Murine Mammary Epithelial Cell Line Research Articles

PFKFB3-Meditated Glycolysis via the Reactive Oxygen Species-Hypoxic Inducible Factor 1α Axis Contributes to Inflammation and Proliferation of Staphylococcus aureus in Epithelial Cells.

Mastitis caused by antibiotic-resistant strains of Staphylococcus aureus is a significant concern in the livestock industry due to the economic losses it incurs. Regulating immunometabolism has emerged as a promising approach for preventing bacterial inflammation. To investigate the possibility of alleviating inflammation caused by S aureus infection by regulating host glycolysis, we subjected the murine mammary epithelial cell line (EpH4-Ev) to S aureus challenge. Our study revealed that S aureus can colonize EpH4-Ev cells and promote inflammation through hypoxic inducible factor 1α (HIF1α)-driven glycolysis. Notably, the activation of HIF1α was found to be dependent on the production of reactive oxygen species (ROS). By inhibiting PFKFB3, a key regulator in the host glycolytic pathway, we successfully modulated HIF1α-triggered metabolic reprogramming by reducing ROS production in S aureus-induced mastitis. Our findings suggest that there is a high potential for the development of novel anti-inflammatory therapies that safely inhibit the glycolytic rate-limiting enzyme PFKFB3.

HOXB7 induces STAT3-mediated transformation and lung metastasis in immortalized mammary gland NMuMG cells.

The homeobox family genes are often dysregulated in various cancer types. Particularly HOXB7 amplification and overexpression correlate with poor prognosis in various cancer such as gastric, pancreatic, and lung cancers. Moreover, HOXB7 is known to contribute to cancer progression by promoting epithelial to mesenchymal transition, anticancer drug resistance, and angiogenesis. In this study, we show that HOXB7 is coamplified with ERBB2 in a subset of breast cancer patients and HOXB7 expression correlates with poor prognosis in HER2-positive breast cancer patients. This clinical observation is supported by the following results-HOXB7 overexpression in an immortalized murine mammary gland epithelial cell line NMuMG induces cellular transformation in vitro, tumorigenesis, and lung metastasis through the activation of JAK-STAT signaling.

Reduction of ROS-HIF1α-driven glycolysis by taurine alleviates Streptococcus uberis infection.

Antibiotic-resistant strains of Streptococcus uberis (S. uberis) frequently cause clinical mastitis in dairy cows resulting in enormous economic losses. The regulation of immunometabolism is a promising strategy for controlling this bacterial infection. To investigate whether taurine alleviates S. uberis infection by the regulation of host glycolysis via HIF1α, the murine mammary epithelial cell line (EpH4-Ev) and C57BL/6J mice were challenged with S. uberis. Our data indicate that HIF1α-driven glycolysis promotes inflammation and damage in response to the S. uberis challenge. The activation of HIF1α is dependent on mTOR-mediated ROS production. These results were confirmed in vivo. Taurine, an intracellular metabolite present in most animal tissues, has been shown to effectively modulate HIF1α-triggered metabolic reprogramming and contributes to a reduction of inflammation, which reduces mammary tissue damage and prevents mammary gland dysfunction in S. uberis-induced mastitis. These data provide a novel putative prophylactic and therapeutic strategy for amelioration of dairy cow mastitis and bacterial inflammation.

17β-Estradiol and ICI182,780 Differentially Regulate STAT5 Isoforms in Female Mammary Epithelium, With Distinct Outcomes.

