Inhibition of Specific NF-κB Activity Contributes to the Tumor Suppressor Function of 14-3-3σ in Breast Cancer

14-3-3σ is frequently lost in human breast cancers by genetic deletion or promoter methylation. We have now investigated the involvement of 14-3-3σ in the termination of NF-κB signal in mammary cells and its putative role in cancer relapse and metastasis. Our results show that 14-3-3σ regulates nuclear export of p65-NF-κB following chronic TNFα stimulation. Restoration of 14-3-3σ in breast cancer cells reduces migration capacity and metastatic abilities in vivo. By microarray analysis, we have identified a genetic signature that responds to TNFα in a 14-3-3σ-dependent manner and significantly associates with different breast and other types of cancer. By interrogating public databases, we have found that over-expression of this signature correlates with poor relapse-free survival in breast cancer patients. Finally, screening of 96 human breast tumors showed that NF-κB activation strictly correlates with the absence of 14-3-3σ and it is significantly associated with worse prognosis in the multivariate analysis. Our findings identify a genetic signature that is important for breast cancer prognosis and for future personalized treatments based on NF-κB targeting

Hematopoietic stem cell development requires transient Wnt/β-catenin activity

Understanding how hematopoietic stem cells (HSCs) are generated and the signals that control this process is a crucial issue for regenerative medicine applications that require in vitro production of HSC. HSCs emerge during embryonic life from an endothelial-like cell population that resides in the aorta-gonad-mesonephros (AGM) region. We show here that β-catenin is nuclear and active in few endothelial nonhematopoietic cells closely associated with the emerging hematopoietic clusters of the embryonic aorta during mouse development. Importantly, Wnt/β-catenin activity is transiently required in the AGM to generate long-term HSCs and to produce hematopoietic cells in vitro from AGM endothelial precursors. Genetic deletion of β-catenin from the embryonic endothelium stage (using VE-cadherin-Cre recombinase), but not from embryonic hematopoietic cells (using Vav1-Cre), precludes progression of mutant cells toward the hematopoietic lineage; however, these mutant cells still contribute to the adult endothelium. Together, those findings indicate that Wnt/β-catenin activity is needed for the emergence but not the maintenance of HSCs in mouse embryos

Impaired embryonic haematopoiesis yet normal arterial development in the absence of the Notch ligand Jagged1

Specific deletion of Notch1 and RBPjκ in the mouse results in abrogation of definitive haematopoiesis concomitant with the loss of arterial identity at embryonic stage. As prior arterial determination is likely to be required for the generation of embryonic haematopoiesis, it is difficult to establish the specific haematopoietic role of Notch in these mutants. By analysing different Notch-ligand-null embryos, we now show that Jagged1 is not required for the establishment of the arterial fate but it is required for the correct execution of the definitive haematopoietic programme, including expression of GATA2 in the dorsal aorta. Moreover, successful haematopoietic rescue of the Jagged1-null AGM cells was obtained by culturing them with Jagged1-expressing stromal cells or by lentiviral-mediated transduction of the GATA2 gene. Taken together, our results indicate that Jagged1-mediated activation of Notch1 is responsible for regulating GATA2 expression in the AGM, which in turn is essential for definitive haematopoiesis in the mouse

Inhibition of specific NF-KB activity contributes to the tumor suppressor function of 14-3-3omega in breast cancer

14-3-3σ is frequently lost in human breast cancers by genetic deletion or promoter methylation. We have now investigated the involvement of 14-3-3σ in the termination of NF-κB signal in mammary cells and its putative role in cancer relapse and metastasis. Our results show that 14-3-3σ regulates nuclear export of p65-NF-κB following chronic TNFα stimulation. Restoration of 14-3-3σ in breast cancer cells reduces migration capacity and metastatic abilities in vivo. By microarray analysis, we have identified a genetic signature that responds to TNFα in a 14-3-3σ-dependent manner and significantly associates with different breast and other types of cancer. By interrogating public databases, we have found that over-expression of this signature correlates with poor relapse-free survival in breast cancer patients. Finally, screening of 96 human breast tumors showed that NF-κB activation strictly correlates with the absence of 14-3-3σ and it is significantly associated with worse prognosis in the multivariate analysis. Our findings identify a genetic signature that is important for breast cancer prognosis and for future personalized treatments based on NF-κB targeting.This work was funded by ISCIII/FEDER-Subdirección General de Evaluación y Fomento de la Investigación (PI07/0778, PI10/01128, PS09/1296 and PS09/01285), AGAUR (2009SGR23 and 2009SGR321) and Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+I), iniciativa Ingenio 2010, Programa Consolider and Instituto de Salud Carlos III (ISCIII)/FEDER (RD06/0020/0098 and RD06/0020/0109). Financial support was provided to RRG by BBVA foundation and Asociación Española Contra el Cáncer. NLB acknowledges funding from the Spanish Ministry of Science and Technology (SAF2009-06954). AJ-S was funded by Science and Education Spanish Ministry (MEC) FPI predoctoral fellowship (BES-2008-001850). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Relapse-free survival analysis.

