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The eleventh annual workshop of the European Network for Breast Development and Cancer, Methods in mammary gland biology and breast cancer, took place on the 16th to 18th of May 2019 in Weggis, Switzerland. The main topics of the meeting were high resolution genomics and proteomics for the study of mammary gland development and cancer, breast cancer signaling, tumor microenvironment, preclinical models of breast cancer, and tissue morphogenesis. Exciting novel findings in, or highly relevant to, mammary gland biology and breast cancer field were presented, with insights into the methods used to obtain them. Among others, the discussed methods included single-cell RNA sequencing, genetic barcoding, lineage tracing, spatial transcriptomics, optogenetics, genetic mouse models and organoids. [ABSTRACT FROM AUTHOR]

Cellular transformation is initiated by the activation of oncogenes and a closely associated developmental reprogramming of the epigenetic landscape. Transcription factors, regulators of chromatin states and microRNAs influence cell fates in development and stabilize the phenotypes of normal, differentiated cells and of cancer cells. The miR-302/367 cluster, predominantly expressed in human embryonic stem cells (hESs), can promote the cellular reprogramming of human and mouse cells and contribute to the generation of iPSC. We have used the epigenetic reprogramming potential of the miR-302/367 cluster to 'de-program' tumor cells, that is, hift their gene expression pattern towards an alternative program associated with more benign cellular phenotypes. Induction of the miR-302/367 cluster in extensively mutated U87MG glioblastoma cells drastically suppressed the expression of transformation related proteins, for example, the reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC, and the transcription factors POU3F2, SALL2 and OLIG2, required for the maintenance of glioblastoma stem-like tumor propagating cells. It also diminished PI3K/AKT and STAT3 signaling, impeded colony formation in soft agar and cell migration and suppressed pro-inflammatory cytokine secretion. At the same time, the miR-302/367 cluster restored the expression of neuronal markers of differentiation. Most notably, miR-302/367 cluster expressing cells lose their ability to form tumors and to establish liver metastasis in nude mice. The induction of the miR-302/367 cluster in U87MG glioblastoma cells suppresses the expression of multiple transformation related genes, abolishes the tumor and metastasis formation potential of these cells and can potentially become a new approach for cancer therapy. [ABSTRACT FROM AUTHOR]

Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl+ K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells, Stat5 is predominantly present in the cytoplasm and the survival of the Jak2(V617F)+ HEL cells is impeded through the inhibition of the cytoplasmic functions of Stat5. [ABSTRACT FROM AUTHOR]

The signal transducer and activator of transcription Stat5 is transiently activated by growth factor and cytokine signals in normal cells, but its persistent activation has been observed in a wide range of human tumors. Aberrant Stat5 activity was initially observed in leukemias, but subsequently also found in carcinomas. We investigated the importance of Stat5 in human tumor cell lines. shRNA mediated downregulation of Stat5 revealed the dependence of prostate and breast cancer cells on the expression of this transcription factor. We extended these inhibition studies and derived a peptide aptamer (PA) ligand, which directly interacts with the DNA-binding domain of Stat5 in a yeast-two-hybrid screen. The Stat5 specific PA sequence is embedded in a thioredoxin (hTRX) scaffold protein. The resulting recombinant protein S5-DBD-PA was expressed in bacteria, purified and introduced into tumor cells by protein transduction. Alternatively, S5-DBD-PA was expressed in the tumor cells after infection with a S5-DBD-PA encoding gene transfer vector. Both strategies impaired the DNA-binding ability of Stat5, suppressed Stat5 dependent transactivation and caused its intracellular degradation. Our experiments describe a peptide based inhibitor of Stat5 protein activity which can serve as a lead for the development of a clinically useful compound for cancer treatment. [ABSTRACT FROM AUTHOR]