Prolactin (PRL) and estrogen cooperate in lobuloalveolar development of the mammary gland and jointly regulate gene expression in breast cancer cells in vitro. Canonical PRL signaling activates STAT5A/B, homologous proteins that have different target genes and functions. Although STAT5A/B are important for physiological mammary function and tumor pathophysiology, little is known about regulation of their expression, particularly of STAT5B, and the consequences for hormone action. In this study, we examined the effect of two estrogenic ligands, 17β-estradiol (E2) and the clinical antiestrogen, ICI182,780 (ICI, fulvestrant) on expression of STAT5 isoforms and resulting crosstalk with PRL in normal and tumor murine mammary epithelial cell lines. In all cell lines, E2 and ICI significantly increased protein and corresponding nascent and mature transcripts for STAT5A and STAT5B, respectively. Transcriptional regulation of STAT5A and STAT5B by E2 and ICI, respectively, is associated with recruitment of estrogen receptor alpha and increased H3K27Ac at a common intronic enhancer 10 kb downstream of the Stat5a transcription start site. Further, E2 and ICI induced different transcripts associated with differentiation and tumor behavior. In tumor cells, E2 also significantly increased proliferation, invasion, and stem cell-like activity, whereas ICI had no effect. To evaluate the role of STAT5B in these responses, we reduced STAT5B expression using short hairpin (sh) RNA. shSTAT5B blocked ICI-induced transcripts associated with metastasis and the epithelial mesenchymal transition in both cell types. shSTAT5B also blocked E2-induced invasion of tumor epithelium without altering E2-induced transcripts. Together, these studies indicate that STAT5B mediates a subset of protumorigenic responses to both E2 and ICI, underscoring the need to understand regulation of its expression and suggesting exploration as a possible therapeutic target in breast cancer.

Lysosomal protein turnover contributes to the acquisition of TGFβ-1 induced invasive properties of mammary cancer cells.

BackgroundNormal epithelial cells and carcinoma cells can acquire invasiveness by epithelial-to-mesenchymal transition (EMT), a process of considerable cellular remodeling. The endosomal/lysosomal compartment is a principal site of intracellular protein degradation. Lysosomal cathepsin proteases are secreted during cancer progression. The established pro-metastatic role of specific cysteine cathepsins has until now been ascribed to their contribution to extracellular matrix remodeling. We hypothesized that cysteine cathepsins affect transforming growth factor β-1 (TGFβ-1)-induced EMT of normal and malignant mammary epithelial cells.MethodsThe role of lysosomal proteolysis in TGFβ-1-induced EMT and invasion was investigated in a normal and a novel malignant murine mammary epithelial cell line. The contribution of cysteine cathepsins was determined by addition of the general cysteine cathepsin inhibitor E64d. Hallmarks of EMT were analyzed by molecular- and cell-biologic analyses including real-time cell migration/invasion assays. A quantitative proteome comparison using stable isotopic labeling with amino acids in culture (SILAC) showed the effect of E64d on TGFβ-1 induced proteome changes. Lysosomal patterning and junctional adhesion molecule A (Jam-a) localization and abundance were analyzed by immunofluorescence.ResultsWe found increased lysosome activity during EMT of malignant mammary epithelial cells. Cysteine cathepsin inhibition had no effect on the induction of the TGFβ-1-induced EMT program on transcriptional level. Protease inhibition did not affect invasion of TGFβ-1 treated normal mammary epithelial cells, but reduced the invasion of murine breast cancer cells. Remarkably, reduced invasion was also evident if E64d was removed 24 h before the invasion assay in order to allow for recovery of cathepsin activity. Proteome analyses revealed a high abundance of lysosomal enzymes and lysosome-associated proteins in cancer cells treated with TGFβ-1 and E64d. An accumulation of those proteins and of lysosomal vesicles was further confirmed by independent methods. Interestingly, E64d caused lysosomal accumulation of Jam-a, a tight junction component facilitating epithelial cell-cell adhesion.ConclusionOur results demonstrate an important role of lysosomal proteolysis in cellular remodeling during EMT and a pivotal contribution of lysosomal cysteine cathepsins to TGFβ-1 induced acquisition of breast cancer cell invasiveness. These findings provide an additional rationale to use cathepsin inhibitors to stall tumor metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0313-5) contains supplementary material, which is available to authorized users.

