Methionine Protects Mammary Cells against Oxidative Stress through Producing S-Adenosylmethionine to Maintain mTORC1 Signaling Activity

The mechanistic target of rapamycin complex 1 (mTORC1) signaling plays pivotal roles in cell growth and diseases. However, it remains mechanistically unclear about how to maintain mTORC1 activity during mammary glands development. Here we showed that mammary glands suffered from aggravated oxidative stress as pregnancy advanced and was accompanied by an increase in H2O2 levels, while the consumption for methionine and S-adenosylmethionine (SAM) rather than S-adenosylhomocysteine (SAH) were promoted in vivo. Likewise, H2O2 promoted SAM synthesis and reduced SAM utilization for methylation depending on H2O2 levels and treatment time in vitro. H2O2 inhibited phosphorylation of S6 kinase Thr 389 (p-S6K1 (T389)), 4E-BP1 Thr 37/46 and ULK1 Ser 757, the downstream of mTORC1, in mammary epithelial cells. However, methionine and SAM were shown to activate mTORC1 under H2O2-exposed condition. Moreover, this effect was not disabled by SGI-1027 which inhibits SAM transmethylation. In conclusion, methionine appeared to protect mammary cells against oxidative stress through producing SAM to maintain mTORC1 signaling activity.

The Voltage-Gated Sodium Channel Beta4 Subunit Maintains Epithelial Phenotype in Mammary Cells

The SCN4B gene, coding for the NaVβ4 subunit of voltage-gated sodium channels, was recently found to be expressed in normal epithelial cells and down-regulated in several cancers. However, its function in normal epithelial cells has not been characterized. In this study, we demonstrated that reducing NaVβ4 expression in MCF10A non-cancer mammary epithelial cells generated important morphological changes observed both in two-dimensional cultures and in three-dimensional cysts. Most notably, the loss of NaVβ4 induced a complete loss of epithelial organisation in cysts and increased proteolytic activity towards the extracellular matrix. Loss of epithelial morphology was associated with an increased degradation of β-catenin, reduced E-cadherin expression and induction of mesenchymal markers N-cadherin, vimentin, and α-SMA expression. Overall, our results suggest that Navβ4 may participate in the maintenance of the epithelial phenotype in mammary cells and that its downregulation might be a determining step in early carcinogenesis.

In vitro–In vivo Hybrid Approach for Studying Modulation of NRF2 in Immortalized Bovine Mammary Cells

Nuclear factor erythroid 2-related factor 2 (NRF2) plays a key role in the response to oxidative stress. Diets containing known NRF2 modulators could be used to minimize oxidative stress in dairy cows. Currently, studies evaluating the activity of NRF2 in bovine have used the classical in vitro approach using synthetic media, which is very different than in vivo conditions. Furthermore, studies carried out in vivo cannot capture the short-term and dynamic response of NRF2. Thus, there is a need to develop new approaches to study NRF2 modulation. The aim of the present study was to establish an in vitro–in vivo hybrid system to investigate activation of NRF2 in bovine cells that can serve as an intermediate model with results closer to what is expected in vivo. To accomplish the aim, we used a combination of a gene reporter assay in immortalized bovine mammary cells, synthetic NRF2 modulators, and blood serum from periparturient cows. Synthetic agonist tert-butylhydroquinone and sulforaphane confirmed to be effective activators of bovine NRF2 with acute and large effect at 30 and 5 μM, respectively, with null response after the above doses due to cytotoxicity. When the agonists were added to blood serum the response was more linear with maximum activation of NRF2 at 100 and 30 μM, respectively, and the cytotoxicity was prevented. High concentration of albumin in blood serum plays an important role in such an effect. Brusatol (100 nM) was observed to be an effective NRF2 inhibitor while also displaying general protein synthesis inhibition and cytotoxicity when added to synthetic media. A consistent inhibition of NRF2 was observed when brusatol was added to the blood serum but the cytotoxicity was reduced. The synthetic inhibitor ML385 had no effect on modulation of bovine NRF2. Hydrogen peroxide activates NRF2 in bovine mammary cells starting from 100 μM; however, strong cytotoxicity was detected starting at 250 μM when cells were cultivated in the synthetic media, while blood serum prevented cytotoxicity. Overall, our data indicated that the use of synthetic media can be misleading in the study of NRF2 in bovine and the use of blood serum appears necessary.

