Cell heterogeneity in human breast cancer

Dos objetivos principales fueron planteados en este trabajo: 1) investigar la heterogeneidad celular en carcinomas mamarios humanos, caracterizando distintas subpoblaciones celulares respecto a proliferación y expresión de diversos marcadores tumorales; 2) obtener anticuerpos monoclonales (AMC) contra estos tumores, con propiedades citotóxicas, que reconozcan células indiferenciadas, de alto potencial proliferativo. Los estudios de heterogeneidad celular fueron realizados utilizando muestras tumorales humanas, la proliferación evaluada mediante incorporación de [3H]Timidina y marcadores por inmunohistoquímica. Los resultados permitieron establecer una secuencia de expresión de marcadores a medida que progresa la diferenciación celular y disminuye la capacidad proliferativa. Pudieron identificarse marcadores asociados a distintas etapas de la diferenciación (NCA90, receptor estroncio, receptor de progesterona, TAG72, CaMBr1, CEA) y otros que comienzan a expresarse en células indiferenciadas, altamente proliferativas, y continuarían durante todo el proceso de diferenciación (Ag2.15, PEM). Se investigaron también las propiedades del AMC FC-2.15 (anti-Ag2.15) desarrollado utilizando como inmunogeno un carcinoma mamario humano indiferenciado. FC-2.15 presento una potente citotoxicidad contra células Ag2.15+ mediando lisis por complemento humano, reconocio un antígeno presente en membrana plasmática en alta densidad (2,8x10 6 /célula) y fue lentamente internalizado. Los resultados presentados en este trabajo sugieren que FC-2.15 podría ser útil en inmunoterapia de pacientes con neoplasias Ag2.15+.The main purposes of this study were: 1) to investigate the cell heterogeneity in human breast cancer and characterize the different cell subpopulations with regard to proliferation and expression of tumor markers; 2) to obtain monoclonal antibodies (MAb) against these tumors which recognize highly undifferentiated (stem) cells, and mediate specific cytotoxicity. Cell heterogeneity was studied in human tumor samples, proliferation was evaluated by [3H]thymidine incorporation and markers by immunohistochemistry. According to the results, it could be defined a regular sequence of marker expression as cell differentiation proceeds and proliferation decreases. Some markers were identified which are expressed in different stages of the differentiation pathway (NCA90, estrogen receptor, progesterone receptor, TAG72, CaMBr1, CEA). Conversely, the expression of other markers (Ag2.15, PEM) would start in undifferentiated-highly proliferative cells and remain throughout the whole differentiation process. It was also investigated the properties of MAb FC-2.15 (anti-Ag2.15) generated using an undifferentiated human breast carcinoma as immunogen. Results showed that FC-2.15 exhibits strong human complement-mediated cytotoxicity against Ag2.15+ cells. It recognizes a high-density antigen (2.8x10 6/cell) in the cell membrane and is slowly internalized. The results obtained in this study suggest that FC-2.15 could be a useful agent for passive immuntherapy.Fil:Ballaré, Cecilia J.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Polycomb Regulates NF-κB Signaling in Cancer through miRNA

The mechanisms leading to the constitutive activation of NF-κB in cancers and the pathways upstream and downstream of this activation are not fully understood. In this issue of Cancer Cell, Yamagishi et al. demonstrate that Polycomb-mediated silencing of miR-31 is implicated in the aberrant activation of NF-κB signaling in tumors

Remodelatge de la cromatina durant la inducció per progesterona del promotor de l'MMTV