<p>Abreviations: DFS, disease free survival; HR, hazard ratio; CI, confidence interval; HER2, human epidermal growth factor receptor 2.</p><p>Relapse free survival analysis of the group of patients that were studied for the presence of nuclear p65. Commonly used clinical predictors such as tumor grade and size, or the number of infiltrated lymph nodes were studied in comparison with the status of p65.</p

Re-expression of 14-3-3σ in MDA-MB-231 cells inhibits bone metastasis in vivo.

<p>(A) Representative bioluminescence images of mice injected with 0.5×10⁶ control or 14-3-3σ-overexpressing MDA-MB-231 cells 4 weeks after intracardiac injection. (B) Scattered plot of the dorsal photon flux (normalized to Day 0) at different times after injection. Each symbol represents one animal (C–D) H&E staining and IHC for 14-3-3σ (C) and IHC for ki-67 (D) of a representative bone-metastasis from a mouse injected with control (MT) or 14-3-3σ-expressing cells. (E–F) Quantification of cells expressing the proliferation marker ki-67 (E) and number of mitotic figures in the H&E staining (F) determined by counting 10 different high power fields per tumor (3 tumors counted for each group).</p

Defective 14-3-3σ is responsible for delayed p65 nuclear export in breast cancer cells.

<p>(A and B) IF analysis of 14-3-3σ expression (A) and p65-NF-κB nuclear translocation (B) in MCF7 control and 14-3-3σ expressing cells. (C) Quantification of p65 subcellular distribution in one representative of 3 independent experiments. (D) Western blot analysis of 14-3-3σ levels in MCF10A pools transduced with different shRNA. (E, F) Representative images of p65 IF (E) and quantification of p65 subcellular distribution in MCF10A cells (F). One representative of three independent experiments is shown. (G) Subcellular fractionation followed by western blot analysis of p65 in the different MCF10A pools transduced with scrambled or 14-3-3σ-shRNAs. Average of densitometric quantification of 3 independent experiments is shown in the lower panel. For representation of p65 distribution in 3c and 3f, two groups have been considered, cytoplasmic: homogenous or cytoplasmic staining and nuclear: preferentially nuclear distribution.</p

14-3-3σ-dependent NF-κB activity correlates with poor prognosis in breast cancer patients.

<p>(A) Representative tumors showing different patterns of 14-3-3σ expression, that was distributed both in cytoplasm and nucleus of tumor cells. Consistently, positive 14-3-3σ staining was observed in myoepithelial cells of normal ducts and ductal carcinoma <i>in situ</i> (arrowheads). (B and E) Kaplan-Meier curves depict cumulative disease-free survival of breast cancer patients stratified by the absence of 14-3-3σ (B) and by the presence of nuclear p65 (E). (C) Double IF of representative breast tumors showing that nuclear p65 distribution was restricted to the 14-3-3σ negative samples (D) Cross-tabulation of 14-3-3σ expression and NF-κB activation (as determined by nuclear p65) in the different tumors analyzed. (F) Kaplan-Meier curve depict relapse-free survival of breast cancer patients stratified by up-regulation of 14-3-3σ-dependent signature in the primary tumors.</p

Hematopoietic stem cell development requires transient Wnt/β-catenin activity

Understanding how hematopoietic stem cells (HSCs) are generated and the signals that control this process is a crucial issue for regenerative medicine applications that require in vitro production of HSC. HSCs emerge during embryonic life from an endothelial-like cell population that resides in the aorta-gonad-mesonephros (AGM) region. We show here that β-catenin is nuclear and active in few endothelial nonhematopoietic cells closely associated with the emerging hematopoietic clusters of the embryonic aorta during mouse development. Importantly, Wnt/β-catenin activity is transiently required in the AGM to generate long-term HSCs and to produce hematopoietic cells in vitro from AGM endothelial precursors. Genetic deletion of β-catenin from the embryonic endothelium stage (using VE-cadherin–Cre recombinase), but not from embryonic hematopoietic cells (using Vav1-Cre), precludes progression of mutant cells toward the hematopoietic lineage; however, these mutant cells still contribute to the adult endothelium. Together, those findings indicate that Wnt/β-catenin activity is needed for the emergence but not the maintenance of HSCs in mouse embryos