RUNX1 and FOXP3 interplay regulates expression of breast cancer related genes

Runx1 participation in epithelial mammary cells is still under review. Emerging data indicates that Runx1 could be relevant for breast tumor promotion. However, to date no studies have specifically evaluated the functional contribution of Runx1 to control gene expression in mammary epithelial tumor cells. It has been described that Runx1 activity is defined by protein context interaction. Interestingly, Foxp3 is a breast tumor suppressor gene. Here we show that endogenous Runx1 and Foxp3 physically interact in normal mammary cells and this interaction blocks Runx1 transcriptional activity. Furthermore we demonstrate that Runx1 is able to bind to R-spondin 3 (RSPO3) and Gap Junction protein Alpha 1 (GJA1) promoters. This binding upregulates Rspo3 oncogene expression and downregulates GJA1 tumor suppressor gene expression in a Foxp3-dependent manner. Moreover, reduced Runx1 transcriptional activity decreases tumor cell migration properties. Collectively, these data provide evidence of a new mechanism for breast tumor gene expression regulation, in which Runx1 and Foxp3 physically interact to control mammary epithelial cell gene expression fate. Our work suggests for the first time that Runx1 could be involved in breast tumor progression depending on Foxp3 availability

Aberrant RET expression impacts on normal mammary gland post-lactation transition enhancing cancer potential

RET is a receptor tyrosine kinase with oncogenic potential in the mammary epithelium. Several receptors with oncogenic activity in the breast are known to participate in specific developmental stages. We found that RET is differentially expressed during mouse mammary gland development: RET is present in lactation and its expression dramatically decreases in involution, the period during which the lactating gland returns to a quiescent state after weaning. Based on epidemiological and pre-clinical findings, involution has been described as tumor promoting. Using the Ret/MTB doxycycline-inducible mouse transgenic system we show that sustained expression of RET in the mammary epithelium during the post-lactation transition to involution is accompanied by alterations in tissue remodeling and an enhancement of cancer potential. Following constitutive Ret expression we observed a significant increase in neoplastic lesions in the post-involuting versus the virgin mammary gland. Furthermore, we show that abnormal RET overexpression during lactation promotes factors that prime involution, including premature activation of Stat3 signaling and, using RNA-seq, an acute-phase inflammatory signature. Our results demonstrate that RET overexpression negatively affects the normal post-lactation transition.Fil: Vallone, Sabrina Aldana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Garcia Sola, Martin Emilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Schere Levy, Carolina Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Meiss, Roberto P.. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Hermida, Gladys Noemí. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Chodosh, Lewis A.. University of Pennsylvania; Estados UnidosFil: Kordon, Edith Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Hynes, Nancy E.. Friedrich Miescher Institute For Biomedical Research; SuizaFil: Gattelli, Albana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Angiotensin-(1-7) counteracts the transforming effects triggered by angiotensin II in breast cancer cells

Angiotensin (Ang) II, the main effector peptide of the renin-angiotensin system, has been implicated in multiple aspects of cancer progression such as proliferation, migration, invasion, angiogenesis and metastasis. Ang-(1-7), is a biologically active heptapeptide, generated predominantly from AngII by the enzymatic activity of angiotensin converting enzyme 2. Previous studies have shown that Ang-(1-7) counterbalances AngII actions in different pathophysiological settings. In this study, we have analysed the impact of Ang( 1-7) on AngII-induced pro-tumorigenic features on normal murine mammary epithelial cells NMuMG and breast cancer cells MDA-MB-231. AngII stimulated the activation of the survival factor AKT in NMuMG cells mainly through the AT1 receptor. This PI3K/AKT pathway activation also promoted epithelial-mesenchymal transition (EMT). Concomitant treatment of NMuMG cells with AngII and Ang-(1-7) completely abolished EMT features induced by AngII. Furthermore, Ang-(1-7) abrogated AngII induced migration and invasion of the MDA-MB-231 cells as well as pro-angiogenic events such as the stimulation of MMP-9 activity and VEGF expression. Together, these results demonstrate for the first time that Ang-(1-7) counteracts tumor aggressive signals stimulated by AngII in breast cancer cells emerging the peptide as a potential therapy to prevent breast cancer progression

Mouse mammary tumors display Stat3 activation dependent on leukemia inhibitory factor signaling

