Murine amniotic fluid stem cells contribute mesenchymal but not epithelial components to reconstituted mammary ducts

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

The inhibition of Stat5 by a peptide aptamer ligand specific for the DNA binding domain prevents target gene transactivation and the growth of breast and prostate tumor cells

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

STAT3 Induction of miR-146b Forms a Feedback Loop to Inhibit the NF-kB to IL-6 Signaling Axis and STAT3-Driven Cancer Phenotypes

Interleukin-6 (IL-6)–mediated activation of signal transducer and activator of transcription 3 (STAT3) is a mechanism by which chronic inflammation can contribute to cancer and is a common oncogenic event. We discovered a pathway, the loss of which is associated with persistent STAT3 activation in human cancer. We found that the gene encoding the tumor suppressor microRNA miR-146b is a direct STAT3 target gene, and its expression was increased in normal breast epithelial cells but decreased in tumor cells. Methylation of the miR-146b promoter, which inhibited STAT3-mediated induction of expression, was increased in primary breast cancers. Moreover, we found that miR-146b inhibited nuclear factor kB (NF-kB)–dependent production of IL-6, subsequent STAT3 activation, and IL-6/STAT3–driven migration and invasion in breast cancer cells, thereby establishing a negative feedback loop. In addition, higher expression of miR-146b was positively correlated with patient survival in breast cancer subtypes with increased IL6 expression and STAT3 phosphorylation. Our results identify an epigenetic mechanism of crosstalk between STAT3 and NF-kB relevant to constitutive STAT3 activation in malignancy and the role of inflammation in oncogenesis

Rapid validation of cancer genes in chimeras derived from established genetically engineered mouse models

Recent technological advances have opened the door for the fast and cost-effective generation of genetically engineered mouse models (GEMMs) to study cancer. We describe here a conceptually novel approach for the generation of chimeric GEMMs based on the controlled introduction of various genetic elements in embryonic stem cells (ESCs) that are derived from existing mouse strains with a predisposition for cancer. The isolation of GEMM-derived ESC lines is greatly facilitated by the availability of the newly defined culture media containing inhibitors that effectively preserve ESC pluripotency. The feasibility of the GEMM-ESC approach is discussed in light of current literature and placed into the context of existing models. This approach will allow for fast and flexible validation of candidate cancer genes and drug targets and will result in a repository of GEMM-ESC lines and corresponding vector collections that enable easy distribution and use of preclinical models to the wider scientific community

Mammary epithelial reconstitution with gene-modified stem cells assigns roles to Stat5 in luminal alveolar cell fate decisions, differentiation, involution and mammary tumor formation

