Mammary Myoepithelial Cells Research Articles

Abstract B012: Wnt/Ror2 signaling regulates myoepithelial cell fate and luminal cell proliferation during mammary development

Abstract Background: The mammary gland exhibits extensive proliferative and differentiation potential throughout development. Wnt signaling has been recognized as a crucial player in multiple stages of mammary gland development, including mammary placode specification, gland branching morphogenesis, stem cell maintenance, and alveolar development. In our laboratory, prior research has revealed the importance of the β-catenin-independent noncanonical Wnt receptor, Ror2, in branching morphogenesis, cell differentiation, and actin-cytoskeletal dynamics within the mammary epithelium. However, the specific roles of Ror2 in the myoepithelial and luminal cell compartments have yet to be fully elucidated. Methods and Results: In this study, we investigated the functional role of Ror2 in these two distinct cell populations by developing mouse models with lineage-specific deletion of Ror2 in myoepithelial versus luminal epithelial compartments. To achieve this, we generated two tamoxifen-inducible, p63- and Keratin 8-specific Ror2 knockout mouse models, which carried a ROSA26-mT/mG fluorescent reporter for mammary myoepithelial cells and luminal cells, respectively. We induced postnatal Ror2 deletion at week 4 or 6 and assessed the mammary glands 2 to 4 weeks after Ror2 deletion. Through whole-mount imaging of mammary glands, we observed a significant increase in branching morphogenesis when Ror2 was deleted in the myoepithelial compartment, which was further confirmed in 3D branching assays in vitro. RNA-sequencing analysis revealed novel transcriptional alterations encompassing altered morphogenesis, cell differentiation, and intermediate filament organization upon Ror2 loss. Additional gene expression validation and immunofluorescence demonstrated altered myoepithelial cell fate specification, leading to a shift to a luminal-like cell fate, along with altered cell-cell adhesion among epithelial populations and abnormal branching morphogenesis within the developing gland. Moreover, luminal-specific deletion of Ror2 resulted in increased proliferation and abnormal lumen formation, both in vivo and in vitro. Conclusion: Our findings from these tissue-specific knockout mouse models highlight the critical role of the noncanonical Wnt receptor, Ror2, in regulating mammary epithelial cell fate, branching morphogenesis, and proliferation. These results also provide intriguing insights into the coordination between canonical and noncanonical Wnt signaling in mammary gland development, with potential implications for understanding breast cancer tumorigenesis and heterogeneity (Supported by NIH CA016303-46 and CPRIT RP210027). Citation Format: Hongjiang Si, Jeffrey Rosen, Chad Creighton, Kevin Roarty. Wnt/Ror2 signaling regulates myoepithelial cell fate and luminal cell proliferation during mammary development [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B012.

Establishment and characterization of a HER2-enriched canine mammary cancerous myoepithelial cell line

BackgroundCanine mammary tumors (CMTs) have a poor prognosis, along with tumor recurrence and metastasis. Cell lines are vital in vitro models for CMT research. Many CMT epithelial cell lines were reported. However, canine mammary myoepithelial cells, the contractile component of the canine mammary tissue were overlooked. This study aimed at establishing such a cell line. CMT-1 cell line was obtained from a canine mammary tumor CMT-1 and characterized molecularly through qPCR, western blotting, immunochemistry and immunofluorescence. Its doubling time, cytogenetic analysis and migration rate were evaluated using growth study, karyotype analysis and wound healing assay respectively. To determine its tumorigenesis, xenograft transplantation was performed.ResultsCMT-1 tumor was a complex canine mammary carcinoma that stained negative to estrogen receptors (ER) and progesterone receptors (PR), but positive to human epidermal growth receptor-2 (HER2), defined as HER2-enriched subtype. In this study, a CMT-1 cell line obtained from CMT-1 tumor was immune-positive to vimentin, α-SMA, p63 and negative to E-cadherin (E-cad), indicating CMT-1 cells were myoepithelial cells. It was successfully cultured for more than 50 passages showing the same immunoreactivity to ER, PR, and HER2 as the primary canine tumor. The doubling time of CMT-1 cell line was 26.67 h. The chromosome number of CMT-1 cells ranged from 31 to 64. A potential spontaneous epithelial to mesenchymal transition (EMT) was noticed during cell cultures. Potential EMT-induced CMT-1 cells showed no significance in migration rate compared to the original CMT-1 cells. CMT-1 cells was able to grow on a 3D culture and formed grape-like, solid, and cystic mammospheres at different time period. Inoculation of CMT-1 cells induced a complex HER2-enriched mammary tumor with metastasis in mice.ConclusionsA canine cancerous HER2-enriched myoepithelial cell line was successfully established and a canine mammosphere developed from myoepithelial cells was documented in this study. We are expecting this novel cell line and its associated mammospheres could be used as a model to elucidate the role of myoepithelial cells in CMT carcinogensis in the future.

