulatory functions of MPNCR were explored in bovine mammary epithelial cells (BMECs). The MTT and EdU assays revealed that lncRNA MPNCR significantly suppressed cell viability and proliferation of BMECs. The subcellular localization indicated that MPNCR had a potential function as a competing endogenous RNA (ceRNA) to regulate the proliferation of BMECs. Bioinformatics analysis showed that lncRNA MPNCR had two binding sites for miR-31, further confirmed by the dual-luciferase report assay. The RT-qPCR result showed that the expression of miR-31 was downregulated following the overexpression of MPNCR in BMECs. Furthermore, the expression of MPNCR was significantly inhibited by miR-31 overexpression and upregulated by miR-31 inhibition. Cell viability and proliferation were significantly suppressed or promoted with the transfection of miR-31 inhibitor or miR-31 mimic into BMECs, respectively, whereas rescue with MPNCR reduced the proliferation of BMECs. The dual-luciferase report assay showed an interaction between miR-31 and the 3' UTR of the target gene CAMK2D. RT-qPCR and western blot further showed that the expression of CAMK2D was significantly increased with the overexpression of miR-31, while its expression was significantly suppressed with the inhibition of miR-31, indicating CAMK2D was a target of miR-31.ConclusionTaken together, these results demonstrate the function and regulatory mechanism of lncRNA MPNCR on the proliferation of BMECs, and that its regulation may contribute to bovine mammary cell population management.

Multiscale imaging of basal cell dynamics in the functionally mature mammary gland

The mammary epithelium is indispensable for the continued survival of more than 5,000 mammalian species. For some, the volume of milk ejected in a single day exceeds their entire blood volume. Here, we unveil the spatiotemporal properties of physiological signals that orchestrate the ejection of milk from alveolar units and its passage along the mammary ductal network. Using quantitative, multidimensional imaging of mammary cell ensembles from GCaMP6 transgenic mice, we reveal how stimulus evoked Ca2+ oscillations couple to contractions in basal epithelial cells. Moreover, we show that Ca2+-dependent contractions generate the requisite force to physically deform the innermost layer of luminal cells, compelling them to discharge the fluid that they produced and housed. Through the collective action of thousands of these biological positive-displacement pumps, each linked to a contractile ductal network, milk begins its passage toward the dependent neonate, seconds after the command.

Ras-transformation reduce FAM20C expression and osteopontin phosphorylation

Family with sequence similarity 20, member C (FAM20C) is the main kinase of secreted phosphoproteins, including the multifunctional protein and cytokine, osteopontin (OPN). The phosphorylation of OPN varies greatly among cell types, tissues and species, and the different phospho-isoforms contribute to the multifunctionality of the protein. Expression of OPN is increased in human malignancies, and less phosphorylated isoforms of the protein have been associated with this phenotype. Here, we compared OPN from ras-transformed fibroblasts with that from their non-transformed parental cells, and found that OPN was less phosphorylated after ras-transformation. Furthermore, we demonstrated that expression of FAM20C mRNA was reduced five-fold in ras-transformed fibroblasts compared with non-transformed fibroblasts. Transfection with FAM20C of the ras-transformed fibroblasts restored the FAM20C mRNA expression but the phosphorylation of OPN was not increased proportionally. Likewise, the mRNA level of FAM20C was reduced in the malignant ras-transformed mammary cell line MCF10ACA1a compared with its non-transformed parental cell line MCF10A. These results suggest that expression of the FAM20C kinase is reduced after oncogenic ras-transformation, which potentially affects the phosphorylation of secreted phosphoproteins.