The stress response resolution assay. I. Quantitative assessment of environmental agent/condition effects on cellular stress resolution outcomes in epithelium

The events or factors that lead from normal cell function to conditions and diseases such as aging or cancer reflect complex interactions between cells and their environment. Cellular stress responses, a group of processes involved in homeostasis and adaptation to environmental change, contribute to cell survival under stress and can be resolved with damage avoidance or damage tolerance outcomes. To investigate the impact of environmental agents/conditions upon cellular stress response outcomes in epithelium, a novel quantitative assay, the "stress response resolution" (SRR) assay, was developed. The SRR assay consists of pretreatment with a test agent or vehicle followed later by a calibrated stress conditions exposure step (here, using 6-thioguanine). Pilot studies conducted with a spontaneously-immortalized murine mammary epithelial cell line pretreated with vehicle or 20 µg N-ethyl-N-nitrososurea/ml medium for 1 hr, or two hTERT-immortalized human bronchial epithelial cell lines pretreated with vehicle or 100 µM zidovudine/lamivudine for 12 days, found minimal alterations in cell morphology, survival, or cell function through 2 weeks post-exposure. However, when these pretreatments were followed 2 weeks later by exposure to calibrated stress conditions of limited duration (for 4 days), significant alterations in stress resolution were observed in pretreated cells compared with vehicle-treated control cells, with decreased damage avoidance survival outcomes in all cell lines and increased damage tolerance outcomes in two of three cell lines. These pilot study results suggest that sub-cytotoxic pretreatments with chemical mutagens have long-term adverse impact upon the ability of cells to resolve subsequent exposure to environmental stressors.

Heparan Sulfate Chains of Syndecan-1 Regulate Ectodomain Shedding

Matrix metalloproteinases release intact syndecan-1 ectodomains from the cell surface giving rise to a soluble, shed form of the proteoglycan. Although it is known that shed syndecan-1 controls diverse pathophysiological responses in cancer, wound healing, inflammation, infection, and immunity, the mechanisms regulating shedding remain unclear. We have discovered that the heparan sulfate chains present on syndecan core proteins suppress shedding of the proteoglycan. Syndecan shedding is dramatically enhanced when the heparan sulfate chains are enzymatically degraded or absent from the core protein. Exogenous heparan sulfate or heparin does not inhibit shedding, indicating that heparan sulfate must be attached to the core protein to suppress shedding. Regulation of shedding by heparan sulfate occurs in multiple cell types, for both syndecan-1 and syndecan-4 and in murine and human syndecans. Mechanistically, the loss of heparan sulfate enhances the susceptibility of the core protein to proteolytic cleavage by matrix metalloproteinases. Enhanced shedding of syndecan-1 following loss of heparan sulfate is accompanied by a dramatic increase in core protein synthesis. This suggests that in response to an increase in the rate of shedding, cells attempt to maintain a significant level of syndecan-1 on the cell surface. Together these data indicate that the amount of heparan sulfate present on syndecan core proteins regulates both the rate of syndecan shedding and core protein synthesis. These findings assign new functions to heparan sulfate chains, thereby broadening our understanding of their physiological importance and implying that therapeutic inhibition of heparan sulfate degradation could impact the progression of some diseases.

Caveolin-1 expression is elevated in claudin-low mammary tumor cells

BackgroundCaveolin-1 is a scaffolding protein found in plasma membrane invaginations known as caveolae. Caveolin-1 can regulate a number of intracellular processes such as signal transduction, cholesterol metabolism and vesicular transport. With respect to breast cancer caveolin-1 has been observed in both tumor cells and stromal cells surrounding tumors however most of the recent research has focused on how the loss of caveolin-1 in the stromal cells surrounding the tumor alters the tumor microenvironment.MethodsCaveolin-1 expression was evaluated in (1) mammary tumors induced by the transgenic overexpression of the type I insulin-like growth factor receptor (IGF-IR), (2) mammary tumors that became independent of IGF-IR signalling and acquired a claudin-low genotype, (3) two murine mammary epithelial tumor cell lines and (4) two murine mammary claudin-low tumor cell lines.ResultsWe found that mammary tumors induced by IGF-IR overexpression expressed low levels of caveolin-1 while mammary tumors that became independent of IGF-IR signalling expressed considerably higher levels of caveolin-1. Interestingly, pockets of caveolin-1 positive cells could be observed in some of the IGF-IR-induced mammary tumors and these caveolin-1 positive cells were associated with tumor cells that expressed basal cytokeratins (cytokeratins 5 and 14). This caveolin-1 expression pattern was maintained in the murine mammary tumor cell lines in that the epithelial mammary tumor cell lines expressed little or no caveolin-1 while the claudin-low cell lines expressed caveolin-1.ConclusionsOur model indicates that mammary tumor cells with epithelial characteristics lack caveolin-1 while mesenchymal tumor cells express caveolin-1 suggesting that caveolin-1 may serve as a marker of mammary tumor cells with mesenchymal characteristics such as claudin-low breast tumors.