Expression of LGR5 in mammary myoepithelial cells and in triple-negative breast cancers

Lineage tracing in mice indicates that LGR5 is an adult stem cell marker in multiple organs, such as the intestine, stomach, hair follicles, ovary, and mammary glands. Despite many studies exploring the presence of LGR5 cells in human tissues, little is known about its expression profile in either human mammary tissue or pathological lesions. In this study we aim to investigate LGR5 expression in normal, benign, and malignant lesions of the human breast using RNA in situ hybridization. LGR5 expression has not been observed in normal lactiferous ducts and terminal duct lobular units, whereas LGR5-positive cells have been specifically observed in the basal myoepithelium of ducts in the regenerative tissues, ductal carcinoma in situ, and in ducts surrounded by invasive cancer cells. These findings suggest LGR5 marks facultative stem cells that are involved in post injury regeneration instead of homeostatic stem cells. LGR5 positivity was found in 3% (9 of 278 cases) of invasive breast cancers (BC), and it showed positive associations with higher histologic grades (P = 0.001) and T stages (P < 0.001), while having negative correlations with estrogen receptor (P < 0.001) and progesterone receptor (P < 0.001) expression. Remarkably, all LGR5-positive BC, except one, belong to triple-negative BC (TNBC), representing 24% (9 of 38 cases) of all of them. LGR5 histoscores have no correlations with EGFR, CK5/6, Ki-67, or P53 expression. Additionally, no β-catenin nuclear localization was observed in LGR5-positive BC, indicating that canonical Wnt pathway activation is less likely involved in LGR5 expression in BC. Our results demonstrate that LGR5 expression is induced in regenerative conditions in the myoepithelium of human mammary ducts and that its expression is only observed in TNBC subtype among all invasive BC. Further studies regarding the functional and prognostic impact of LGR5 in TNBC are warranted.

Cell-specific expression of functional glucose transporter 8 in mammary gland

Differentiated mammary epithelial cells are responsible for milk synthesis during lactation, supporting early postnatal life in mammals. These cells are found in the terminal alveoli of a secretory epithelium, which is surrounded by myoepithelial cells and a stroma rich in fatty tissue. The aim of this study was to explore the cell-specific expression of the glucose transporter GLUT8 in mammary gland and evaluate its functionality for glucose transport, in order to confirm its role in lactose synthesis. Our histological results revealed that GLUT8 is expressed in adipocytes and the epithelial and myoepithelial cells in mammary gland, with a predominant intracellular granular pattern. Colocalization studies of endogenous and green fluorescent protein fused GLUT8 revealed their expressions in lysosome and Golgi, respectively, with Pearson's coefficient correlations of 0.82 ± 0.05 and 0.68 ± 0.16. Functional studies of dileucine to dialanine mutant of GLUT8 showed a fructose-sensitive 2-deoxy glucose uptake at a rate of 83.3 pmoles/(min∗106 cells), 7 folds over empty vector, with a 60 ± 4 and 72 ± 6% decline in 2-deoxy glucose in the presence of 20 and 50 mM fructose, respectively.We concluded that functional GLUT8 is expressed in mammary gland, localizing in mammary epithelial and myoepithelial cells, and adipocytes. In lactation, GLUT8 is expressed mainly in luminal epithelial cells, at the compartments of the endomembrane system. It is necessary to explore the physiological/pathological functions of GLUT8 in mammary gland, including its role in lactation.

Breast milk apelin level increases with maternal obesity and high-fat feeding during lactation.

