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

Integrative study of the regulatory and epigenomic programs involved in cancer development

El càncer ha estat tradicionalment considerat una malaltia fonamentalment genètica, però recentment s'està fent palès que la desregulació de mecanismes epigenètics contribueix en gran manera al desenvolupament tumoral. Al bell mig de la intersecció entre la genètica i l'epigenètica s'hi troben els factors reguladors de la cromatina (CRFs, en anglès), que són un focus important de recerca a causa de la seva potencial utilitat en teràpies contra el càncer. En aquesta tesi, determino l'estat transcriptòmic de cèl·lules normals i tumorals basant-me en informació epigenètica i regulatòria, i descric l'existència d'una sincronització global de l'expresió gènica en què la regulació controlada per Polycomb es manifesta com a un dels dos components principals. Presento una anàlisi sobre com la baixa expressió dels gens regulats per Polycomb contribueix a l'avenç del càncer de mama i a la transició entre epitel·li i mesènquima. A més, identifico aquesta baixa expressió com a factor valuós de pronòstic independent. Aprofitant les dades genòmiques de càncer que han estat posades a la disposició del públic recentment, també avaluo l'estat mutacional dels CRFs en molts tumors humans provinents de diferents teixits i línies cel·lulars de càncer. Els resultats indiquen que 39 CRFs són potencialment conductors del procès cancerígen en almenys un teixit, malgrat que molts d'ells es torben mutats en freqüències relativament baixes. Finalment, presento un recurs per a visualitzar i analitzar alteracions genòmiques entre línies cel·lulars de càncer en el context de la resistència a fàrmacs i de la informació sobre alteracions deCancer has traditionally been regarded as a genetic disease, but recently it is becoming apparent that the deregulation of epigenetic mechanisms greatly contributes to tumour development. At the crossing of genetics and epigenetics lie chromatin regulatory factors (CRFs), which are the focus of intense research due to their potential usefulness in anticancer therapy. In this thesis, I determine the transcriptomic state of normal and tumour cells based on epigenetic and regulatory information, and describe the existence of a global synchronisation of gene expression in which Polycomb regulation arises as one of the two main components. I present an analysis on how the under-expression of Polycomb regulated genes contributes to breast cancer progression and epithelial to mesenchymal transition. Furthermore, I identify this under-expression as a valuable independent prognostic factor. Taking advantage on the wealth of cancer genomics data made available recently, I also evaluate the mutational status of CRFs across many human tumours from different tissues and cancer cell lines, and find that 39 CRFs are potential cancer drivers in at least one tissue, even though most of them are mutated at relatively low frequencies. Finally, I present a resource to visualise and analyse genomic alterations across cancer cell lines in the context of drug sensitivity/resistance and the information on somatic tumour alterations

The mutational landscape of chromatin regulatory factors across 4,623 tumor samples

Background: Chromatin regulatory factors are emerging as important genes in cancer development and are regarded as interesting candidates for novel targets for cancer treatment. However, we lack a comprehensive understanding of the role of this group of genes in different cancer types. Results: We have analyzed 4,623 tumor samples from thirteen anatomical sites to determine which chromatin regulatory factors are candidate drivers in these different sites. We identify 34 chromatin regulatory factors that are likely drivers in tumors from at least one site, all with relatively low mutational frequency. We also analyze the relative importance of mutations in this group of genes for the development of tumorigenesis in each site, and in different tumor types from the same site. Conclusions: We find that, although tumors from all thirteen sites show mutations in likely driver chromatin regulatory factors, these are more prevalent in tumors arising from certain tissues. With the exception of hematopoietic, liver and kidney tumors, as a median, the mutated factors are less than one fifth of all mutated drivers across all sites analyzed. We also show that mutations in two of these genes, MLL and EP300, correlate with broad expression changes across cancer cell lines, thus presenting at least one mechanism through which these mutations could contribute to tumorigenesis in cells of the corresponding tissues.We acknowledge funding from the Spanish Ministry of Economy and Competitivity (grant numbers SAF2009-06954 and SAF2012-36199) and the Spanish National Institute of Bioinformatics. AJ-S is supported by an FPI fellowshi

The mutational landscape of chromatin regulatory factors across 4,623 tumor samples

Background: Chromatin regulatory factors are emerging as important genes in cancer development and are regarded as interesting candidates for novel targets for cancer treatment. However, we lack a comprehensive understanding of the role of this group of genes in different cancer types. Results: We have analyzed 4,623 tumor samples from thirteen anatomical sites to determine which chromatin regulatory factors are candidate drivers in these different sites. We identify 34 chromatin regulatory factors that are likely drivers in tumors from at least one site, all with relatively low mutational frequency. We also analyze the relative importance of mutations in this group of genes for the development of tumorigenesis in each site, and in different tumor types from the same site. Conclusions: We find that, although tumors from all thirteen sites show mutations in likely driver chromatin regulatory factors, these are more prevalent in tumors arising from certain tissues. With the exception of hematopoietic, liver and kidney tumors, as a median, the mutated factors are less than one fifth of all mutated drivers across all sites analyzed. We also show that mutations in two of these genes, MLL and EP300, correlate with broad expression changes across cancer cell lines, thus presenting at least one mechanism through which these mutations could contribute to tumorigenesis in cells of the corresponding tissues.We acknowledge funding from the Spanish Ministry of Economy and Competitivity (grant numbers SAF2009-06954 and SAF2012-36199) and the Spanish National Institute of Bioinformatics. AJ-S is supported by an FPI fellowshi

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

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

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

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

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