Breast cancer patients with disseminated metastatic disease still have a very unfavorable prognosis. Investigations into the molecular mechanisms that underlie metastasis formation have a high priority and can possibly result in improved therapeutic interventions. The process of oncogenic epithelial to mesenchymal transition (EMT) has recently become a focus in cancer research because it encompasses many of the phenotypic traits characteristic of metastatic cells, e.g., increased motility, invasion, anoikis resistance, immunosuppression, and cancer stem cell potential. A number of central cellular signaling pathways and transcription factors have been implied in the control of EMT and metastasis formation, among them signal originating from the activation of the transforming growth factor β (TGFβ), epithelial growth factor, Wnt, Notch, and Hedgehog pathways. We have investigated the contribution of TGFβ signaling to metastasis-related cellular properties. TGFβ signaling can have tumor-suppressive and -promoting effects depending on the tumor type and the stage of tumor progression. TGFβ can inhibit the proliferation of mammary epithelial cells (MECs), but it can also induce EMT, invasion, and metastasis, possibly through Smad-independent signaling events. We investigated the effects of TGFβ pathway inhibition on the proliferation, differentiation, and invasion of both normal and malignant MECs. shRNA-mediated downregulation of the Smad4 protein in non-tumorigenic HC11 and tumorigenic 4T1 cells promotes the invasiveness of both cell lines. Mammary gland reconstitution studies, with primary MECs expressing shSmad4, resulted only in subtle effects on the glandular morphogenesis. Orthotopic transplantation of shSmad4-transduced 4T1 tumor cells caused the accelerated growth of mammary tumors and enhanced colonization and macroscopic lung metastases when compared to control cells. Surprisingly, the expression of Smad4 was restored, and a strong activation of Stat3 was found in the metastatic lesions present in the lungs. These lesions express metastatic factors, such as angiopoietin-like-4 and the inhibitor of DNA binding/differentiation 1. We suggest that the downregulation of Smad4 inhibits the tumor-suppressive effects of TGFβ signaling and enhances tumor growth. The downregulation, however, was only transient, and the reactivation of Smad4 expression caused the reversal of EMT, mesenchymal to epithelial transition, and thereby promoted metastasis formation in the lungs. [ABSTRACT FROM AUTHOR]

MicroRNAs are small noncoding RNAs that carry out post-transcriptional regulation of the expression of their target genes. However, their roles in mammalian organogenesis are only beginning to be understood. Here we show that the microRNA-212/132 family (which comprises miR-212 and miR-132) is indispensable during the development of the mammary glands in mice, particulary for the regulation of the outgrowth of the epithelial ducts. Mammary transplantation experiments revealed that the function of the miR-212/132 family is required in the stroma but not in the epithelia. Both miR-212 and miR-132 are expressed exclusively in mammary stroma and directly target the matrix metalloproteinase MMP-9. In glands that lack miR-212 and miR-132, MMP-9 expression increases and accumulates around the ducts. This may interfere with collagen deposition and lead to hyperactivation of the tumor growth factor-β signaling pathway, thereby impairing ductal outgrowth. Our results identify the miR-212/132 family as one of the main regulators of the epithelial-stromal interactions that are required for proper pubertal development of the mammary gland. [ABSTRACT FROM AUTHOR]

The mammary gland represents a unique model system to study gene functions in adult stem cells. Mammary stem cells (MaSCs) can regenerate a functional epithelium on transplantation into cleared fat pads. We studied Ihe consequences of distinct genetic modifications of MaSCs on their repopulation and differentiation ability. The reconstitution of ductal trees was used as a stem ceil selection procedure and the nearly quantitative lentiviral infection efficiency of the primary mammary epithelial cells (MECs) rendered the enrichment of MaSCs hefore their transplantation unnecessary. The repopulation frequency of transduced MaSCs was nearly 100% in immunodeficient recipients and the resulting transgenic ducts homogeneously expressed the virally encoded fluorescent marker proteins. Transplantation of a mixture of MECs, expressing different fluorescent proteias, resulted in a distinct pattern of ductal outgrowths originating from a small number of individually transduced MaSCs. We used genetically modified MECs lo define multiple functions of Stat5 during mammary gland development and differentiation. Stat5-downregulation in MaSCs did not affect primary ductal outgrowth, hut impaired side branching and the emergence of mature alveolar cells from luminal progenitors during pregnancy. Conversely, the expression of a constitutively active variant of Stat5 (cS5-F) caused epithelial hyperproliferation, thickening of the ducts and precocious, functional alveoli formation in virgin mice. Expression of cS5-E also prevented involution and caused the formation of estrogen and progesterone receptor positive (ER+PR+) adenocarcinomas. The tumors expressed activated Stat5 and Stat3 and contained a small fraction of CD44+ cells, possibly indicative of cancer stem cells. [ABSTRACT FROM AUTHOR]

