Characterization of a P-Rex1 gene signature in breast cancer cells

The Rac nucleotide Exchange Factor (Rac-GEF) P-Rex1 is highly expressed in breast cancer, specifically in the luminal subtype, and is an essential mediator of actin cytoskeleton reorganization and cell migratory responses induced by stimulation of ErbB and other tyrosine-kinase receptors. Heregulin (HRG), a growth factor highly expressed in mammary tumors, causes the activation of P-Rex1 and Rac1 in breast cancer cells via ErbB3, leading to a motile response. Since there is limited information about P-Rex1 downstream effectors, we carried out a microarray analysis to identify genes regulated by this Rac-GEF after stimulation of ErbB3 with HRG. In T-47D breast cancer cells, HRG treatment caused major changes in gene expression, including genes associated with motility, adhesion, invasiveness and metastasis. Silencing P-Rex1 expression from T-47D cells using RNAi altered the induction and repression of a subset of HRG-regulated genes, among them genes associated with extracellular matrix organization, migration, and chemotaxis. HRG induction of MMP10 (matrix metalloproteinase 10) was found to be highly sensitive both to P-Rex1 depletion and inhibition of Rac1 function by the GTPase Activating Protein (GAP) β2-chimaerin, suggesting the dependence of the P-Rex1/Rac1 pathway for the induction of genes critical for breast cancer invasiveness. Notably, there is a significant association in the expression of P-Rex1 and MMP10 in human luminal breast cancer, and their coexpression is indicative of poor prognosis.Facultad de Ciencias MédicasCentro de Investigaciones Inmunológicas Básicas y Aplicada

Characterization of a P-Rex1 gene signature in breast cancer cells

The Rac nucleotide Exchange Factor (Rac-GEF) P-Rex1 is highly expressed in breast cancer, specifically in the luminal subtype, and is an essential mediator of actin cytoskeleton reorganization and cell migratory responses induced by stimulation of ErbB and other tyrosine-kinase receptors. Heregulin (HRG), a growth factor highly expressed in mammary tumors, causes the activation of P-Rex1 and Rac1 in breast cancer cells via ErbB3, leading to a motile response. Since there is limited information about P-Rex1 downstream effectors, we carried out a microarray analysis to identify genes regulated by this Rac-GEF after stimulation of ErbB3 with HRG. In T-47D breast cancer cells, HRG treatment caused major changes in gene expression, including genes associated with motility, adhesion, invasiveness and metastasis. Silencing P-Rex1 expression from T-47D cells using RNAi altered the induction and repression of a subset of HRG-regulated genes, among them genes associated with extracellular matrix organization, migration, and chemotaxis. HRG induction of MMP10 (matrix metalloproteinase 10) was found to be highly sensitive both to P-Rex1 depletion and inhibition of Rac1 function by the GTPase Activating Protein (GAP) β2-chimaerin, suggesting the dependence of the P-Rex1/Rac1 pathway for the induction of genes critical for breast cancer invasiveness. Notably, there is a significant association in the expression of P-Rex1 and MMP10 in human luminal breast cancer, and their coexpression is indicative of poor prognosis.Facultad de Ciencias MédicasCentro de Investigaciones Inmunológicas Básicas y Aplicada

Transcriptional regulation of oncogenic protein kinase Cε (PKCε) by STAT1 and Sp1 proteins