Introduction: It has been demonstrated that leukemia inhibitory factor (LIF) induces epithelium apoptosis through Stat3 activation during mouse mammary gland involution. In contrast, it has been shown that this transcription factor is commonly activated in breast cancer cells, although what causes this effect remains unknown. Here we have tested the hypothesis that locally produced LIF can be responsible for Stat3 activation in mouse mammary tumors. Methods: The studies were performed in different tumorigenic and non-tumorigenic mammary cells. The expression of LIF and LIF receptor was tested by RT-PCR analysis. In tumors, LIF and Stat3 proteins were analyzed by immunohistochemistry, whereas Stat3 and extracellular signal-regulated kinase (ERK)1/2 expression and phosphorylation were studied by Western blot analysis. A LIF-specific blocking antibody was used to determine whether this cytokine was responsible for Stat3 phosphorylation induced by conditioned medium. Specific pharmacological inhibitors (PD98059 and Stat3ip) that affect ERK1/2 and Stat3 activation were used to study their involvement in LIF-induced effects. To analyze cell survival, assays with crystal violet were performed. Results: High levels of LIF expression and activated Stat3 were found in mammary tumors growing in vivo and in their primary cultures. We found a single mouse mammary tumor cell line, LM3, that showed low levels of activated Stat3. Incidentally, these cells also showed very little expression of LIF receptor. This suggested that autocrine/paracrine LIF would be responsible for Stat3 activation in mouse mammary tumors. This hypothesis was confirmed by the ability of conditioned medium of mammary tumor primary cultures to induce Stat3 phosphorylation, activity that was prevented by pretreatment with LIF-blocking antibody. Besides, we found that LIF increased tumor cell viability. Interestingly, blocking Stat3 activation enhanced this effect in mammary tumor cells. Conclusion: LIF is overexpressed in mouse mammary tumors, where it acts as the main Stat3 activator. Interestingly, the positive LIF effect on tumor cell viability is not dependent on Stat3 activation, which inhibits tumor cell survival as it does in normal mammary epithelium. © 2007 Quaglino et al.; licensee BioMed Central Ltd.Fil:Quaglino, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Schere-Levy, C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Romorini, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Kordon, E.C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Búsqueda de factores involucrados en el origen y la evolución de tumores mamarios murinos, su relevancia enla biología de la mama normal : participación del LIF en estos procesos