P2-01-10: Blockage of Autocrine TGF-b Signaling Reduces Tumorigenecity and Lung Metastasis in a Murine Breast Cancer Cell Line, NMuMG-ST.

Abstract Transforming growth factor beta (TGF-b) is known to have a dualistic role in breast cancer cells, acting as a tumor suppressor or a tumor promoter. This dualistic property varies in different cells and at different progression stages. Studies show that autocrine TGF-b is necessary for the growth and survival of MDA-MB-231 cells, and was also known to enhance the survival of MCF-7 cells. However, the role of autocrine TGF-b in spontaneously transformed cells is still less understood. We have retrovirally transduced a dominant negative TGF-b Type II receptor (DN RII) into a spontaneously transformed murine mammary gland epithelial cell line NMuMG-ST (DN RII cells). The expression of DN RII reduces TGF-b sensitivity of NMuMG-ST cells in a gene transcription assay and a growth inhibition assay. Interestingly, the autocrine TGF-b supports the survival of NMuMG-ST cells, although the exogenous TGF-b inhibits the growth of NMuMG-ST cells. Moreover, more apoptosis were observed in the DN RII cells compared to the control cells in both in vivo and in vitro experiment. We found that AKT and ERK pathway mediates autocrine TGF-b's survival signal. Results of western immunoblot for several stem cell markers and mammosphere formation assay suggest that autocrine TGF-b signaling is essential for the maintenance of stem like cell subpopulation in NMuMG-ST cells. By using immunocytostaining, we found that the DN RII cells expressed more E-cadherin and less Vimentin when compared to the control NMuMG-ST cells, which indicate that blocking autocrine TGF-b signaling pathway could inhibit the process of EMT in NMuMG-ST cells. Notably, when the cells were inoculated orthotopically in nude mouse mammary glands, tumors formed by DN RII cells grew at a similar rate as those formed by control cells, but caused fewer lung metastases. Our finding identified that the autocrine TGF-b signaling is important for the survival and metastatic ability of NMuMG-ST, which could provide an important foundation for further investigation on the role of autocrine TGF-b signaling in breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-10.

Mammary gland specific expression of Brk/PTK6 promotes delayed involution and tumor formation associated with activation of p38 MAPK

IntroductionProtein tyrosine kinases (PTKs) are frequently overexpressed and/or activated in human malignancies, and regulate cancer cell proliferation, cellular survival, and migration. As such, they have become promising molecular targets for new therapies. The non-receptor PTK termed breast tumor kinase (Brk/PTK6) is overexpressed in approximately 86% of human breast tumors. The role of Brk in breast pathology is unclear.MethodsWe expressed a WAP-driven Brk/PTK6 transgene in FVB/n mice, and analyzed mammary glands from wild-type (wt) and transgenic mice after forced weaning. Western blotting and immunohistochemistry (IHC) studies were conducted to visualize markers of mammary gland involution, cell proliferation and apoptosis, as well as Brk, STAT3, and activated p38 mitogen-activated protein kinase (MAPK) in mammary tissues and tumors from WAP-Brk mice. Human (HMEC) or mouse (HC11) mammary epithelial cells were stably or transiently transfected with Brk cDNA to assay p38 MAPK signaling and cell survival in suspension or in response to chemotherapeutic agents.ResultsBrk-transgenic dams exhibited delayed mammary gland involution and aged mice developed infrequent tumors with reduced latency relative to wt mice. Consistent with delayed involution, mammary glands of transgenic animals displayed decreased STAT3 phosphorylation, a marker of early-stage involution. Notably, p38 MAPK, a pro-survival signaling mediator downstream of Brk, was activated in mammary glands of Brk transgenic relative to wt mice. Brk-dependent signaling to p38 MAPK was recapitulated by Brk overexpression in the HC11 murine mammary epithelial cell (MEC) line and human MEC, while Brk knock-down in breast cancer cells blocked EGF-stimulated p38 signaling. Additionally, human or mouse MECs expressing Brk exhibited increased anchorage-independent survival and resistance to doxorubicin. Finally, breast tumor biopsies were subjected to IHC analysis for co-expression of Brk and phospho-p38 MAPK; ductal and lobular carcinomas expressing Brk were significantly more likely to express elevated phospho-p38 MAPK.ConclusionsThese studies illustrate that forced expression of Brk/PTK6 in non-transformed mammary epithelial cells mediates p38 MAPK phosphorylation and promotes increased cellular survival, delayed involution, and latent tumor formation. Brk expression in human breast tumors may contribute to progression by inducing p38-driven pro-survival signaling pathways.

