Putting the brakes on mammary tumorigenesis

Interferone sind wichtige Signalmoleküle des Immunsystems die es den Zellen eines Organismus ermöglichen zielgerecht auf Pathogene wie Viren, Parasiten, Bakterien aber auch Tumorzellen zu reagieren. Der Transkriptionsfaktor STAT1 (signaltransducer and activator of transcription 1) spielt eine zentrale Rolle in der Signalübertragung durch Interferone und ist daher Voraussetzung für ein voll funktionstüchtiges Immunsystem. STAT1 wird ebenfalls für die effiziente zytotoxische Aktivität von T Zellen (CTLs) sowie natürliche Killerzellen (NK Zellen) benötigt. Funktionelle Deletionen oder Mutationen des STAT1 Genes in Mäusen aber auch Menschen äußern sich in einer Hypersensibilität gegenüber viralen und bakteriellen Infektionen. Abgesehen von seiner Schlüsselfunktion im Immunsystem wird STAT1 aber auch eine wichtige Rolle als Tumor-Suppressor zugeschrieben. Eine Brustkrebsstudie an Frauen konnte eine hohe Expression und Aktivität von STAT1 mit einem längeren und rezidivfreien Überleben der Patientinnen positiv korrelieren. Mausmodelle mit ErbB2-induzierten Brusttumoren zeigten außerdem, dass die totale oder die gewebsspezifische Deletion von STAT1 zu einer verkürzten Latenz der Tumorentstehung führt. Diese Erkenntnisse weisen darauf hin, dass STAT1 eine wichtige Rolle in der Brustkrebsentstehung besitzt. Die genaue Funktion von STAT1 im Brustgewebe und der Mechanismus, wie STAT1 die Brustkrebsentstehung unterdrückt, konnten bislang jedoch noch nicht aufgeklärt werden. Im Rahmen meiner Dissertation konnte ich anhand von Mausmodellen zeigen, dass der Verlust von STAT1 ausreicht, um Brustkrebs auszulösen. Stat1-/- Mäuse entwickelten spontane schwangerschaftsvermittelte Brusttumore mit einer generell höheren Inzidenz und verkürzten Latenz als Stat1+/+ Kontrolltiere. Die Wichtigkeit von STAT1 in der Tumorentstehung wurde auch dadurch bestätigt, da STAT1-positive Brusttumore der Kontrollgruppe eine mosaikartige Expression und einen teilweisen Verlust des STAT1 Proteins aufwiesen. Stat1-/- Tumore zeigten eine heterogene Histopathologie und kein einheitliches Expressionsmuster des Estrogenrezeptors (ER) oder des humanen epithelialen Wachstumsfaktors 2 (HER2). Dies bedeutet, dass sich die tumorsuppressive Funktion von STAT1 nicht auf einen bestimmten onkogenen Treiber beschränkt, sondern dass STAT1 als globaler Brusttumorsuppressor wirkt. Die Transplantation von Stat1-/- Brustgewebe in Stat1+/+ Mäuse und vice versa zeigte, dass STAT1 im Immunsystem UND im Brustgewebe wichtig für die Unterdrückung der Krebsentstehung ist. Zytotoxische T Zellen wurden als Hauptmediatoren in der Überwachung von Brusttumoren identifiziert; NK Zellen spielen hierbei nur eine untergeordnete Rolle. Zusätzlich konnte ich zeigen, dass STAT1 im Brustepithel für die Aufrechterhaltung der Wachstumskontrolle zuständig ist - ein Effekt der über IRF1, einen Downstream-Faktor von STAT1 vermittelt wird. Sowohl transformierte als auch primäre Zellen des Brustepithels wiesen eine erhöhte Teilungsrate im Vergleich zu Wildtyp-Kontrollen auf. In 3D Kulturen bildeten Stat1-/- und Irf1-/- Brustepithelzellen unstrukturierte Mammospheren. Übereinstimmend damit fanden sich auffällig häufig sogenannte “mammary intraepithelial neoplasias” (MINs) in den Stat1-/- Mäusen. MINs zeichnen sich durch abnormes Zellwachstum aus und stellen ein präkarzinogenes Stadium dar, welches das Potential besitzt sich in invasiven Brustkrebs weiterzuentwickeln. Zusammenfassend kam ich durch meine Forschungsergebnisse zu dem eindeutigen Schluss, dass der Transkriptionsfaktor STAT1 die Brustkrebsentstehung über zwei unabhängige