Transcription Factor RFX3 Stabilizes Mammary Basal Cell Identity

The myoepithelial cell compartment of the murine postnatal mammary gland is generated from basal cap cells in the terminal end bud and maintained by self-renewal. Transdifferentiation to the luminal lineage does not normally occur but can be induced by DNA damage, luminal cell death or transplantation into a recipient mammary fat pad. Myoepithelial cells cultivated in vitro can also transdifferentiate towards the luminal lineage. Little is known about the molecular mechanisms and gene regulatory networks underlying this plasticity. Using a transgenic mouse (Tg11.5kb-GFP) that marks cap cells with GFP, we discovered that mature myoepithelial cells placed in culture begin to express GFP within ~24 hrs and later express the Keratin 8 (K8) luminal marker. Cell tracking showed that most K8+ cells arose from GFP+ cells, suggesting that myoepithelial cells de-differentiate towards a progenitor state before changing lineage. Differential gene expression analysis, comparing pure GFP+ cap cells with mature myoepithelial cells, identified multiple transcription factors that iRegulon predicted might regulate the myoepithelial to cap cell transition. Knockout of one of these genes, Regulatory Factor 3 (Rfx3), significantly reduced the population of GFP+ cells and increased differentiation to the K8+ luminal lineage. Rfx3 knockout also reduced mammosphere growth and mammary gland regeneration efficiency in a transplantation assay, but had no effect on proliferation in vitro. Together, these data support a key role for Rfx3 in the stabilization of the mammary basal cell lineages.

Long Intergenic Non-Coding RNAs in the Mammary Parenchyma and Fat Pad of Pre-Weaning Heifer Calves: Identification and Functional Analysis

Enhanced plane of nutrition at pre-weaning stage can promote the development of mammary gland especially heifer calves. Although several genes are involved in this process, long intergenic non-coding RNAs (lincRNAs) are regarded as key regulators in the regulated network and are still largely unknown. We identified and characterized 534 putative lincRNAs based on the published RNA-seq data, including heifer calves in two groups: fed enhanced milk replacer (EH, 1.13 kg/day, including 28% crude protein, 25% fat) group and fed restricted milk replacer (R, 0.45 kg/day, including 20% crude protein, 20% fat) group. Sub-samples from the mammary parenchyma (PAR) and mammary fat pad (MFP) were harvested from heifer calves. According to the information of these lincRNAs’ quantitative trait loci (QTLs), the neighboring and co-expression genes were used to predict their function. By comparing EH vs R, 79 lincRNAs (61 upregulated, 18 downregulated) and 86 lincRNAs (54 upregulated, 32 downregulated) were differentially expressed in MFP and PAR, respectively. In MFP, some differentially expressed lincRNAs (DELs) are involved in lipid metabolism pathways, while, in PAR, among of DELs are involved in cell proliferation pathways. Taken together, this study explored the potential regulatory mechanism of lincRNAs in the mammary gland development of calves under different planes of nutrition.

Comparing syngeneic and autochthonous models of breast cancer to identify tumor immune components that correlate with response to immunotherapy in breast cancer

Background: The heterogeneity of the breast tumor microenvironment (TME) may contribute to the lack of durable responses to immune checkpoint blockade (ICB), however, mouse models to test this are currently lacking. Proper selection and use of preclinical models are necessary for rigorous, preclinical studies to rapidly move laboratory findings into the clinic to treat patients.Methods: We compared 3 versions of a common syngeneic and autochthonous mouse model to elucidate how tumor latency and TME heterogeneity contributes to ICB resistance. We performed comprehensive characterization of the TME using quantitative flow-cytometry and RNA expression analysis (NanoString) utilizing three distinct syngeneic breast cancer models, all derived from the MMTV-PyMT autochthonous model. A commonly used protocol was used to obtain tumor cells from MMTV-PyMT mice and 1E6, 1E5 or 1E4 cells were immediately injected into the mammary fat pad of FVB/NJ wild type mice. We then performed deep immunophenotyping and tested ICB efficacy in the 3 syngeneic models compared to the autochthonous model. Results: The 4 models had vastly different TMEs that correlated to ICB responses. We found that the number of cells used to generate syngeneic tumors significantly influences tumor latency, infiltrating leukocyte population and response to ICB. Compared to the autochthonous model, all 3 syngeneic models had significantly more tumor infiltrating lymphocytes (TILs; CD3+, CD4+, and CD8+) and higher proportions of PD-L1 positive myeloid cells, whereas the MMTV-PyMT model had the highest frequency of myeloid cells out of total leukocytes. Increased TILs correlated with response to anti-PD-L1 and anti-CTLA-4 therapy; but PD-L1expression on tumor cells or PD-1 expression of T-cells did not.Conclusions: These studies reveal that the commonly used syngeneic models have low concordance with the autochthonous model. We have identified ICB-sensitive and resistant syngeneic breast cancer models, generated from the same tumor cell inoculum, and find that only the 1E4 syngeneic model is representative of the slow growing, autochthonous model. Given the lack of benefit from ICB in breast cancer, the identification of robust murine models presented here provides the opportunity to further interrogate the TME for breast cancer treatment and provide novel insights into therapeutic combinations to overcome ICB resistance.