Abstract 1456: Prostaglandin E2 receptor EP1 as a metastasis suppressor in breast cancer

Abstract The cyclooxygenase-2 enzyme (COX-2) is highly expressed in breast cancer and contributes to the metastatic process. Inhibition of COX-2 can decrease breast tumor growth and metastasis. Prostaglandin E2 (PGE2), the chief COX-2 product in tumors, has been identified as a predominant protumorigenic prostanoid. PGE2 mediates biological effects by binding to each of four EP receptors (EP1-4). EP1 is coupled to calcium mobilization and PKC; whereas, EP2 and EP4 activate PKA/cAMP, PI3K and ERK pathways. EP3 generally inhibits cAMP levels. EP4, expressed on malignant breast cells, promotes metastasis, but a role for EP1 in metastasis has not been investigated. Using a combination of immunohistochemistry, pharmacologic and genetic approaches, we studied EP1 expression in human breast tissue and function in a murine model of breast cancer metastasis. Our published studies indicate that EP1 was detected by immunohistochemistry in the cytoplasm and nucleus of benign ducts and malignant cells in invasive ductal carcinomas. Overall survival for women with tumors that were negative for nuclear EP1 was significantly worse than for women with EP1 expression. Additionally, pharmacologic antagonism and reduction of EP1 expression by shRNA increased metastatic capacity in our murine model of metastatic breast cancer. These data suggested that EP1 may function as a metastasis suppressor. We now report that murine mammary tumor cell lines 410, 410.4 and 66.1 have decreased mRNA levels of EP1 compared to normal murine mammary epithelial cell line EpH4. The EP1 receptor is normally coupled to calcium mobilization. Even though EP1 expression is decreased in mammary tumor cell lines, preliminary studies suggest the existence of functional EP1 receptor as PGE2 is capable of inducing calcium mobilization in 410.4 cells. In addition, treatment of 410 and 66.1 cells with 5-azacytidine is capable of increasing EP1 mRNA levels which may identify a clinically relevant strategy to increase expression of this protective receptor. We have also identified the presence of a variant EP1 (EP1v) transcript in this murine metastatic breast cancer model. EP1v has been identified previously in murine mast cell line MC/9 and in rat uterus; however, this is the first report that EP1v has been identified in malignant cells. Like EP1, mRNA expression of EP1v is also decreased in 410, 410.4 and 66.1 cell lines compared to EpH4 cells. Our published studies show that EP1 acts to suppress metastasis and we are currently exploring the contribution of EP1v to this mechanism. These findings suggest that EP1 has the potential to be a new therapeutic target in reducing breast cancer metastasis and increasing overall cancer survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1456. doi:10.1158/1538-7445.AM2011-1456

Abstract 2434: CXCR3-mediated cellular and molecular events involved in breast cancer metastasis