The mammary gland develops mainly postnatally and contains a small fraction of mammary stem cells (MaSCs), required for the generation and maintenance of its epithelium. The mouse mammary gland represents a unique model system to study gene functions in adult, solid-organ stem cells. In addition, breast cancer is thought to originate from cancer stem cells (CSCs) emerging from transformed MaSCs or progenitor cells with acquired selfrenewal capacity. The identification of the genes and signaling pathways regulating these processes in MaSCs is limited by the lack of specific markers, which would allow the purification of these cells. In spite of this limitation, stem cell activity can be measured by the ability to reconstitute an entire functional mammary epithelium upon transplantation of primary mammary epithelial cells (MECs) into cleared fat pads. The optimization of lentiviral transduction procedure for adherent primary MECs is presented in this work. It resulted in very efficient transduction of MECs including the stem cell fraction. Gene modified MECs were introduced into cleared fat pads of immunodeficient Rag2-/-γc-/- mice to investigate the functional potential of MaSCs. The frequency of MaSC repopulation was mouse strain dependent and reached nearly 100% in immunodeficient recipients. These animals lack an immune response to the encoded fluorescent marker proteins. Transplantation of a mixture of MECs expressing different fluorescent proteins resulted in distinct areas of ductal outgrowths expressing single fluorescent proteins. These ducts grew in a territorially restricted fashion and most likely originated from individual transduced MaSCs. Loss and gain of function studies of the signal transducer and activator of transcription 5 (Stat5) using genetic alteration of MaSCs and mammary epithelial reconstitution allowed insights into the role of Stat5 in mammary gland development and tumor formation. Freshly isolated mouse MECs were modified by transduction with lentiviral vectors encoding either a short hairpin RNA able to downregulate both Stat5 isoforms (shStat5) or a constitutively active variant of Stat5a (cS5-F). The cS5-F mutant carries a point mutation (S710F) and is constitutively tyrosine phosphorylated in the absence of inducing cytokine signals. The downregulation of Stat5 in MaSCs and their progenies did not affect outgrowth of primary ducts, but caused the formation of thinner ducts with reduced side-branching in virgin glands. A more pronounced phenotype was observed during pregnancy when Stat5 knockdown impaired alveologenesis. Conversely, constitutive activation of Stat5a in virgin glands fully replaced the function of lactogenic hormones and caused hyperproliferation of luminal cells, thickening of the ducts and precocious development of functional alveoli. Characterization of the different cell populations present in these transgenic grafts indicated that Stat5 activity regulates proliferation and/or survival of luminal progenitor cells and is sufficient for the emergence of mature alveolar cells from these progenitors. The persistent activation of Stat5 during the involution stage prevented apoptosis of terminally differentiated mammary secretory cells and induced tumorigenic transformation post lactationally with short latencies. These tumors were classified as mammary adenocarcinomas and could be serially transplanted into secondary virgin hosts. The primary and secondary tumors were highly proliferative, expressed activated Stat5 and Stat3. They contained a fraction of Lin-CD24lowCD44+ cells, markers present in CSCs. Microarray analysis of cS5-F-expressing ducts and tumors revealed the enhanced expression of Stat5 target genes. Previously identified target genes and many novel genes, regulated downstream of activated Stat5, were found. These genes are candidates with putative roles in gland development or in malignant transformation. The method established in this work allows the genetic manipulation of stem cells ex vivo.The consequences of genetic alterations can be studied in progenitor and differentiated cells upon transplantation in vivo. This represents a practical alternative to conventional transgenic mouse models to decipher the function of genes during mammary gland development and tumorigenesis.Die Brustdrüse (glandula mammaris) bietet ein einzigartiges Modellsystem zum Studium der adulten Stammzellen und der molekularen Signalwege, welche die Selbsterneuerung dieserZellen sowie die Proliferation und Differenzierung der Vorläuferzellen