Roles of Adiponectin and Leptin Signaling Related microRNAs in The Preventive Effects of Calorie Restriction in Mammary Tumor Development

Calorie restriction (CR) is suggested preventing development of mammary tumors (MT) however the mechanism remains to be clarified. We aimed to determine the miRNA profile in mice applied to two different CR protocols; chronic (CCR) and intermittent (ICR) and follow the MT development. In addition, the roles of miRNAs involved in adiponectin and/or leptin signaling pathways were investigated. Mice were enrolled into ad-libitum (AL), CCR, or ICR which was three-weeks AL feeding followed by one-week of 60% CR in cyclic manner. Blood and tissue collection were performed at weeks 10, 17/18, 49/50 and 81/82. Long-term CCR provided better protection compared to ICR for MT development with a delay in the MT occurrence. Adiponectin expression in mammary fat pad were significantly higher in CCR group compared to AL. Using GeneChip™ Array, 250 of 3,195 miRNAs were differentially expressed among the dietary groups. 13 of 250 miRNAs were related to adiponectin and/or leptin signaling genes. Results were verified by RT-PCR. Specifically, miR-326-3p, miR-500-3p and miR-129-5p which are adiponectin and/or leptin signaling related may play important roles in the preventive effects of CR in MT development and in ageing. Thus, these miRNAs might be putative biomarkers to target for diagnostic and treatment purposes. Novelty Bullets • Type of Calorie restriction and micro RNA interaction is related to ageing. • miR-326-3p, miR-500-3p and miR-129-5p expression levels were differentially expressed in MT development and in ageing. • The adiponectin and/or leptin signaling pathways related genes are regulated by certain miRNAs in the protective effects of CR

Visualizing tumour self-homing with magnetic particle imaging

Iron labeled CTCs home to previously established mammary fat pad tumours and can be visualized using magnetic particle imaging and magnetic resonance imaging.

PSVII-19 Fatty acid profiles in subcutaneous adipose and the mammary fat pad of Holstein calves receiving different estradiol treatments

Estradiol administration increases mammary parenchyma growth and can increase the mass of the mammary fat pad (MFP) in calves. Estradiol can affect fatty acid metabolism, but it is unknown how estradiol affects the fatty acid profile of the MFP and if these effects are consistent across adipose stores. The objective of this investigation was to determine if fatty acid profiles were affected by estradiol administration and if this response is adipose tissue type specific. Holstein heifer calves were reared on a common diet and administered 12 daily injections prior to euthanasia at 82 days of age. Injections were either daily injections of corn oil (n = 4; CON), 9 injections of corn oil followed by 3 injections of estradiol (n = 4; SHORT), or 12 injections of estradiol (n = 4; LONG). Fatty acids were extracted from MFP and subcutaneous adipose tissues samples and analyzed using gas chromatography. Data were analyzed using a mixed model considering the effect of treatment, adipose tissue depot, their interaction, and the random effect of animal. Only significant treatment effects and treatment-adipose type interactions are presented here (Table 1). Estradiol administration (SHORT and LONG) increased total C18:1 fatty acids (P = 0.05), and tended to increase non-C18:2 t10 c12 CLAs, C18:3, and total omega-3 fatty acids (P ≤ 0.1) in the MFP and subcutaneous adipose tissues relative to CON. There was a treatment by tissue interaction for C18:1 trans-10 (P = 0.01), and a tendency for C18:1 trans-11 and C18:2 trans-10 cis-12 (P ≤ 0.1) indicating that estradiol’s effects on fatty acids are tissue specific. Together, these results indicate that the MFP responds differently to estradiol than subcutaneous adipose tissues and that these alterations are associated with different periods of induced mammary growth via estradiol.