Introduction: Amniotic fluid harbors cells indicative of all three germ layers, and pluripotent fetal amniotic fluid stem cells (AFSs) are considered potentially valuable for applications in cellular therapy and tissue engineering. We investigated whether it is possible to direct the cell fate of AFSs in vivo by transplantation experiments into a particular microenvironment, the mammary fat pad. This microenvironment provides the prerequisites to study stem cell function and the communication between mesenchymal and epithelial cells. On clearance of the endogenous epithelium, the ductal tree can be reconstituted by the transfer of exogenously provided mammary stem cells. Analogously, exogenously provided stem cells from other tissues can be investigated for their potential to contribute to mammary gland regeneration. Methods: We derived pluripotent murine AFSs, measured the expression of stem cell markers, and confirmed their in vitro differentiation potential. AFSs were transplanted into cleared and non cleared fat pads of immunocompromised mice to evaluate their ability to assume particular cell fates under the instructive conditions of the fat-pad microenvironment and the hormonal stimulation during pregnancy. Results: Transplantation of AFSs into cleared fat pads alone or in the presence of exogenous mammary epithelial cells caused their differentiation into stroma and adipocytes and replaced endogenous mesenchymal components surrounding the ducts in co-transplantation experiments. Similarly, transplantation of AFSs into fat pads that had not been previously cleared led to AFS-derived stromal cells surrounding the elongating endogenous ducts. AFSs expressed the marker protein α-SMA, but did not integrate into the myoepithelial cell layer of the ducts in virgin mice. With pregnancy, a small number of AFS-derived cells were present in acinar structures. Conclusions: Our data demonstrate that the microenvironmental cues of the mammary fat pad cause AFSs to participate in mammary gland regeneration by providing mesenchymal components to emerging glandular structures, but do not incorporate or differentiate into ductal epithelial cells. [ABSTRACT FROM AUTHOR]

Background: Signal transducer and activator of transcription 3 and 5 (Stat3 and Stat5) play important roles in cell differentiation, proliferation, apoptosis and inflammation. They are transiently activated by ligand-receptor interactions in normal cells but are often found to be constitutively active in cancer cells. Analysis of their activation pattern is therefore important for the description of developmental processes and the understanding of cellular transformation. Materials and methods: To visualize Stat3 and Stat5 transactivation activity in different cell types, we designed novel reporter constructs. These constructs comprise Stat3 or Stat5 specific promoter elements and reporter genes encoding bgalactosidase or fluorescent proteins. These constructs were integrated into lentiviral gene transfer vectors facilitating efficient transduction of most cell types. Results: The lentiviral reporter constructs were used to infect different cell types and their inducibility by activated Stat3 or Stat5 was measured. The Stat3-mCherry reporter was active in transduced tumor cells, which exhibit high levels of phosphorylated Stat3 and it was inducible in HepG2 liver cells by interleukin-6 treatment. The Stat5-LacZ reporter was active in cultured cells upon hormone induction of Stat5 and in primary mammary epithelial cells transplanted into cleared fat pads of mice during late pregnancy. Conclusion: These novel reporter constructs are valuable tools to investigate and to distinguish between Stat3 and Stat5 activity in primary cells and cancer cells. They will also be useful in the discovery of drugs targeting Stat3 or Stat5. They can also be employed to generate transgenic mice and track Stat activity during development. [ABSTRACT FROM AUTHOR]

The mouse mammary gland represents a unique model for the study of growth, differentiation and the control of organogenesis. It consists of branching epithelial ducts that are embedded in the fat pad displaying functional differentiation during pregnancy and lactation [1]. The transplantation of isolated mammary epithelial cells into cleared fat pads results in the formation of ductal trees and functional alveoli [2]. The focus of our investigation is the contribution of individual genes to the functional development of the epithelial compartment in this organ. The signal transducer and activator of transcription 5 (Stat5) is a key regulatory transcription factor in mammary gland development and differentiation [3]. However, the contribution of Stat5 for the self-renewal capacity and the repopulation potential of mammary epithelial cells and its role in breast cancer remains poorly understood. Freshly isolated mouse mammary epithelial cells were genetically modified by transduction with lentiviral gene-transfer vectors encoding a Stat5 specific small hairpin RNA (shStat5) or a constitutively active variant of Stat5 (cS5F). This mutant is constitutively phosphorylated on Y964 and activated in the absence of inducing cytokine signals [4]. This approach allowed us to target a small fraction of stem cells within the total population of isolated mammary epithelial cells. The transduced cells were transplanted into the mammary fat pads of 3-weeks old mice cleared of endogenous epithelium. The consequences of the downregulation or upregulation of Stat5 in mammary epithelial stem cells on their repopulation capacity and functional differentiation was assessed in virgin and pregnant mice. Stat5-deficient cells gave rise to ductal structures, similar to the ones observed in virgin mice, but appeared narrower with less branching. More pronounced differences were observed during pregnancy, where Stat5 deficient cells failed to undergo lobulo-alveolar differentiation. Conversely, the upregulation of Stat5 caused a dramatic thickening of the primary ducts in virgin mice and precocious development of alveoli. In conclusion, we have shown that the lentiviral vector mediated loss of function and gain of function of specific genes in mammary stem cells permits rapid and efficient analysis of gene function in mammary gland development. [ABSTRACT FROM AUTHOR]