Abstract We previously detected chemokine receptor CXCR3 in malignant epithelium of early stage breast cancers by immunohistochemistry and the expression levels were positively correlated with significantly poorer survival. We also showed that CXCR3 contributes to metastatic potential in a murine model of metastatic breast cancer. The goal of the present study is to determine the cellular and molecular mechanisms underlying CXCR3-mediated breast cancer metastasis. Treatment of the human breast cancer cell line MDA-MB-231 with the CXCR3 ligand CXCL10 induced calcium mobilization and Rac activation; both responses are essential for tumor cell chemotaxis. Not surprisingly, CXCL10 enhanced migration and invasion of MDA-MB-231 cells. We also showed that CXCL10 promoted anchorage independent growth of both human malignant mammary epithelial cells (MDA-MB-231 and MDA-MB-468) and murine malignant mammary epithelial cell line 66.1 in an ERK- dependent manner. In tumor cells, ERK1/2 is robustly activated after CXCR3 ligand stimulation, whereas the same pathway is inhibited or not changed in normal cells. In addition, CXCR3 activation provided some degree of anoikis resistance to 66.1 cells. There are two isoforms of CXCR3 in human cells. The “classical” CXCR3 receptor (CXCR3-A) mediates proliferation and migration of various cell lines, while CXCR3-B, a splice variant of CXCR3-A, has opposing functions. We determined the expression pattern of these two CXCR3 isoforms in a panel of human breast cancer cell lines by real-time quantitative PCR and showed that the CXCR3-B/CXCR3-A mRNA expression ratio is decreased (0.16-0.63) compared with normal epithelial cell line MCF-10A (2.16). Taken together, these data indicate that multiple molecular and cellular mechanisms may contribute to CXCR3-mediated breast cancer metastasis. Due to the distinct functions of CXCR3-A and CXCR3-B, the relative amounts of CXCR3-A and CXCR3-B may dictate cell behavior. Further studies will investigate how the balance between CXCR3-A and CXCR3-B regulates the metastatic potential of breast cancer cells and allow us to optimize therapeutic strategies targeting CXCR3. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2434. doi:10.1158/1538-7445.AM2011-2434

ETV6-NTRK3–Mediated Breast Epithelial Cell Transformation Is Blocked by Targeting the IGF1R Signaling Pathway

The insulin-like growth factor (IGF) 1 receptor (IGF1R) is an important therapeutic target under study in many cancers. Here, we describe a breast cancer model based on expression of the ETV6-NTRK3 (EN) chimeric tyrosine kinase that suggests novel therapeutic applications of IGF1R inhibitors in secretory breast cancers. Originally discovered in congenital fibrosarcomas with t(12;15) translocations, EN was identified subsequently in secretory breast carcinoma (SBC) which represent a variant of invasive ductal carcinoma. Because fibroblast transformation by EN requires the IGF1R axis, we hypothesized a similar dependency may exist in mammary cells and, if so, that IGF1R inhibitors might be useful to block EN-driven breast oncogenesis. In this study, we analyzed EN expressing murine and human mammary epithelial cell lines for transformation properties. Various IGF1R signaling inhibitors, including the dual specificity IGF1R/insulin receptor (INSR) inhibitor BMS-536924, were then tested for effects on three-dimensional Matrigel cell growth, migration, and tumor formation. We found that EN expression increased acinar size and luminal filling in Matrigel cultures and promoted orthotopic tumor growth in mice. Tumors were well differentiated and nonmetastatic, similar to human SBC. The known EN effector pathway, PI3K-Akt, was activated in an IGF1- or insulin-dependent manner. BMS-536924 blocked EN transformation in vitro, whereas BMS-754807, another IGIFR/INSR kinase inhibitor currently in clinical trials, significantly reduced tumor growth in vivo. Importantly, EN model systems mimic the clinical phenotype observed in human SBC. Moreover, EN has a strict requirement for IGF1R or INSR in breast cell transformation. Thus, our findings strongly encourage the evaluation of IGF1R/INSR inhibitors to treat EN-driven breast cancers.