Recent evidence indicates that levels of breast milk (BM) hormones such as leptin can fluctuate with maternal adiposity, suggesting that BM hormones may signal maternal metabolic and nutritional environments to offspring during postnatal development. The hormone apelin is highly abundant in BM but its regulation during lactation is completely unknown. Here, we evaluated whether maternal obesity and overnutrition impacted BM apelin and leptin levels in clinical cohorts and lactating rats. BM and plasma samples were collected from normal-weight and obese breastfeeding women, and from lactating rats fed a control or a high fat (HF) diet during lactation. Apelin and leptin levels were assayed by ELISA. Mammary gland (MG) apelin expression and its cellular localization in lactating rats was measured by quantitative RT-PCR and immunofluorescence, respectively. BM apelin levels increased with maternal BMI, whereas plasma apelin levels decreased. BM apelin was also positively correlated with maternal insulin and C-peptide levels. In rats, maternal HF feeding exclusively during lactation was sufficient to increase BM apelin levels and decrease its plasma concentration without changing body weight. In contrast, BM leptin levels increased with maternal BMI in humans, but did not change with maternal HF feeding during lactation in rats. Apelin is highly expressed in the rat MG during lactation and was mainly localized to mammary myoepithelial cells. We found that MG apelin gene expression was up-regulated by maternal HF diet and positively correlated with BM apelin content and maternal insulinemia. Our study indicates that BM apelin levels increase with long- and short-term overnutrition, possibly via maternal hyperinsulinemia and transcriptional upregulation of MG apelin expression in myoepithelial cells. Apelin regulates many physiological processes, including energy metabolism, digestive function, and development. Further studies are needed to unravel the consequences of such changes in offspring development.

The Adipose Microenvironment Dysregulates the Mammary Myoepithelial Cells and Could Participate to the Progression of Breast Cancer.

Breast cancer is the most common cancer among women worldwide. Overweight and obesity are now recognized as established risk factors for this pathology in postmenopausal women. These conditions are also believed to be responsible for higher recurrence and mortality rates. Reciprocal interactions have been described between adipose and cancer cells. An adipose microenvironment favors a greater proliferation of cancer cells, their invasion and even resistance to anti-cancer treatments. In addition, the chronic low-grade inflammation observed in obese individuals is believed to amplify these processes. Among the cell types present in the breast, myoepithelial cells (MECs), located at the interface of the epithelial cells and the stroma, are considered “tumor suppressor” cells. During the transition from ductal carcinoma in situ to invasive cancer, disorganization or even the disappearance of MECs is observed, thereby enhancing the ability of the cancer cells to migrate. As the adipose microenvironment is now considered as a central actor in the progression of breast cancer, our objective was to evaluate if it could be involved in MEC functional modifications, leading to the transition of in situ to invasive carcinoma, particularly in obese patients. Through a co-culture model, we investigated the impact of human adipose stem cells from women of normal weight and obese women, differentiated or not into mature adipocytes, on the functionality of the MECs by measuring changes in viability, apoptosis, gene, and miRNA expressions. We found that adipose cells (precursors and differentiated adipocytes) could decrease the viability of the MECs, regardless of the original BMI. The adipose cells could also disrupt the expression of the genes involved in the maintenance of the extracellular matrix and to amplify the expression of leptin and inflammatory markers. miR-122-5p and miR-132-3p could also be considered as targets for adipose cells. The metabolite analyses revealed specific profiles that may be involved in the growth of neoplastic cells. All of these perturbations could thus be responsible for the loss of tumor suppressor status of MECs and promote the transition from in situ to invasive carcinoma.

Impact of estrogen population pharmacokinetics on a QSP model of mammary stem cell differentiation into myoepithelial cells

&lt;abstract&gt; &lt;p&gt;Stem cell differentiation cascades are critical components of healthy tissue maintenance. Dysregulation in these systems can lead to serious diseases, including cancer. Myoepithelial mammary cells are produced from differentiated mammary stem cells in processes regulated, in part, by estrogen signalling and concentrations. To quantify and predict the production of mammary myoepithelial cell production by estrogen, we developed a mechanistic, quantitative systems pharmacology (QSP) model that includes the explicit characterization of free and unbound estrogen concentrations in circulation. Linking this model to a previously developed population pharmacokinetics model for ethinyl estradiol, a synthetic form of estrogen included in oral contraceptives, we predicted the effects of estrogen on myoepithelial cell development. Interestingly, pharmacokinetic intraindividual variability alone did not significantly impact on our modelos predictions, suggesting that combinations of physiological and pharmacokinetic variability drive heterogeneity in mechanistic QSP models. Our model is one component of an improved understanding of mammary myoepithelial cell production and development, and our results support the call for mechanistically constructed systems models for disease and pharmaceutical modelling.&lt;/p&gt; &lt;/abstract&gt;

Mex3c mutation affects lactation through impairing milk ejection in female mice.