Per a comprendre els mecanismes que governen l'expressió dels gens inclosos en els genomes eucariòtics és necessari prendre en consideració la manera en què les regions reguladores d'aquests gens estan organitzades en la cromatina. Les diferències en l'organització de la cromatina i en les modificacions químiques dels components de la cromatina expliquen els diferents patrons de l'expressió gènica en diversos tipus de cèll. ules i la seva resposta específica a senyals externs. Basant-nos en els nostres estudis sobre la inducció hormonal del promotor del mouse mammary tumour virus (MMTV), podem concloure que la seqüència nucleotídica primària determina no solament la manera en què la doble hèlix de DNA envolta l'octàmer d'histona, i així l'accessibilitat de punts d'unió per als factors de transcripció, sinó també la manera en què aquests factors estableixen sinergismes i la naturalesa del remodelatge de la cromatina dependent d'ATP. A més, la senyalització via crosstalk amb cascades de cinases citoplasmàtiques canvia l'estructura de la cromatina en els gens diana i és fonamental per a la correcta regulació a través de receptors d'hormones esteroidees.Understanding the mechanisms governing the expression of the genes encompassed in the eukaryotic genomes requires a careful consideration of the way regulatory regions of these genes are packaged in chromatin. Differences in the chromatin organization and in the chemical modifications of chromatin components account for the different patterns of gene expression in various cell types and for their specific response to external signals. Based on our studies on the hormonal induction of mouse mammary tumour virus (MMTV) promoter we conclude that the primary nucleotide sequence determines not only the way theDNAdouble helix wraps around the histone octamer, and so the accessibility of binding sites for transcription factors, but also the way these factors synergize and the nature of the ATP-dependent chromatin remodelling. Moreover, signalling via crosstalk with cytoplasmic kinase cascades changes the chromatin structure of target genes and is essential for proper regulation by steroid hormone receptors

Polycomb Factor PHF19 Controls Cell Growth and Differentiation Toward Erythroid Pathway in Chronic Myeloid Leukemia Cells

Leucèmia mieloide crònica; Diferenciació eritroïdal; PolycombLeucemia mieloide crónica; Diferenciación eritroide; PolycombChronic myeloid leukemia; Erythroid differentiation; PolycombPolycomb group (PcG) of proteins are a group of highly conserved epigenetic regulators involved in many biological functions, such as embryonic development, cell proliferation, and adult stem cell determination. PHD finger protein 19 (PHF19) is an associated factor of Polycomb repressor complex 2 (PRC2), often upregulated in human cancers. In particular, myeloid leukemia cell lines show increased levels of PHF19, yet little is known about its function. Here, we have characterized the role of PHF19 in myeloid leukemia cells. We demonstrated that PHF19 depletion decreases cell proliferation and promotes chronic myeloid leukemia (CML) differentiation. Mechanistically, we have shown how PHF19 regulates the proliferation of CML through a direct regulation of the cell cycle inhibitor p21. Furthermore, we observed that MTF2, a PHF19 homolog, partially compensates for PHF19 depletion in a subset of target genes, instructing specific erythroid differentiation. Taken together, our results show that PHF19 is a key transcriptional regulator for cell fate determination and could be a potential therapeutic target for myeloid leukemia treatment.This work was supported by the Di Croce Laboratory is supported by grants from the Spanish Ministry of Science and Innovation (BFU2016-75008-P and PID2019-108322GB-100), “Fundación Vencer El Cancer” (VEC), the European Regional Development Fund (FEDER), and from AGAUR (SGR 2017-2019). We acknowledge the support of the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme/Generalitat de Catalunya. PV was supported by the Fundación Científica de la Asociación Española Contra el Cáncer. SA was funded by the Ramon y Cajal program of the Ministerio de Ciencia, Innovación y Universidades, the European Social Fund under the reference number RYC-2018-025002-I, and the Instituto de Salud Carlos III-FEDER (PI19/01814)

CDC2 mediates progestin initiated endometrial stromal cell proliferation: a PR signaling to gene expression independently of its binding to chromatin