MYBsuppresses differentiation and apoptosis of human breast cancer cells

IntroductionMYB is highly expressed in estrogen receptor positive (ER + ve) breast tumours and tumour cell lines. We recently demonstrated that MYB is essential for the proliferation of ER + ve breast cancer cells, and have now investigated its role in mammary epithelial differentiation.MethodsMCF-7 breast cancer cells were treated with sodium butyrate, vitamin E succinate or 12-O-tetradecanoylphorbol-13-acetate to induce differentiation as measured by Nile Red staining of lipid droplets and β-casein expression. The non-tumorigenic murine mammary epithelial cell (MEC) line, HC11, was induced to differentiate with lactogenic hormones. MYB levels were manipulated by inducible lentiviral shRNA-mediated knockdown and retroviral overexpression.ResultsWe found that MYB expression decreases following chemically-induced differentiation of the human breast cancer cell line MCF-7, and hormonally-induced differentiation of a non-tumorigenic murine mammary epithelial cell (MEC) line, HC11. We also found that shRNA-mediated MYB knockdown initiated differentiation of breast cancer cells, and greatly sensitised them to the differentiative and pro-apoptotic effects of differentiation-inducing agents (DIAs). Sensitisation to the pro-apoptotic effects DIAs is mediated by decreased expression of BCL2, which we show here is a direct MYB target in breast cancer cells. Conversely, enforced expression of MYB resulted in the cells remaining in an undifferentiated state, with concomitant suppression of apoptosis, in the presence of DIAs.ConclusionsTaken together, these data imply that MYB function is critical in regulating the balance between proliferation, differentiation, and apoptosis in MECs. Moreover, our findings suggest MYB may be a viable therapeutic target in breast cancer and suggest specific approaches for exploiting this possibility.

Abstract 2076: Mammary stem cell-like miRNA expression and relation to cancer

Abstract Mammary stem cells are suggested to be the origin of breast cancer, but are elusive and vaguely characterized. Cancer may be sustained by cancer stem cells, that are resistant to common treatments like irradiation and chemotherapy. To determine a possible association between mammary stem cells and breast cancer, we have previously made a detailed characterization of the transcriptome in murine mammary epithelial stem-like cell line (HC11) and made a thorough investigation of global gene-expression changes during stepwise differentiation using dual-color comparative microarray technique. Subsequently, we performed a cross-species comparison to reveal conserved gene expression between stem cells and subtype-specific and prognosis gene signatures. Our analysis provided a unique map of the transcriptomic changes and a number of novel mammary stem cell markers. Our comparisons showed a relationship between breast cancers of poor prognosis and stem-like gene expression. In particular the specific subtypes of basal like tumors and Luminal C types, which both have a poor prognosis, showed stem cell like gene expression pattern. We are subsequently investigating regulations of miRNAs during differentiation. We have found very strong miRNA regulation during differentation of the stem cell like cells, and are investigating the functions of key regulated miRNAs. We imply the same miRNAs could be affecting poor prognosis breast cancers. By defining the association between mammary stem cell like cells and stem cell like tumors, we can start to understand the pathways that will need to be targeted for successful treatment of poor prognosis breast cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2076.

Metabolomic Changes Accompanying Transformation and Acquisition of Metastatic Potential in a Syngeneic Mouse Mammary Tumor Model

Breast cancer is the most common cancer type for women in the western world. Despite decades of research, the molecular processes associated with breast cancer progression are still inadequately defined. Here, we focus on the systematic alteration of metabolism by using the state of the art metabolomic profiling techniques to investigate the changes of 157 metabolites during the progression of normal mouse mammary epithelial cells to an isogenic series of mammary tumor cell lines with increasing metastatic potentials. Our results suggest a two-step metabolic progression hypothesis during the acquisition of tumorigenic and metastatic abilities. Metabolite changes accompanying tumor progression are identified in the intracellular and secreted forms in several pathways, including glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, fatty acid and nucleotide biosynthesis, and the GSH-dependent antioxidative pathway. These results suggest possible biomarkers of breast cancer progression as well as opportunities of interrupting tumor progression through the targeting of metabolic pathways.

Mammary Stem Cell-Like Gene Expression and Relation to Cancer.

Abstract Mammary stem cells are bipotential and suggested to be the origin of breast cancer development, but are elusive and vaguely characterized.Breast tumors can be divided into subgroups, each one requiring specific treatment. To determine a possible association between mammary stem cells and breast cancer, we have previously made a detailed characterization of the transcriptome in murine mammary epithelial stem-like cell line (HC11) and made a thorough investigation of global gene-expression changes during stepwise differentiation using dual-color comparative microarray technique.Subsequently, we performed a cross-species comparison to reveal conserved gene expression between stem cells and subtype-specific and prognosis gene signatures, and correlated gene expression to in vivo mammary gland development. Our analysis provided a unique map of the transcriptomic changes and a number of novel mammary stem cell markers. Interestingly, our comparisons show that specific subtypes of breast cancers with poor prognosis and metastasizing capabilities show resemblance to stem-like gene expression. We are now investigating several marker candidates and regulations of miRNAs during differentiation. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1162.