Mechanismen unterdrückt: Einerseits durch die Aufrechterhaltung einer effizienten Tumorüberwachung mittels zytotoxischer T Zellen, und andererseits durch die Wachstumskontrolle von Zellen des Brustepithels, welche über den Transkriptionsfaktor IRF1 vermittelt wird.Interferons are important signaling molecules of the immune system that allow cells of an organism to react specifically to pathogens such as viruses, parasites, bacteria but also tumor cells. The transcription factor STAT1 (signal transducer and activator of transcription 1) is an essential mediator of interferon signaling and therefore a prerequisite for a fully functional immune system. STAT1 is also required for the efficient cytotoxic activity of T cells (CTLs) and natural killer (NK) cells. Functional deficiency or mutation of STAT1 leads to hypersensitivity to viral and bacterial infections in both mice and men. Besides being a key factor in immune regulation, STAT1 is also considered as an important tumor suppressor. A breast cancer study correlated a high expression and activation of STAT1 positively with an overall longer and relapse-free survival of cancer patients. In mouse models of ErbB2-induced breast cancer the complete but also the tissue specific deletion of STAT1 results in a decreased latency of tumor formation. These observations indicate STAT1 as important safeguard in mammary cancer development. However, the exact role of STAT1 in the mammary gland tissue and the mechanism how STAT1 confers mammary tumor suppression had not yet been clarified. During my PhD thesis I revealed that loss of STAT1 suffices to promote mammary tumor formation in mice. Stat1-/- mice developed spontaneous pregnancy-induced mammary tumors with an overall higher incidence and shorter latency when compared to Stat1+/+ controls. The importance of STAT1 in tumor suppression was further confirmed as STAT1-positive tumors of the control group showed a mosaic expression and partially down-regulation of STAT1 protein. Stat1-/- tumors displayed a heterogeneous histopathology and did not show a particular pattern of expression of the estrogen receptor (ER) or human epidermal growth factor receptor 2 (HER2). This identifies STAT1 as a global mammary tumor suppressor that acts independent of a distinct oncogenic driver. The transplantation of Stat1-/- mammary tissue into wild type mice and vice versa linked the tumor suppressing role of STAT1 to both, the immune system AND the mammary tissue. Cytotoxic T cells were defined as the main executors of mammary tumor surveillance, whereas NK cells play only a marginal role. I further demonstrated that STAT1 is responsible for maintaining growth control in the mammary epithelium - an effect which is mediated by IRF1, a downstream-factor of STAT1. Both transformed and primary Stat1-/- mammary epithelial cells displayed increased growth rates compared to wild type controls. Further, STAT1- and IRF1-deficiency resulted in the formation of unstructured mammospheres in 3D cultures. Consistently, Stat1-/- mice developed mammary intraepithelial neoplasias (MINs) with high frequency. MINs are characterized by abnormal growth of mammary epithelial cells and represent a potential pre-carcinogenic stage as they have a high potential to develop into invasive breast cancer. In summary I unequivocally conclude from my data, that STAT1 controls mammary tumor formation in a dual way: On the one hand by sustaining efficient cytotoxic T cell surveillance and on the other hand by maintaining growth control of mammary epithelial cells. The growth inhibitory effect of STAT1 might be exerted via the transcription factor IRF1