El virus del tumor mamario murino (MMTV),es un retrovirus de tipo B que induce el desarrollo de tumores de mama, en ratones suceptibles a la infección, mediante mutagénesis insercional en genes de células somáticas. En este trabajo se utilizaron ratones de la cepa BALB/c infectados con la variante viral MMTV(LA),la cual fue encontrada previamente en nuestro laboratorio. Se ha descripto anteriormente que los ratones infectados con esta variante viral, poseen una incidencia de tumores de mama del 90%. Estos tumores presentan inicialmente un crecimiento preñez-dependiente (HD)que luego se independiza del control hormonal para mostrar un fenotipo hormono-independiente (HI). Para determinar si el compartimiento lobulo-alveolar es o no un factor relevante en la inducción de tumores por el MMTV(LA),se infectaron crías transgénicas WAP-TGFβ1 con esta variante viral mediante el amamantamiento utilizando madres infectadas con el virus. Estos ratones transgénicos expresan TGFBl bajo el control del promotor de la proteína WAP. Este promotor pertenece a una proteína de la leche abundante en murinos, es específica del epitelio mamario y se activa principalmente durante la preñez tardía y la lactancia. Ha sido demostrado previamente, que las hembras transgénicas para WAP-TGFβ1 son incapaces de amamantar a sus crías. Esto se debe a que la sobre-expresión de este factor durante la preñez, desencadena un incremento de la apoptosis en las células alveolares responsables de la producción y secreción de leche. Otra consecuencia de la expresión del transgen es la senescencia prematura de las células progenitoras (stem cells) mamarias, puesta en evidencia por la pérdida de la capacidad clonogénica de las mismas. Los resultados obtenidos en el presente trabajo indican que la actividad pro-apoptótica del TGFβ1 sobre las células lobulo-alveolares mamarias, no redujo la capacidad tumorigénica del MMTV(LA). Por otro lado, encontramos que la infección de las hembras WAPTGFβ1 con el MMTV(LA)revirtió la senescencia prematura de las células progenitores inducida por el transgen. El hecho que la infección con MMTV induzca el restablecimiento de la capacidad clonogénica de las “stem cells” mamarias transgénicas, sugiere que un evento temprano de la oncogénesis inducida por este vian podría ser la inmortalizaciónde las células progenitoras mamarias. Estos resultados (junto con los reportados por otros autores) sugieren que las "stem cells" mamarias podrían ser un blanco preferencial para la actividad oncogénica del MMTV. Esto implicaría un origen y crecimiento clonal para estos tumores. En relación con esta hipótesis se estudió, por análisis de Southern blot, el DNA de las inserciones virales exógenas en los pasajes de tumores mamarios inducidos por MMTV(LA). Estos experimentos se basan en el hecho que sólo puede encontrarse un patrón de bandas especifico y estable del DNA viral en todos los pasajes tumorales, si los tumores originales poseen un origen clonal u oligoclonal. El análisis de los resultados de 17 líneas tumorales in vivo generadas por pasajes singenéicos, muestran que los tumores HD se originan frecuentemente a partir de distintas células mamarias infectadas, mientras que los tumores HI surgen a partir de una sola célula. En todos los casos analizados, se encontraron nuevas inserciones de MMTV(LA) durante la progresión de tumores policlonales HD a tumores monoclonales HI. Por lo tanto, en este modelo, la progresión tumoral se produce asociada al surgimiento de clones que contienen inserciones ausentes o minoritarias en los tumores HD originales. Se ha reportado que la expresión del LIF tanto en el útero como en algunas líneas celulares de cáncer de mama puede ser regulada por estrógeno y progesterona. Sin embargo, no existían evidencias en la literatura sobre la expresión ni la actividad del LIF en la glándula mamaria normal. Por otro lado, se conoce bien que la presencia del LIF es necesaria para mantener a las “stem cells" embrionarias en estado pluripotente. Dado que la glándula mamaria está regulada por hormonas ováricas y que existen células progenitoras mamarias especificas involucradas en el desarrollo mamario normal y tumoral, nos propusimos investigar la participación del LIF en la biologia de la glándula mamaria. Nuestros resultados muestran que este factor se expresa durante el desarrollo de la glándula mamaria del ratón en mamas de hembras vírgenes y preñadas. Sin embargo, hallamos que su expresión máxima se induce durante la fase temprana de la involución mamaria postlactancia. En esta etapa, se habia demostrado que la acumulación de la leche causada por la falta de succión induce la liberación de factores locales que desencadenan la activación del factor de transcripción Stat3 y apoptosis del epitelio mamario. Aunque se sabía que dicho factor cumple un rol fundamental en desencadenar apoptosis durante esta fase, se desconocian los factores involucrados en su activación. Los resultados mostrados aquí indican que el LIF se libera como un factor local durante la fase temprana de la involución debido a la acumulación de la leche en la mama e induce apoptosis en el epitelio. Además detenninamos que el LIF es capaz de inducir la activación de Stat3 en el epitelio mamario in vivo. Por otro lado, nuestros resultados muestran que todos los tumores mamarios inducidos por MMTV(LA) poseen altos niveles de expresión de LIF y de activación de Stat3 y sugieren que el primero sería al menos parcialmente responsable de la activación del segundo. Si bien hemos encontrado el rol biológico de este camino de señalización (LIF-Stat3) en la glándula mamaria normal, queda todavía por dilucidar la significancia de este mecanismo en los tumores mamarios. En conclusión, los resultados presentados aquí refuerzan la idea que las células pluripotenciales y los factores asociados a prevenir su diferenciación, pueden cumplir un rol relevante en la biología de la glándula mamaria normal y tumoral.Mouse mammary tumor virus (MMTV) is a type B retrovirus that causes mammary tumors in susceptible mice by insertional mutagenesis. In the present work it has been used the MMTV(LA),which has been first found in our laboratory. It has been previously showed that mice infected with this variant have a mammary tumor incidence of 90%. These tumors initiain show a hormone-dependent (HD) behavior, but evolve afterwards to a hormone-independent (HI)phenotype. In order to determine whether or not the mammary gland lobulo-alveolar compartiment is relevant for MMTV(LA)-induced tumorigenesis, WAP-TGFβ1 transgenic mice were nursed with MMTV(LA) infected mothers. This transgenic mice, express TGFβ1 under the control of the whey acidic protein (WAP) promoter. This promoter corresponds to a compicuos murine milkprotein, is mammary epithelium-specific and most active during late pregnancy and lactation. It has been demonstrated that TGFβ1 transgenic female mice are unable to lactate. This is because the ectopic expression of this factor in the lobular compartment of the mammary gland during pregnancy induces an increased rate of alveolar apoptosis. Another consequence of the WAP-TGFβ1 transgene expression is the premature senescence of the mammary specific epithelial stem cells manifested in a significant decreased capacity of WAP-TGFβ1 mammary transplant to grow and repopulate a cleared normal fat pad. The results showed herein indicate that WAP-TGFβ1 expression and the consequent lower number of lobulo-alveolar cells in the transgenic animals did not abrogate MMTV(LA) capacity to induce mammary tumors. On the other hand, we have found that MMTV(LA) infection reverted the proviously observed early senecence of WAP-TGFβ1 transgenic mammary epithelium. Since MMTV infection induces a recovery in the clonogenic capacity of the transgenic mammary stem cells, it raises the possibility that mammary stem cells inmortalization would be an early event in MMTV(LA)induced tumorigenesis. Our results show that mammary stem cells could be a preferencial target for MMTV-induced tumorigenesis. This implies a clonal origin for these tumors. Then, to test this hypothesis, Southern blot analysis of MMTV insertions in tumor cell DNA was performed. These studies were carried out based on the fact that a specific and reproducible pattem of bands can only be found in a mono or oligoclonal cell populations. Analysis from 17 tumor lines generated from pregnant MMTV(LA) infected females indicated that HD tumors frequently arise from different mammary infected cells while all HI tumor variants arise from a single cell. In all the cases analyzed herein, new MMTV(LA) insertions were found during progression from policlonal HD to monoclonal HI tumors. It has been reported that leukemia inhibitory factor (LIF) expression could be regulated by estrogen and progesterone in uterus and in some mammary tumor cell lines. However, there were no reports about LIF expression or activity in the normal mammary gland. On the other hand, it is very well known the importance of the LIF presence in order to maintain pluripotent embryonic stem cells. Since mammary gland is regulated by ovaric hormone and there are mammary specific stem cells involved in normal and neoplastic development. We decided to investigate LIF participation in the mammary gland biology. Our results show that LIF is expressed during mammary development in Virgin and pregnant females. However, LIF expression picks shortly after weaning during the early phase of mammary involution. It has been demonstrated that during this period, milk stasis caused by the lack of suckling results in the induction of local factors that lead to the activation of Stat3 transcription factor and mammary epithelium apoptosis. The results presented herein demonstrate that LIF is secreted as a mammary local factor in response to milk stasis and induces mammary ephitelium apoptosis. Moreover, we also demonstrate that LIF induces Stat3 phosphorylation in the mammary gland in vivo. On the other hand, we have found high LIF expression and Stat3 activation in all the MMW(LA) induced HD and HI mammary tumors and our results suggest that LIF would be at least partialliy responsible of that activation. While the biological role of this pathway in the nomal mammary gland has been very reciently uncover, its significance in mammary neoplastic glands remains to be elucidated. In conclusion, the results showed herein support the relevant role of mammary stem cells and factors possibly associated with the prevention of their differentiation, in the biology of normal and neoplastic mammary gland.Fil:Schere Levy, Carolina P.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Morphological and immunohistochemical analysis of MMTV(LA)-induced HIT and LM3 mammary tumors growing