Mouse Mex3c encodes RNA-binding proteins of variant length through alternative splicing. Its mutation results in multiple defects including growth retardation, perturbed energy balance, and defective antiviral innate immunity. Here we report that Mex3c mutation affects mammary gland development and lactation in female mice. Pups of Mex3c mutant dams die of starvation soon after birth. Milk contents are present in the alveoli but deficient in the ducts of the mammary glands in mutant mice. Mutant mice do not show prolactin or oxytocin deficiency. They also develop myoepithelial cells in the mammary glands. Mex3c is expressed in the mammary gland epithelium. Our data suggest that functional defects in mammary gland epithelium or myoepithelial cells could cause lactation defects.

Is it ductal carcinoma in situ with microinvasion or "Ductogenesis"? The role of myoepithelial cell markers.

Mammary myoepithelial cells have been under-recognized for many years since they were considered less important in breast cancer tumorigenesis compared to luminal epithelial cells. However, in recent years with advances in genomics, cell biology, and research in breast cancer microenvironment, more emphasis has been placed on better understanding of the role that myoepithelial cells play in breast cancer progression. As the result, it has been recognized that the presence or absence of myoepithelial cells play a critical role in the assessment of tumor invasion in diagnostic breast pathology. In addition, advances in screening mammography and breast imaging has resulted in increased detection of ductal carcinoma in situ and consequently more diagnosis of ductal carcinoma in situ with microinvasion. In the present review, we discuss the characteristics of myoepithelial cells, their genomic markers and their role in the accurate diagnosis of ductal carcinoma in situ with microinvasion. We also share our experience with reporting of various morphologic features of ductal carcinoma in situ that may mimic microinvasion and introduce the term of ductogenesis.

Physiological and pharmacological evaluation of oxytocin-induced milk ejection in mice.

Oxytocin, a posterior pituitary hormone, causes the contraction of the mammary myoepithelial cells that surround the acini. This ejects milk from the acini into the primary mammary ducts. The milk ejection responses by oxytocin have not yet been exactly evaluated in mice. Thus, we present a novel method for quantitatively evaluating oxytocin-induced milk ejection in anesthetized lactating mice. We cannulated the mammary duct, administered oxytocin intraperitoneally or intravenously, and collected and measured the ejected milk. Intraperitoneal oxytocin administration (150 mU) induced continuous but oscillatory milk ejection. Repeated intravenous administration of 1.5 mU of oxytocin elicited repeated transient milk ejection. The volume of the ejected milk as a proportion of the stored volume just before each ejection (rather than ejection volume itself) was an expedient and reliable parameter representing the potency of ejection. The oxytocin sensitivity of mice at day 18 of lactation was determined from a sigmoidal dose–response curve as ED50 ≈ 2.69 mU. Based on this dose–response relationship, the specific activity of the oxytocin receptor agonists (Thr4, Gly7)-oxytocin and WAY 267464 were estimated as 976 and 6.87 U/mg, respectively. The assay presented here could be useful for physiological and pharmacological investigations of oxytocin-induced milk ejection.

Preeclampsia-Like Features and Partial Lactation Failure in Mice Lacking Cystathionine γ-Lyase—An Animal Model of Cystathioninuria