Although non-genomic steroid receptor pathways have been studied over the past decade, little is known about the direct gene expression changes that take place as a consequence of their activation. Progesterone controls proliferation of rat endometrial stromal cells during the peri-implantation phase of pregnancy. We showed that picomolar concentration of progestin R5020 mimics this control in UIII endometrial stromal cells via ERK1-2 and AKT activation mediated by interaction of Progesterone Receptor (PR) with Estrogen Receptor beta (ERb) and without transcriptional activity of endogenous PR and ER. Here we identify early downstream targets of cytoplasmic PR signaling and their possible role in endometrial stromal cell proliferation. Microarray analysis of global gene expression changes in UIII cells treated for 45 min with progestin identified 97 up- and 341 down-regulated genes. The most over-represented molecular functions were transcription factors and regulatory factors associated with cell proliferation and cell cycle, a large fraction of which were repressors down-regulated by hormone. Further analysis verified that progestins regulate Ccnd1, JunD, Usf1, Gfi1, Cyr61, and Cdkn1b through PR-mediated activation of ligand-free ER, ERK1-2 or AKT, in the absence of genomic PR binding. ChIP experiments show that progestin promoted the interaction of USF1 with the proximal promoter of the Cdc2 gene. Usf1 knockdown abolished Cdc2 progestin-dependent transcriptional regulation and cell proliferation, which also blocked Cdc2 knockdown. We conclude that progestin-induced proliferation of endometrial stromal cells is mediated by ERK1-2 and AKT dependent early regulation of USF1, which directly induces Cdc2. To our knowledge, this is the first description of early target genes of progestin-activated classical PR via crosstalk with protein kinases and independently of hormone receptor binding to the genomic targets.Fil: Vallejo, Griselda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: la Greca, Alejandro Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Tarifa Reischle, Inti C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Mestre Citrinovitz, Ana Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Ballaré, Cecilia. Centro de Regulación Genómica; EspañaFil: Beato, Miguel. Centro de Regulación Genómica; España. Universitat Pompeu Fabra; EspañaFil: Saragüeta, Patricia Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentin

Progesterone Receptor induces bcl-x expression through intragenic binding sites favoring RNA Polymerase II elongation

Steroid receptors were classically described for regulating transcription by binding to target gene promoters. However, genome-wide studies reveal that steroid receptors-binding sites are mainly located at intragenic regions. To determine the role of these sites, we examined the effect of pro- gestins on the transcription of the bcl-x gene, where only intragenic progesterone receptor-binding sites (PRbs) were identified. We found that in response to hormone treatment, the PR is recruited to these sites along with two histone acetyltransferases CREB-binding protein (CBP) and GCN5, leading to an increase in histone H3 and H4 acetylation and to the binding of the SWI/SNF complex. Concomitant, a more relaxed chromatin was detected along bcl-x gene mainly in the regions sur- rounding the intragenic PRbs. PR also mediated the recruitment of the positive elongation factor pTEFb, favoring RNA polymerase II (Pol II) elongation activity. Together these events promoted the re-dis- tribution of the active Pol II toward the 30-end of the gene and a decrease in the ratio between proximal and distal transcription. These results suggest a novel mechanism by which PR regulates gene ex- pression by facilitating the proper passage of the polymerase along hormone-dependent genes.Fil: Bertucci, Paola Yanina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Nacht, Ana Silvina. Universitat Pompeu Fabra; España. Centro de Regulación Genómica; EspañaFil: Alló, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Rocha Viegas, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Ballaré, Cecilia. Universitat Pompeu Fabra; España. Centro de Regulación Genómica; EspañaFil: Soronellas, Daniel. Centro de Regulación Genómica; España. Universitat Pompeu Fabra; EspañaFil: Castellano, Giancarlo. Centro de Regulación Genómica; España. Universitat Pompeu Fabra; EspañaFil: Zaurin, Roser. Centro de Regulación Genómica; España. Universitat Pompeu Fabra; EspañaFil: Kornblihtt, Alberto Rodolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Beato, Miguel. Centro de Regulación Genómica; España. Universitat Pompeu Fabra; EspañaFil: Vicent, Guillermo. Centro de Regulación Genómica; España. Universitat Pompeu Fabra; EspañaFil: Pecci, Adali. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Mutational analysis of progesterone receptor functional domains in stable cell lines delineates sets of genes regulated by different mechanisms