kontrollieren. Die Brustdrüse besteht aus dem Brustepithel und dem Stroma, das zum größten Teil aus dem Fettgewebe gebaut ist. Es enthält auch andere Zelltypen z. B. Fibroblasten und Makrophagen. Die Entwicklung der Brustdrüse findet hauptsächlich nach der Geburt, während der Pubertät, Schwangerschaft und Laktation statt. Ein funktionelles Brustepithel wird während der aufeinander folgenden Zyklen von Schwangerschaft, Laktation und Abstillen auf- und wieder abgebaut. Diese Regenerations-Kapazität kann für die Organrekonstitution genutzt werden. Die Transplantation der kleinen Anzahl von Brustepithelzellen oder des Drüsenfragments in das Fettgewebe einer Empfängermaus, deren eigenes Brustepithel entfernt wurde (cleared fat pad), führt zur vollständigen Epithelregeneration. Die zyklische Entwicklung und Regenerations-Fähigkeit des Epithelgewebes lässt auf die Existenz von Stammzellen schließen, die im Verbund der Epithelzellen überdauern. Diese gewebespezifischen Stammzellen sind in der Lage sich durch asymmetrische Zellteilung zu erneuern (self-renewal) und gleichzeitig die differenzierenden Vorläuferzellen zu bilden. Die während der Pubertät und Schwangerschaft erhöhten systemischen Hormone, lokalen Wachstumsfaktoren und Zytokine kontrollieren die Stammzellen-Proliferation und die Differenzierung der Vorläuferzellen in den verschiedenen Brustepithel-Zelllinien: Myoepithel-, Luminal- und Alveolarzellen. Aufgrund der Tatsache, dass die Entstehung von Brustkrebs mit aberranten Proliferations- und Differenzierungsprogrammen in malignanten Stamm-/ Vorläuferzellen (cancer stem cells) einhergeht, ist die Identifizierung der Signalwege, die diese Prozesse regulieren, für die Stammzellen- und Krebs-Forschung sehr bedeutend. Basierend auf diesen Erkenntnissen wurden im Rahmen des vorliegenden Projektes die Methoden zur genetischen Manipulation von nicht-angereicherten Brustdrüsen-Stammzellen entwickelt. Durch effiziente lentivirale Transduktion von adhärenten Primärzellen wurden nahezu 90% der Zellen, einschließlich der Stammzellen, transduziert. Diese Optimierung erfolgte durch 1) die Anwendung von konzentrierten Lentiviren mit hoher Qualität, 2) Passagierung der Primärzellen und Entfernung von Gewebeklumpen von den VIII Primärzellkulturen, und besonders 3) durch die Reduzierung der Zelldichte während der viralen Transduktion. Für Brust-Stammzellen sind keine spezifischen Oberflächen-Marker bekannt und daher ist ihre Isolierung deutlich erschwert. Man konnte sie bis jetzt nur anhand der moderaten Expression von CD24 (hitzestabilen Antigen) und hoher Expression von CD49f oder CD29 (α6- oder β1-Integrin) ungefähr 10-fach anreichern. Allerdings haben andere Studien gezeigt, dass die Transplantation der FACS-sortierten Stammzellen zu einer Schädigung der Stammzellen und folglich zu einer Reduktion der Repopulation-Frequenz führen kann. Aus diesem Grund wurden die genetisch modifizierten Stammzellen nicht sortiert. Durch die Transplantationen der transduzierten Primärzellen wurde ihr Stammzellen-Anteil in ihrer natürlichen Nische (cleared fat pad) selektiert. Die transplantierten Stammzellen sind in der Lage duktale Auswüchse zu entwickeln. Mit dieser Strategie konnten Transplantate mit homogener Expression von Fluoreszenz-Markergenen, wie z. B. GFP, erzielt werden. FACS Analysen der Zellen, die aus Transplantaten isoliert werden, haben gezeigt, dass alle drei Brustepithelzell-Populationen, nämlich Luminal-, Basal- und Stammzellen, transduziert waren und GFP exprimierten und daher aus transduzierten Zellen hervor gingen. Die Transplantationen einer Mischung der unterschiedlich fluoreszenzmarkierten Stammzellen ergaben einzelne verzweigte Auswüchse, in denen jeweils nur ein Fluoreszenz-Markerprotein exprimiert wurde. Sie stammen sehr wahrscheinlich von einzelnen transduzierten Stammzellen ab und wachsen jeweils in einem begrenzten Bereich des Brustfettgewebes aus. Die Immun-Antwort der Empfängermäuse gegen Fluoreszenz- Markerproteine könnte das Auswachsen der Transplantate inhibieren. Brustepithelium-Rekonstitutionen waren daher in den Rag2-/-γc-/- Empfängermäusen mit geschwächtem Immunsystem besonders effiziert. Die lentivirale Manipulation von Stammzellen und deren Einsatz in Brustepithelium-Rekonstitutionen kann als alternative Methode zur gewebsspezifischen Knockout-Technik angesehen werden. Für die Etablierung dieser Methode wurde im Rahmen dieser Arbeit ein zentraler Transkriptionsfaktor in der Brustentwicklung, signal transducer and activator of transcription 5 (Stat5), untersucht. (...