Elevated plasma homocysteine levels are considered as a risk factor for cardiovascular diseases as well as preeclampsia—a pregnancy disorder characterized by hypertension and proteinuria. We previously generated mice lacking cystathionine γ-lyase (Cth) as cystathioninuria models and found them to be with cystathioninemia/homocysteinemia. We investigated whether Cth-deficient (Cth−/−) pregnant mice display any features of preeclampsia. Cth−/− females developed normally but showed mild hypertension (~10 mmHg systolic blood pressure elevation) in late pregnancy and mild proteinuria throughout development/pregnancy. Cth−/− dams had normal numbers of pups and exhibited normal maternal behavior except slightly lower breastfeeding activity. However, half of them could not raise their pups owing to defective lactation; they could produce/store the first milk in their mammary glands but not often provide milk to their pups after the first ejection. The serum oxytocin levels and oxytocin receptor expression in the mammary glands were comparable between wild-type and Cth−/− dams, but the contraction responses of mammary gland myoepithelial cells to oxytocin were significantly lower in Cth−/− dams. The contraction responses to oxytocin were lower in uteruses isolated from Cth−/− mice. Our results suggest that elevated homocysteine or other unknown factors in preeclampsia-like Cth−/− dams interfere with oxytocin that regulates milk ejection reflex.

Brown adipocytes can display a mammary basal myoepithelial cell phenotype in vivo

ObjectivePrevious work has suggested that white adipocytes may also show a mammary luminal secretory cell phenotype during lactation. The capacity of brown and beige/brite adipocytes to display a mammary cell phenotype and the levels at which they demonstrate such phenotypes in vivo is currently unknown. MethodsTo investigate the putative adipocyte origin of mammary gland cells, we performed genetic lineage-labeling experiments in BAT and the mammary glands. ResultsThese studies indicated that the classic brown adipocytes (Ucp1+) and subcutaneous beige/brite adipocytes (Ucp1−/+) were found in the mammary gland during lactation, when they exhibited a mammary myoepithelial phenotype. Up to 2.5% of the anterior dorsal interscapular mammary myoepithelial cell population had a brown adipocyte origin with an adipose and myoepithelial gene signature during lactation. Eliminating these cells, along with all the brown adipocytes, significantly slowed offspring growth, potentially demonstrating their functional importance. Additionally, we showed mammary epithelial lineage Mmtv+ and Krt14+ cells expressed brown adipocyte markers after weaning, demonstrating that mammary gland cells can display an adipose phenotype. ConclusionsThe identification of a brown adipocyte origin of mammary myoepithelial cells provides a novel perspective on the interrelationships between adipocytes and mammary cells with implications for our understanding of obesity and breast cancer.

Contractility Assay for Established Myoepithelial Cell Lines.

The capacity of mammary myoepithelial cells to contract in response to suckling stimuli is essential for lactation. We describe here a protocol for studying the contractile activity of myoepithelial cells in vitro. This protocol includes the establishment of stable myoepithelial cell lines from mouse mammary glands and quantitative evaluation of the contraction and subsequent relaxation of cultured myoepithelial cells in response to oxytocin. It can be used for analyses of mouse mutants with gene deletions or overexpression altering myoepithelial cell function.

Numb and Numbl act to determine mammary myoepithelial cell fate, maintain epithelial identity, and support lactogenesis.

Mammary epithelium is comprised of an inner layer of luminal epithelial cells and an outer layer of contractile myoepithelial cells with mesenchymal properties. These two compartments interact throughout mammary morphogenesis to form branching ducts during puberty and terminate in secretory alveoli during lactation. It is not known how the myoepithelial cell lineage is specified, nor how signals in myoepithelial cells contribute to lactogenesis. Here, we show that Numb and Numbl are enriched in mammary myoepithelial cells, with their expression peaking during pregnancy. We use conditional Numb- and Numbl-knockout mouse models to demonstrate that loss of Numb/Numbl compromised the myoepithelial layer and expanded the luminal layer, led epithelial cells to undergo epithelial-to-mesenchymal transition, and resulted in lactation failure as a result of abnormal alveolar formation during pregnancy. Numb and Numbl function via repression of the Notch signaling pathway and of the p53-p21 axis during mammary gland development. These findings highlight the importance of Numb and Numbl in the control of myoepithelial cell fate determination, epithelial identity, and lactogenesis.-Zhang Y., Li, F., Song, Y., Sheng, X., Ren, F., Xiong, K., Chen, L., Zhang, H., Liu, D., Lengner, C. J., Xue, L., Yu, Z. Numb and Numbl act to determine mammary myoepithelial cell fate, maintain epithelial identity, and support lactogenesis.

Gene expression in breastmilk cells is associated with maternal and infant characteristics.