Gene expression in murine mammary epithelial stem cell-like cells shows similarities to human breast cancer gene expression

IntroductionMammary stem cells are bipotential and suggested to be the origin of breast cancer development, but are elusive and vaguely characterized. Breast tumors can be divided into subgroups, each one requiring specific treatment. To determine a possible association between mammary stem cells and breast cancer, a detailed characterization of the transcriptome in mammary stem cells is essential.MethodsWe have used a murine mammary epithelial stem-like cell line (HC11) and made a thorough investigation of global gene-expression changes during stepwise differentiation using dual-color comparative microarray technique. Subsequently, we have performed a cross-species comparison to reveal conserved gene expression between stem cells and subtype-specific and prognosis gene signatures, and correlated gene expression to in vivo mammary gland development.ResultsOur analysis of mammary stem-like and stepwise cell differentiation, and an in-depth description of our findings in a breast cancer perspective provide a unique map of the transcriptomic changes and a number of novel mammary stem cell markers. We correlate the alterations to in vivo mammary gland differentiation, and describe novel changes in nuclear receptor gene expression. Interestingly, our comparisons show that specific subtypes of breast cancers with poor prognosis and metastasizing capabilities show resemblance to stem-like gene expression.ConclusionsThe transcriptional characterization of these mammary stem-like cells and their differentiation-induced gene expression patterns is here made widely accessible and provides a basis for research on mammary stem-like cells. Our comparisons suggest that some tumors are more stem-like than others, with a corresponding worse prognosis. This information would, if established, be important for treatment decisions. We also suggest several marker candidates valuable to investigate further.

Selective Response to Insulin Versus Insulin-Like Growth Factor-I and -II and Up-Regulation of Insulin Receptor Splice Variant B in the Differentiated Mouse Mammary Epithelium

The terminal differentiation of the mouse mammary gland epithelium during lactation has been shown to require IGFs and/or superphysiological levels of insulin. It has been suggested that IGF receptor I (IGF-IR), in addition to its well-established role in the mammary gland during puberty and pregnancy, serves as the principal mediator of IGFs at this stage of development. However, our analysis of the expression levels of IGF-IR and the two insulin receptor (IR) splice variants, IR-A and IR-B, has revealed a 3- to 4-fold up-regulation of IR-B transcripts and a 6-fold down-regulation of IGF-IR transcripts and protein during terminal differentiation in the developing mammary gland. IR-B expression was also more than 10-fold up-regulated in murine mammary epithelial cell line HC11 during differentiation in vitro. As already described for the human form, murine IR-B cloned from HC11 exhibited selectivity for insulin as compared with IGFs. When differentiated HC11 cells were stimulated by 10 nm insulin, a concentration that is unable to activate IGF-IR, induction of milk protein and lipid synthetic enzyme gene expression, lactate production, and phosphorylation of Akt were observed. In contrast, on differentiated HC11 cells 10 nm IGF-I or 10 nm IGF-II were able to exert growth-promoting effects only. The lack of response of differentiated cells to low levels of IGFs could not be explained by inactivation of IGFs by IGF binding proteins. Our results suggest a previously unrecognized predominant role for IR-B in the differentiated mammary epithelium.

Positive effect of silymarin on cell growth and differentiation in bovine and murine mammary cells

Silymarin, a naturally acknowledged hepatoprotector used in humans to treat liver diseases has been tested in murine (HC11) and bovine (BME-UV) mammary epithelial cell lines to evaluate a possible direct effect on cell growth and differentiation in mammary gland. Silymarin enhanced cell proliferation (p < 0.05) from 10 to 1000 ng/ml in association with growth factors, (up to 20%) or alone (up to 15%) versus controls. Furthermore, silymarin (100 ng/ml) was able to increase (p < 0.05) beta-casein gene expression alone or in association with prolactin (5 microg/ml). These effects may be related with protein kinase B (AKT) activation induced by silymarin treatment (p < 0.05) and/or by a dose-related inhibitory effect (p < 0.05) on caspase-3 activity related to a protective role in cell apoptosis. These data suggest that silymarin should be considered a candidate to support mammary gland activity during a lactogenetic state.