<p><b>Copyright information:</b></p><p>Taken from "Mouse mammary tumors display Stat3 activation dependent on leukemia inhibitory factor signaling"</p><p>http://breast-cancer-research.com/content/9/5/R69</p><p>Breast cancer research : BCR 2007;9(5):R69-R69.</p><p>Published online 10 Oct 2007</p><p>PMCID:PMC2242666.</p><p></p> Hematoxylin/eosin-stained tumors. Moderately differentiated hormone-independent tumors (HIT). Papillary formations into lumen of cystic ducts and less differentiated zones with small glandular structures. Original magnification ×100; inset ×400. LM3 poorly differentiated adenocarcinoma with abundant infiltrated vascular stroma. Areas with sarcomatous appearance and apoptotic images (black arrowheads). Original magnification in (a, b) ×100; inset ×400. Leukemia inhibitory factor (LIF) immunohistochemistry. HIT: heterogenous and regular distribution mainly in poorly differentiated areas with cytoplasmic and nucleic positive staining; intense cytoplasmic staining increasing peripherally indicates a secretory pattern (arrowhead). LM3 tumor: patchy LIF staining. Inset: positive cytoplasmic staining; the ill-defined cell membrane pattern with granular cytoplasmic staining indicates LIF secretion (arrowhead). Involuting (48-hour) and lactating mammary glands were used as positive and negative controls, respectively, for LIF staining. Original magnification in (c-f) 250×; inset: 400× Stat3 (signal transduction and activators of transcription 3) immunohistochemistry. HIT: stromal and predominantly epithelial positive nuclear staining in solid areas. Inset: stained nuclei were also seen in glandular regions. LM3 tumor: intense cytoplasmic positive staining in stromal and epithelial cells. The inset shows a negative gland surrounded by positive stromal cells. Original magnification in (g, h) 250×; inset: 400