Breastmilk is a rich source of cells with a heterogeneous composition comprising early-stage stem cells, progenitors and more differentiated cells. The gene expression profiles of these cells and their associations with characteristics of the breastfeeding mother and infant are poorly understood. This study investigated factors associated with the cellular dynamics of breastmilk and explored variations amongst women. Genes representing different breastmilk cell populations including mammary epithelial and myoepithelial cells, progenitors, and multi-lineage stem cells showed great variation in expression. Stem cell markers ESRRB and CK5, myoepithelial marker CK14, and lactocyte marker α-lactalbumin were amongst the genes most highly expressed across all samples tested. Genes exerting similar functions, such as either stem cell regulation or milk production, were found to be closely associated. Infant gestational age at delivery and changes in maternal bra cup size between pre-pregnancy and postpartum lactation were associated with expression of genes controlling stemness as well as milk synthesis. Additional correlations were found between genes and dyad characteristics, which may explain abnormalities related to low breastmilk supply or preterm birth. Our findings highlight the heterogeneity of breastmilk cell content and its changes associated with characteristics of the breastfeeding dyad that may reflect changing infant needs.

A Mutation in the Carbohydrate Recognition Domain Drives a Phenotypic Switch in the Role of Galectin-7 in Prostate Cancer.

The observation that galectin-7 (gal-7) is specifically expressed in mammary myoepithelial (basal) cells prompted us to investigate whether this protein is expressed in the basal cells of other tissues. Given that breast and prostate cancer have remarkable underlying biological similarities and given the important roles of basal cells in prostate cancer, we examined the expression patterns and role of gal-7 in human prostate cancer. Using tissue microarray, we found that although gal-7 is readily expressed in basal cells in normal prostate tissue, it is downregulated in prostate cancer (PCa) cells. De novo expression of gal-7 in prostate cancer cells increases their sensitivity to apoptosis in response to etoposide and cisplatin. The assessment of a carbohydrate-recognition domain (CRD)-defective mutant form of gal-7 (R7S) showed that the ability of this protein to modulate apoptosis was independent of its CRD activity. This activity was also independent of its ability to translocate to the mitochondrial and nuclear compartments. However, CRD activity was necessary to inhibit the invasive behaviors of prostate cancer cells. In vivo, gal-7 overexpression in PCa cells led to a modest yet significant reduction in tumor size, while its CRD-defective mutant form significantly increased tumor growth compared to controls. Taken together, these results suggest that although de novo expression of gal-7 may be an interesting means of increasing the tumorigenic phenotypes of PCa cells, alterations in the CRD activity of this protein drive a phenotypic switch in its role in PCa cells. This CRD-independent activity represents a paradigm shift in our understanding of the functions of galectin. The R74S model will be useful to distinguish CRD-dependent and CRD-independent functions of gal-7 in cancer progression.

Mouse mammary stem cells express prognostic markers for triple-negative breast cancer.

IntroductionTriple-negative breast cancer (TNBC) is a heterogeneous group of tumours in which chemotherapy, the current mainstay of systemic treatment, is often initially beneficial but with a high risk of relapse and metastasis. There is currently no means of predicting which TNBC will relapse. We tested the hypothesis that the biological properties of normal stem cells are re-activated in tumour metastasis and that, therefore, the activation of normal mammary stem cell-associated gene sets in primary TNBC would be highly prognostic for relapse and metastasis.MethodsMammary basal stem and myoepithelial cells were isolated by flow cytometry and tested in low-dose transplant assays. Gene expression microarrays were used to establish expression profiles of the stem and myoepithelial populations; these were compared to each other and to our previously established mammary epithelial gene expression profiles. Stem cell genes were classified by Gene Ontology (GO) analysis and the expression of a subset analysed in the stem cell population at single cell resolution. Activation of stem cell genes was interrogated across different breast cancer cohorts and within specific subtypes and tested for clinical prognostic power.ResultsA set of 323 genes was identified that was expressed significantly more highly in the purified basal stem cells compared to all other cells of the mammary epithelium. A total of 109 out of 323 genes had been associated with stem cell features in at least one other study in addition to our own, providing further support for their involvement in the biology of this cell type. GO analysis demonstrated an enrichment of these genes for an association with cell migration, cytoskeletal regulation and tissue morphogenesis, consistent with a role in invasion and metastasis. Single cell resolution analysis showed that individual cells co-expressed both epithelial- and mesenchymal-associated genes/proteins. Most strikingly, we demonstrated that strong activity of this stem cell gene set in TNBCs identified those tumours most likely to rapidly progress to metastasis.ConclusionsOur findings support the hypothesis that the biological properties of normal stem cells are drivers of metastasis and that these properties can be used to stratify patients with a highly heterogeneous disease such as TNBC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0539-6) contains supplementary material, which is available to authorized users.