Characterization of Gene Expression Signatures for the Identification of Cellular Heterogeneity in the Developing Mammary Gland

The developing mammary gland depends on several transcription-dependent networks to define cellular identities and differentiation trajectories. Recent technological advancements that allow for single-cell profiling of gene expression have provided an initial picture into the epithelial cellular heterogeneity across the diverse stages of gland maturation. Still, a deeper dive into expanded molecular signatures would improve our understanding of the diversity of mammary epithelial and non-epithelial cellular populations across different tissue developmental stages, mouse strains and mammalian species. Here, we combined differential mammary gland fractionation approaches and transcriptional profiles obtained from FACS-isolated mammary cells to improve our definitions of mammary-resident, cellular identities at the single-cell level. Our approach yielded a series of expression signatures that illustrate the heterogeneity of mammary epithelial cells, specifically those of the luminal fate, and uncovered transcriptional changes to their lineage-defined, cellular states that are induced during gland development. Our analysis also provided molecular signatures that identified non-epithelial mammary cells, including adipocytes, fibroblasts and rare immune cells. Lastly, we extended our study to elucidate expression signatures of human, breast-resident cells, a strategy that allowed for the cross-species comparison of mammary epithelial identities. Collectively, our approach improved the existing signatures of normal mammary epithelial cells, as well as elucidated the diversity of non-epithelial cells in murine and human breast tissue. Our study provides a useful resource for future studies that use single-cell molecular profiling strategies to understand normal and malignant breast development.

Hypo-osmotic Stress Induces ATP Release via Volume-regulated Anion Channels in Undifferentiated Mammary Cells

The high interstitial ATP concentration in the cancer microenvironment is a major source of adenosine, which acts as a strong immune suppressor. However, the source of ATP release has not been elucidated. We measured the ATP release during hypotonic stress using a real-time ATP luminescence imaging system in primary cultured mammary cells and in breast cell lines. In primary cultured cells, ATP was intermittently released with transient-sharp peaks, while in breast cell lines ATP was released with a slowly rising diffuse pattern. The diffuse ATP release pattern was changed to a transient-sharp pattern by cholera toxin treatment and the reverse change was induced by transforming growth factor (TGF) β treatment. DCPIB, an inhibitor of volume-regulated anion channels (VRACs), only suppressed the diffuse pattern. The inflammatory mediator sphingosine-1-phosphate (S1P) induced a diffuse ATP release pattern isovolumetrically. The knockdown of A isoform of leucine-rich repeat-containing protein 8 (LRRC8A), the essential molecular entity of VRACs, using shRNA suppressed the diffuse pattern. These results suggest that abundantly expressed VRACs are a conduit of ATP release in undifferentiated cells, including cancer cells.

EFA6B regulates a stop signal for collective invasion in breast cancer

Cancer is initiated by somatic mutations in oncogenes or tumor suppressor genes. However, additional alterations provide selective advantages to the tumor cells to resist treatment and develop metastases. Their identification is of paramount importance. Reduced expression of EFA6B (Exchange Factor for ARF6, B) is associated with breast cancer of poor prognosis. Here, we report that loss of EFA6B triggers a transcriptional reprogramming of the cell-to-ECM interaction machinery and unleashes CDC42-dependent collective invasion in collagen. In xenograft experiments, MCF10 DCIS.com cells, a DCIS-to-IDC transition model, invades faster when knocked-out for EFA6B. In addition, invasive and metastatic tumors isolated from patients have lower expression of EFA6B and display gene ontology signatures identical to those of EFA6B knock-out cells. Thus, we reveal an EFA6B-regulated molecular mechanism that controls the invasive potential of mammary cells; this finding opens up avenues for the treatment of invasive breast cancer.