Overexpression of primary microRNA 221/222 in acute myeloid leukemia

BACKGROUND: Acute myeloid leukemia (AML) is a hematopoietic malignancy with a dismal outcome in the majority of cases. A detailed understanding of the genetic alterations and gene expression changes that contribute to its pathogenesis is important to improve prognostication, disease monitoring, and therapy. In this context, leukemia-associated misexpression of microRNAs (miRNAs) has been studied, but no coherent picture has emerged yet, thus warranting further investigations. METHODS: The expression of 636 human miRNAs was compared between samples from 52 patients with AML and 13 healthy individuals by highly specific locked nucleic acid (LNA) based microarray technology. The levels of individual mature miRNAs and of primary miRNAs (pri-miRs) were determined by quantitative reverse transcriptase (qRT) PCR. Transfections and infections of human cell lines were performed using standard procedures. RESULTS: 64 miRNAs were significantly differentially expressed between AML and controls. Further studies on the clustered miRNAs 221 and 222, already known to act as oncogenes in other tumor types, revealed a deficiency of human myeloid cell lines to process vector derived precursor transcripts. Moreover, endogenous pri-miR-221/222 was overexpressed to a substantially higher extent than its mature products in most primary AML samples, indicating that its transcription was enhanced, but processing was rate limiting, in these cells. Comparison of samples from the times of diagnosis, remission, and relapse of AML demonstrated that pri-miR-221/222 levels faithfully reflected the stage of disease. CONCLUSIONS: Expression of some miRNAs is strongly regulated at the posttranscriptional level in AML. Pri-miR-221/222 represents a novel molecular marker and putative oncogene in this disease

Conditional Stat1 Ablation Reveals the Importance of Interferon Signaling for Immunity to Listeria monocytogenes Infection

Signal transducer and activator of transcription 1 (Stat1) is a key player in responses to interferons (IFN). Mutations of Stat1 cause severe immune deficiencies in humans and mice. Here we investigate the importance of Stat1 signaling for the innate and secondary immune response to the intracellular bacterial pathogen Listeria monocytogenes (Lm). Cell type-restricted ablation of the Stat1 gene in naïve animals revealed unique roles in three cell types: macrophage Stat1 signaling protected against lethal Lm infection, whereas Stat1 ablation in dendritic cells (DC) did not affect survival. T lymphocyte Stat1 reduced survival. Type I IFN (IFN-I) signaling in T lymphocytes reportedly weakens innate resistance to Lm. Surprisingly, the effect of Stat1 signaling was much more pronounced, indicating a contribution of Stat1 to pathways other than the IFN-I pathway. In stark contrast, Stat1 activity in both DC and T cells contributed positively to secondary immune responses against Lm in immunized animals, while macrophage Stat1 was dispensable. Our findings provide the first genetic evidence that Stat1 signaling in different cell types produces antagonistic effects on innate protection against Lm that are obscured in mice with complete Stat1 deficiency. They further demonstrate a drastic change in the cell type-dependent Stat1 requirement for memory responses to Lm infection

CDK6 as a key regulator of hematopoietic and leukemic stem cell activation

The cyclin-dependent kinase 6 (CDK6) and CDK4 have redundant functions in reg- ulating cell-cycle progression. We describe a novel role for CDK6 in hematopoietic and leukemic stem cells (hematopoietic stem cells [HSCs] and leukemic stem cells [LSCs]) that exceeds its function as a cell-cycle regulator. Although hematopoiesis appears normal under steady-state conditions, Cdk62/2 HSCs do not efficiently repopulate upon competitive transplantation, and Cdk6-deficient mice are significantly more sus- ceptible to 5-fluorouracil treatment. We find that activation of HSCs requires CDK6, which interferes with the transcription of key regulators, including Egr1. Transcrip- tional profiling of HSCs is consistent with the central role of Egr1. The impaired repopulation capacity extends to BCR-ABLp2101 LSCs. Transplantation with BCR- ABLp2101–infected bone marrow from Cdk62/2 mice fails to induce disease, although recipient mice do harbor LSCs. Egr1 knock-down in Cdk62/2 BCR-ABLp2101 LSKs significantly enhances the potential to form colonies, underlining the importance of the CDK6-Egr1 axis. Our findings define CDK6 as an important regulator of stem cell activation and an essential component of a transcriptional complex that suppresses Egr1 in HSCs and LSCs.Depto. de Bioquímica y Biología MolecularFac. de Ciencias BiológicasTRUEpu