Abstract 209: Pharmacological inhibition of protein kinase C alpha (PKCα) and all trans retinoic acid (ATRA) synergize to inhibit the proliferation, migration and cancer stem-like properties of a triple-negative mammary cancer model

Abstract To study the interaction between PKCα and retinoic acid pathways on the biology of breast cancer, we employed a murine mammary triple negative cell model (LM38-LP, composed by luminal (LEP), myoepithelial (MEP) and cancer stem cells (CSC). We proposed to: A) Study the effect of ATRA (1μM) on the expression of PKCα and Retinoic Acid Receptors (RARs) in LEP, MEP and CSC (mammospheres); B) Evaluate the combined effect of ATRA and a PKCα pharmacological inhibitor (1 uM Go6976) on cell proliferation employing a method to determine the interaction type; C) Analyze the effect of ATRA/PKCα inhibitor on migration and MMP activity on LM38-LP;. D) Analyze the effect of ATRA/PKCα inhibitor on proliferation, self-renewal and differentiation of CSC; E) Study the expression profile of pluripotent genes in CSC and their modulation by treatments. By RT-PCR we found that ATRA (48 h) induced a decrease in PKCα in LEP, MEP and CSC. The same treatment increased RARβ2 and RARγ2 in CSC, and only RARβ2 in LEP. ATRA and PKCα inhibitor interaction results from the proliferation assay were analyzed by Chou-Talalay´s method. We found that the inhibitory effect exerted by ATRA/PKCα inhibitor was synergistic with CI=0.59 (Synergistic with CI&amp;lt;0.7). ATRA/PKCα inhibitor treatment reduced LM38-LP migration more efficiently than each treatment alone, showing a synergic effect (% migration: Control: 80,1±6,9, ATRA: 55,4±5,6, PKCα inhibitor: 37,3±9,5, ATRA/PKCα inhibitor: 20,0±1,7; p≤0.05). Furthermore, MMP2 activity was strongly reduced by the combined treatment. Interestingly, we observed that only the combined treatment induced a decrease of RARγ2 expression in the highly migratory MEP cells. ATRA/PKCα inhibitor treatment synergized to reduce mammospheres growth (Diameter in µm at 96h: Control: 176±8, ATRA: 129±10; PKCα inhibitor: 130±11 ATRA/PKCα inhibitor: 103±5 p≤0.05). Pre-treatment with ATRA/PKCα inhibitor for 96h dramatically affected CSC self-renewal (Number of secondary mammospheres: Control: 313±19; ATRA/PKCα inhibitor: 69±21; p≤0.05). While in a 3D matrigel culture assay ATRA-pretreated CSC formed organized colonies with presence of lumen, the combined treatment led to the formation of small undifferentiated structures and evidence of cell death. By RT-PCR we determined that CSC expressed higher levels of pluripotent genes, such as Nanog, Sox2, Slug and Sox9, than the parental LM38-LP cell line. Besides, only the combined treatment for 96 h induced a decrease in the levels of Nanog and Slug in CSC. Our findings suggest that the pharmacological inhibition of PKCα and ATRA synergize to inhibit proliferation and migration possibly through the decrease of RARγ and the inhibition of MMP2 activity. Furthermore, the blockage of CSC expansion by the combined treatment, possibly occurred through the down regulation of Nanog and Slug. Citation Format: Damian E. Berardi, Maria I. Diaz Bessone, Carolina Flumian, Stefano M. Cirigliano, Elisa D. Bal de Kier Joffe, Alejandro J. Urtreger, Laura B. Todaro. Pharmacological inhibition of protein kinase C alpha (PKCα) and all trans retinoic acid (ATRA) synergize to inhibit the proliferation, migration and cancer stem-like properties of a triple-negative mammary cancer model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 209. doi:10.1158/1538-7445.AM2014-209