iRGD is a derivative of the integrin-binding peptide RGD, which selectively increases the penetrability of tumor tissue to various coadministered substances in several preclinical models. In this study, we investigated the ability of iRGD to improve the delivery of sorafenib and doxorubicin therapy in hepatocellular carcinoma (HCC) using established mouse models of the disease. A contrast-enhanced MRI method was developed in parallel to assess the in vivo effects of iRGD in this setting. We found that iRGD improved the delivery of marker substances to the tumors of HCC-bearing mice about three-fold without a parallel increase in normal tissues. Control peptides lacking the critical CendR motif had no effect. Similarly, iRGD also selectively increased the signal intensity from tumors in Gd-DTPA-enhanced MRI. In terms of antitumor efficacy, iRGD coadministration significantly augmented the individual inhibitory effects of sorafenib and doxorubicin without increasing systemic toxicity. Overall, our results offered a preclinical proof of concept for the use of iRGD coadministration as a strategy to widen the therapeutic window for HCC chemotherapy, as monitored by Gd-DTPA-enhanced MRI as a noninvasive, clinically applicable method to identify iRGD-reactive tumors. [ABSTRACT FROM AUTHOR]

Maturation of dendritic cells (DCs) is required to induce T cell immunity, whereas immature DCs can induce immune tolerance. Although the transcription factor STAT5 is suggested to participate in DC maturation, its role in this process remains unclear. In this study, we investigated the effect of STAT5 inhibition on LPS-induced maturation of human monocyte-derived DCs (Mo-DCs). We inhibited STAT5 by treating Mo-DCs with JQ1, a selective inhibitor of BET epigenetic readers, which can suppress STAT5 function. We found that JQ1 inhibits LPS-induced STAT5 phosphorylation and nuclear accumulation, thereby attenuating its transcriptional activity in Mo-DCs. The diminished STAT5 activity results in impaired maturation of Mo-DCs, as indicated by defective upregulation of costimulatory molecules and CD83, as well as reduced secretion of IL-12p70. Expression of constitutively activated STATS in JQl-treated Mo-DCs overcomes the effects of JQ1 and enhances the expression of CD86, CD83, and IL-12. The activation of STATS in Mo-DCs is mediated by GM-CSF produced following LPS stimulation. Activated STAT5 then leads to increased expression of both GM-CSF and GM-CSFR, triggering an autocrine loop that further enhances STAT5 signaling and enabling Mo-DCs to acquire a more mature phenotype. JQ1 decreases the ability of Mo-DCs to induce allogeneic CD4+ and CDS T cell proliferation and production of proinflammatory cytokines. Furthermore, JQ1 leads to a reduced generation of inflammatory CD8+ T cells and decreased Thl differentiation. Thus, JQ1 impairs LPS-induced Mo-DC maturation by inhibiting STAT5 activity, thereby generating cells that can only weakly stimulate an adaptive-immune response. Therefore, JQ1 could have beneficial effects in treating T cell-mediated inflammatory diseases. [ABSTRACT FROM AUTHOR]

Using a retrovirus-mediated cDNA expression cloning approach, we identified the grainyhead-like 2 (GRHL2) transcription factor as novel protooncogene. Overexpression of GRHL2 in NIH3T3 cells induced striking morphological changes, an increase in cell proliferation, anchorage-independent growth, and tumor growth in vivo.By combining a microarray analysis and a phylogenetic foot printing analysis with various biochemical assays, we identified the epidermal growth factor receptor family member Erbb3 as a novel GRHL2 target gene. In breast cancer cell lines, shRNA-mediated knockdown ofGRHL2 expression or functional inactivation ofGRHL2 using dominant negativeGRHL2 proteins induces down-regulation of ERBB3 gene expression, a striking reduction in cell proliferation, and morphological and phenotypical alterations characteristic of an epithelial-to-mesenchymal transition (EMT), thus implying contradictory roles of GRHL2 in breast carcinogenesis. Interestingly, we could further demonstrate that expression of GRHL2 is directly suppressed by the transcription factor zinc finger enhancer-binding protein 1 (ZEB1), which in turn is a direct target for repression by GRHL2, suggesting that the EMT transcription factors GRHL2 and ZEB1 form a double negative regulatory feedback loop in breast cancer cells. Finally, a comprehensive immunohistochemical analysis of GRHL2 expression in primary breast cancers showed loss ofGRHL2 expression at the invasive front of primary tumors. A pathophysiological relevance of GRHL2 in breast cancer metastasis is further demonstrated by our finding of a statistically significant association between loss of GRHL2 expression in primary breast cancers and lymph node metastasis. We thus demonstrate a crucial role of GRHL2 in breast carcinogenesis. [ABSTRACT FROM AUTHOR]