Bacterial load and survival in mice with cell-type specific Stat1 ablation.

<p>Stat1flfl, Stat1−/− and LysMCreStat1flfl mice were infected for 72 h with 1×10∧5 Lm i.p and bacterial load of spleen (A) and liver (B) was determined after 72 h or survival was monitored over 10 days (n = 11–14) (C). Stat1flfl, Stat1−/−, CD11cCreStat1flfl and LckCreStat1flfl mice were infected with 1×10∧5 Lm i.p and bacterial load was determined after 72 h in spleen (D) or liver (E). The survival of i.v. infected animals was monitored over 10 days with different doses of Lm; 2×10∧3 (F); 4×10∧3 (G), 1×10∧4 (H). Stat1flfl and LckCreStat1flfl mice were infected with 1×10∧6 Lm and survival was monitored over 10 days (n = 11–13) (I). LckCreStat1flfl and control mice (J) and LckCreIfnar1flfl and respective control mice (K; L) were infected with 1×10∧6 Lm and the CFU of spleen and liver was determined after 72 h. Survival was monitored for 10 days (n = 6–15) after infection with 5×10∧6 Lm i.p. (M). Representative results of at least two independent experiments are shown.</p

Adaptive immunity to <i>Listeria monocytogenes</i> in mice with tissue-restricted Stat1 ablation.

<p>Immunised and naïve mice were infected i.v. with 1×10∧5 Lm and bacterial load was determined 48 h after infection (n = 4–5) (7A) or survival of 7 mice of each genotype was monitored (7B). Serum of immunised and naïve mice was analysed for the presence of IFNγ (7C). One representative result of at least two independently performed experiments is shown (n = 4–5).</p

Analysis of serum cytokines in mice with cell-type specific Stat1 ablation 72 hrs after infection with Lm.

<p>Mice with Stat1 ablation in different cell compartments were infected with 1×10∧5 Lm and serum was collected over the course of three days after infection. Indicated cytokines (MCP1, IL6, IFNγ, IL12p70, MCP3, Rantes, MIP1β, IL22) were analysed (4). Mean values of cumulative data out of two experiments (n = 8) are depicted with standard deviations. Significant differences are indicated using asterisks.</p

Peritoneal inflammation 48 hrs after infection.

<p>Chemokines (MCP1, Mip1α, Rantes) were determined in the peritoneal lavage fluid of mice (n = 6) after 48 hrs of infection (6A). Peritoneal exudate cells were isolated 48 h after i.p. infection with 1×10∧6 Lm, adherent cells were enriched and RNA prepared and subjected to qPCR for IL12p40, IL6 and IL10 (6B). Data from 3–5 experiments (n = 10–15) are pooled and means with standard deviation are shown. 48 h after i.p infection peritoneal exudate cells were isolated and cytospins stained with Wright-Giemsa solution. The amount of cells (6C), the percentage of macrophages (6D) and the percentage of neutrophils (6E) were counted and depicted (n = 9).</p

Apoptotic cell death in the spleen.

<p>WT, LckCreStat1flfl, LckCreIfnar1flfl, Ifnar1−/− and Stat1−/− were infected with 1×10∧6 Lm and spleens were isolated 48 h after infection. TUNEL positive cells are visible in dark red; hematoxyline counterstaining indicates the structure of the spleen (3).</p