Treatment with Angiotensin-(1-7) Prevents Development of Oral Papilloma Induced in K-ras Transgenic Mice

Oral Squamous Cell Carcinoma (OSCC) is the most common malignant cancer affecting the oral cavity. It is characterized by high morbidity and very few therapeutic options. Angiotensin (Ang)-(1-7) is a biologically active heptapeptide, generated predominantly from AngII (Ang-(1-8)) by the enzymatic activity of angiotensin-converting enzyme 2 (ACE 2). Previous studies have shown that Ang-(1-7) counterbalances AngII pro-tumorigenic actions in different pathophysiological settings, exhibiting antiproliferative and anti-angiogenic properties in cancer cells. However, the prevailing effects of Ang-(1-7) in the oral epithelium have not been established in vivo. Here, we used an inducible oral-specific mouse model, where the expression of a tamoxifen-inducible Cre recombinase (CreERtam), which is under the control of the cytokeratin 14 promoter (K14-CreERtam), induces the expression of the K-ras oncogenic variant KrasG12D (LSLK-rasG12D). These mice develop highly proliferative squamous papilloma in the oral cavity and hyperplasia exclusively in oral mucosa within one month after tamoxifen treatment. Ang-(1-7) treated mice showed a reduced papilloma development accompanied by a significant reduction in cell proliferation and a decrease in pS6 positivity, the most downstream target of the PI3K/Akt/mTOR signaling route in oral papilloma. These results suggest that Ang-(1-7) may be a novel therapeutic target for OSCC

Origin and progression of pregnancy-dependent mammary tumors induced by new mouse mammary tumor virus variants

In order to study mechanisms of progression of mouse mammary tumor virus (MMTV)-induced pregnancy-dependent mammary lesions, we removed and serially transplanted 17 small tumors detected in MMTV-infected pregnant females. This gave rise to the same number of 'in vivo' tumor lines. Hormone-dependency of the passages was determined by comparing tumor development in multiparous versus virgin hosts. We found that the first passages of most of these lesions (11/17) required pregnancy to grow. However, all these tumor lines lost their hormone-dependence through successive passages. The original pregnancy-dependent lesions were mostly multiclonal and showed high levels of estrogen and progesterone receptors. Alternatively, pregnancy-independent tumors arose as clonal dominant populations exhibiting a lower hormone receptor content. Our data show that the progression of hormone-dependent MMTV-induced mammary tumors is an irreversible process associated with the appearance of additional MMTV insertional events as well as alterations in the composition of the tumor cell population.Fil: Buggiano, Valeria Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Leucemia Experimental; ArgentinaFil: Schere Levy, Carolina Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Leucemia Experimental; ArgentinaFil: Gattelli, Albana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Leucemia Experimental; ArgentinaFil: Cirio, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Leucemia Experimental; ArgentinaFil: Marfil, Mariana. Academia Nacional de Medicina de Buenos Aires. Instituto de Leucemia Experimental; ArgentinaFil: Nepomnaschy, Irene. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Leucemia Experimental; ArgentinaFil: Piazzon, Margarita Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Leucemia Experimental; ArgentinaFil: Helguero, luisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Vanzulli, Silvia. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Kordon, Edith Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Leucemia Experimental; Argentin