Steroid hormone receptors act directly in the nucleus on the chromatin organization and transcriptional activity of several promoters. Furthermore, they have an indirect effect on cytoplasmic signal transduction pathways, including MAPK, impacting ultimately on gene expression. We are interested in distinguishing between the two modes of action of progesterone receptor (PR) on the control of gene expression and cell proliferation. For this, we have stably expressed, in PR-negative breast cancer cells, tagged forms of the PR isoform B mutated at regions involved either in DNA binding (DNA-binding domain) or in its ability to interact with the estrogen receptor and to activate the c-Src/MAPK/Erk/Msk cascade (estrogen receptor-interacting domain). Both mutants impair PR-mediated activation of a well-understood model promoter in response to progestin, as well as hormone-induced cell proliferation. Additional mutants affecting transactivation activity of PR (activation function 2) or a zinc-finger implicated in dimerization (D-box) have also been tested. Microarrays and gene expression experiments on these cell lines define the subsets of hormone-responsive genes regulated by different modes of action of PR isoform B, as well as genes in which the nuclear and nongenomic pathways cooperate. Correlation between CCND1 expression in the different cell lines and their ability to support cell proliferation confirms CCND1 as a key controller gene. Copyright © 2009 by The Endocrine Society.This work was supported by grants from the Catalan Department for Universities, Research and the Information Society, and the Spanish Ministry of Science and Technology and Fondo Europeo de desarrollo regional (SAF2002-03320, BFU2008-00359). A.J. was recipient of a ‘Ramón y Cajal’ appointment from the Spanish Ministry of Science and Technology. I.Q. was recipient of a Formación personal universitario predoctoral fellowship from the Spanish Ministry of Education. L.M-A. was recipient of a predoctoral fellowship funded by Fundación para la investigación y prevención del SIDA en EspañaPeer Reviewe

Role of PRC2-associated factors in stem cells and disease

This work was supported by grants from the Spanish “Ministerio de Educación y Ciencia” (SAF2013-48926-P), from AGAUR, “Fundació a Marató” (20131110), and from the European Commission's 7th Framework Program 4DCellFate grant number 277899 to L.D.CThe Polycomb group (PcG) of proteins form chromatin-binding complexes with histone-modifying activity. The two main PcG repressive complexes studied (PRC1 and PRC2) are generally associated with chromatin in its repressed state. PRC2 is responsible for methylation of histone H3 at lysine 27 (H3K27me3), an epigenetic mark that is linked with numerous biological processes, including development, adult homeostasis and cancer. The core canonical complex PRC2, which contains the EZH1/2, SUZ12 and EED proteins, may be extended and functionally manipulated through interactions with several other proteins. In this review, we focus on these PRC2-associated proteins. As PRC2 functions are diverse, the variability conferred by these sub-stoichiometrically associated members may help to understand specific changes in PRC2 activity, chromatin recruitment and distribution required for gene repression

Progesterone Induction of the 11β-Hydroxysteroid Dehydrogenase Type 2 Promoter in Breast Cancer Cells Involves Coordinated Recruitment of STAT5A and Progesterone Receptor to a Distal Enhancer and Polymerase Tracking▿ †

Steroid hormone receptors regulate gene expression, interacting with target DNA sequences but also activating cytoplasmic signaling pathways. Using the human 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) gene as a model, we have investigated the contributions of both effects on a human progesterone-responsive promoter in breast cancer cells. Chromatin immunoprecipitation has identified two different mechanisms of hormone-induced progesterone receptor (PR) recruitment to the 11β-HSD2 promoter: (i) direct PR binding to DNA at the proximal promoter, abrogated when PR contains a mutated DNA binding domain (DBD), and (ii) STAT5A (signal transducer and activator of transcription 5A)-mediated recruitment of PR to an upstream distal region, impaired by AG490, a JAK/STAT pathway inhibitor. The JAK/STAT inhibitor, as well as expression of dominant-negative STAT5A, impairs hormone induction of 11β-HSD2. On the other hand, the DBD-mutated PR fully supports 11β-HSD2 expression. These results, along with data from a deletion analysis, indicate that the distal region is crucial for hormone regulation of 11β-HSD2. We show active RNA polymerase II tracking from the distal region upon PR and STAT5A binding, concomitant with synthesis of noncoding, hormone-dependent RNAs, suggesting that this region works as a hormone-dependent transcriptional enhancer. In conclusion, coordination of PR transcriptional effects and cytoplasmic signaling activation, in particular the JAK/STAT pathway, are critical in regulating progestin-induced endogenous 11β-HSD2 gene expression in breast cancer cells. This is not unique to this promoter, as AG490 also alters the expression of other progesterone-regulated genes