The Dog as a Natural Animal Model for Study of the Mammary Myoepithelial Basal Cell Lineage and its Role in Mammary Carcinogenesis

Basal-like tumours constitute 2-18% of all human breast cancers (HBCs). These tumours have a basal myoepithelial phenotype and it has been hypothesized that they originate from either myoepithelial cells or mammary progenitor cells. They are heterogeneous in morphology, clinical presentation, outcome and response to therapy. Canine mammary carcinomas (CMCs) have epidemiological and biological similarities to HBCs, are frequently biphasic and are composed of two distinct neoplastic populations (epithelial and myoepithelial). The present study evaluates the potential of CMCs as a natural model for basal-like HBCs. Single and double immunohistochemistry was performed on serial sections of 10 normal canine mammary glands and 65 CMCs to evaluate expression of cytokeratin (CK) 8/18, CK5, CK14, α-smooth muscle actin (SMA), calponin (CALP), p63 and vimentin (VIM). The tumours were also evaluated for Ki67 and human epidermal growth factor receptor (HER)-2 expression. A hierarchical model of cell differentiation was established, similar to that for the human breast. We hypothesized that progenitor cells (CK5(+), CK14(+), p63(+) and VIM(+)) differentiate into terminally-differentiated luminal glandular (CK8/18(+)) and myoepithelial (CALP(+), SMA(+) and VIM(+)) cells via intermediary luminal glandular cells (CK5(+), CK14(+) and CK8/CK18(+)) and intermediary myoepithelial cells (CK5(+), CK14(+), p63(+), SMA(+), CALP(+) and VIM(+)). Neoplastic myoepithelial cells in canine complex carcinomas had labelling similar to that of terminally-differentiated myoepithelial cells, while those of carcinomas-and-malignant myoepitheliomas with a more aggressive biological behaviour (i.e. higher frequency of vascular/lymph node invasion and visceral metastases and higher risk of tumour-related death) were comparable with intermediary myoepithelial cells and had significantly higher Ki67 expression. The majority of CMCs examined were negative for expression of HER-2. The biphasic appearance of CMCs with involvement of the myoepithelial component in different stages of cell differentiation may help to define the role of myoepithelial cells in the mammary carcinogenetic process and the heterogeneous nature of basal-like HBCs.

The CCAAT/Enhancer-Binding Protein Beta-2 Isoform (CEBPβ-2) Upregulates Galectin-7 Expression in Human Breast Cancer Cells

Galectin-7 is considered a gene under the control of p53. However, elevated expression of galectin-7 has been reported in several forms of cancer harboring an inactive p53 pathway. This is especially true for breast cancer where galectin-7 expression is readily expressed in a high proportion in basal-like breast cancer tissues, conferring cancer cells with increased resistance to cell death and metastatic properties. These observations suggest that other transcription factors are capable of inducing galectin-7 expression. In the present work, we have examined the role of CCAAT/enhancer-binding protein beta (C/EBPβ) in inducing expression of galectin-7. C/EBP proteins have been shown to contribute to breast cancer by upregulating pro-metastatic genes. We paid particular attention to C/EBPβ-2 (also known as LAP2), the most transcriptionally active of the C/EBPβ isoforms. Our results showed that ectopic expression of C/EBPβ-2 in human breast cancer cells was sufficient to induce expression of galectin-7 at both the mRNA and protein levels. In silico analysis further revealed the presence of an established CEBP element in the galectin-7 promoter. Mutation of this binding site abolished the transcriptional activity of the galectin-7 promoter. Chromatin immunoprecipitation analysis confirmed that C/EBPβ-2 binds to the endogenous galectin-7 promoter. Analysis of galectin-7 protein expression in normal epithelia and in breast carcinoma by immunohistochemistry further showed the expression pattern of C/EBPβ closely micmicked that of galectin-7, most notably in mammary myoepithelial cells and basal-like breast cancer where galectin-7 is preferentially expressed. Taken together, our findings suggest that C/EBPβ is an important mediator of galectin-7 gene activation in breast cancer cells and highlight the different transcriptional mechanisms controlling galectin-7 in cancer cells.