MicroRNA-941 regulates the proliferation of breast cancer cells by altering histone H3 Ser 10 phosphorylation

Breast cancer including triple negative breast cancer (TNBC) represents an important clinical challenge, as these tumours often develop resistance to conventional chemotherapeutics. MicroRNAs play a crucial role in cell-cycle regulation, differentiation, apoptosis, and migration. Herein, we performed Affymetrix Gene Chip miRNA 4.0 microarray and observed differential regulation of miRNAs (75 upregulated and 199 downregulated) in metastatic MDA-MB-231 cells as compared to immortalized human non-tumorigenic breast epithelial (MCF-10A) cells. MicroRNA-941 was significantly upregulated in MDA-MB-231 cells (almost nine-fold increase) in comparison to MCF-10A cells. Transfection of MiRNA-941 inhibitor significantly decreased the proliferation and migration of MDA-MB-231 cells by altering the expressions of p21, Cyclin D1, PP2B-B1, E-cadherin and MMP-13. Interestingly, we provide first evidence that inhibiting miR-941 prevents cell proliferation and phosphorylation of histone H3 at Ser10 residue. Xenograft model of breast cancer was developed by subcutaneous injection of MDA-MB-231 cells into the mammary fat pad of female athymic nude mice (Crl:NU-Foxn1nu). The tumours were allowed to grow to around 60 mm3, thereafter which we divided the animals into seven groups (n = 5). Notably, intratumoral injection of miR-941 inhibitor significantly abolished the tumour growth in MDA-MB-231 xenograft model. 5-Fluorouracil (10 mg/kg, i.p.) was used as positive control in our study. To the best of our knowledge, we report for the first time that targeting miR-941 improves the sensitivity of MDA-MB-231 cells to 5-fluorouracil. This can be of profound clinical significance, as it provides novel therapeutic approach for treating variety of cancers (overexpressing miRNA-941) in general and breast cancers in particular.

Dexamethasone Attenuates X-Ray-Induced Activation of the Autotaxin-Lysophosphatidate-Inflammatory Cycle in Breast Tissue and Subsequent Breast Fibrosis

We recently showed that radiation-induced DNA damage in breast adipose tissue increases autotaxin secretion, production of lysophosphatidate (LPA) and expression of LPA1/2 receptors. We also established that dexamethasone decreases autotaxin production and LPA signaling in non-irradiated adipose tissue. In the present study, we showed that dexamethasone attenuated the radiation-induced increases in autotaxin activity and the concentrations of inflammatory mediators in cultured human adipose tissue. We also exposed a breast fat pad in mice to three daily 7.5 Gy fractions of X-rays. Dexamethasone attenuated radiation-induced increases in autotaxin activity in plasma and mammary adipose tissue and LPA1 receptor levels in adipose tissue after 48 h. DEX treatment during five daily fractions of 7.5 Gy attenuated fibrosis by ~70% in the mammary fat pad and underlying lungs at 7 weeks after radiotherapy. This was accompanied by decreases in CXCL2, active TGF-β1, CTGF and Nrf2 at 7 weeks in adipose tissue of dexamethasone-treated mice. Autotaxin was located at the sites of fibrosis in breast tissue and in the underlying lungs. Consequently, our work supports the premise that increased autotaxin production and lysophosphatidate signaling contribute to radiotherapy-induced breast fibrosis and that dexamethasone attenuated the development of fibrosis in part by blocking this process.

Genomic DNA PCR analysis to assess xenograft development in mouse mammary gland

The mouse transplantation model remains the most relevant methodology to assess the functional capacities of mammary cells and is particularly appropriate for investigations regarding mammary stem cells, whatever the species studied. Following xenotransplantation in mice mammary fat pad, the development of the xenograft is commonly evaluated by immunohistology. Here, we present a simple and rapid method to control the species specificity of a xenograft based on genomic DNA PCR amplification. DNA is extracted from the fixed samples intended for histology, thus allowing the reuse of precious samples. Standard and digital droplet PCR (requiring low DNA quantities) methods have been used to make the present method suitable for the analysis of xenotransplanted samples.