Overexpression of PKCε, a kinase associated with tumor aggressiveness and widely implicated in malignant transformation and metastasis, is a hallmark of multiple cancers, including mammary, prostate, and lung cancer. To characterize the mechanisms that control PKCε expression and its up-regulation in cancer, we cloned an ∼1.6-kb promoter segment of the human PKCε gene (PRKCE) that displays elevated transcriptional activity in cancer cells. A comprehensive deletional analysis established two regions rich in Sp1 and STAT1 sites located between -777 and-105 bp (region A) and-921 and-796 bp (region B), respectively, as responsible for the high transcriptional activity observed in cancer cells. A more detailed mutagenesis analysis followed by EMSA and ChIP identified Sp1 sites in positions -668/-659 and-269/-247 as well as STAT1 sites in positions -880/-869 and- 793/-782 as the elements responsible for elevated promoter activity in breast cancer cells relative to normal mammary epithelial cells. RNAi silencing of Sp1 and STAT1 in breast cancer cells reduced PKCε mRNA and protein expression, as well as PRKCE promoter activity. Moreover, a strong correlation was found between PKCε and phospho-Ser-727 (active) STAT1 levels in breast cancer cells. Our results may have significant implications for the development of approaches to target PKCε and its effectors in cancer therapeutics.Centro de Investigaciones Inmunológicas Básicas y AplicadasFacultad de Ciencias Médica

PKCα modulates epithelial-to-mesenchymal transition and invasiveness of breast cancer cells through ZEB1

ZEB1 is a master regulator of the Epithelial-to-Mesenchymal Transition (EMT) program. While extensive evidence confirmed the importance of ZEB1 as an EMT transcription factor that promotes tumor invasiveness and metastasis, little is known about its regulation. In this work, we screened for potential regulatory links between ZEB1 and multiple cellular kinases. Exploratory in silico analysis aided by phospho-substrate antibodies and ZEB1 deletion mutants led us to identify several potential phospho-sites for the family of PKC kinases in the N-terminus of ZEB1. The analysis of breast cancer cell lines panels with different degrees of aggressiveness, together with the evaluation of a battery of kinase inhibitors, allowed us to expose a robust correlation between ZEB1 and PKCα both at mRNA and protein levels. Subsequent validation experiments using siRNAs against PKCα revealed that its knockdown leads to a concomitant decrease in ZEB1 levels, while ZEB1 knockdown had no impact on PKCα levels. Remarkably, PKCα-mediated downregulation of ZEB1 recapitulates the inhibition of mesenchymal phenotypes, including inhibition in cell migration and invasiveness. These findings were extended to an in vivo model, by demonstrating that the stable knockdown of PKCα using lentiviral shRNAs markedly impaired the metastatic potential of MDA-MB-231 breast cancer cells. Taken together, our findings unveil an unforeseen regulatory pathway comprising PKCα and ZEB1 that promotes the activation of the EMT in breast cancer cells.Fil: Llorens, María Candelaria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Rossi, Fabiana Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: García, Iris Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Cooke, Mariana. University of Pennsylvania; Estados UnidosFil: Abba, Martín Carlos. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Inmunológicas Básicas y Aplicadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Lopez Haber, Cynthia. University of Pennsylvania; Estados UnidosFil: Barrio Real, Laura. University of Pennsylvania; Estados UnidosFil: Vaglienti, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Rossi, Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Bocco, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Kazanietz, Marcelo Gabriel. University of Pennsylvania; Estados UnidosFil: Soria, Ramiro Gaston. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentin

Transcriptional regulation of oncogenic protein kinase Cε (PKCε) by STAT1 and Sp1 proteins

Overexpression of PKCε, a kinase associated with tumor aggressiveness and widely implicated in malignant transformation and metastasis, is a hallmark of multiple cancers, including mammary, prostate, and lung cancer. To characterize the mechanisms that control PKCε expression and its up-regulation in cancer, we cloned an ∼1.6-kb promoter segment of the human PKCε gene (PRKCE) that displays elevated transcriptional activity in cancer cells. A comprehensive deletional analysis established two regions rich in Sp1 and STAT1 sites located between -777 and-105 bp (region A) and-921 and-796 bp (region B), respectively, as responsible for the high transcriptional activity observed in cancer cells. A more detailed mutagenesis analysis followed by EMSA and ChIP identified Sp1 sites in positions -668/-659 and-269/-247 as well as STAT1 sites in positions -880/-869 and- 793/-782 as the elements responsible for elevated promoter activity in breast cancer cells relative to normal mammary epithelial cells. RNAi silencing of Sp1 and STAT1 in breast cancer cells reduced PKCε mRNA and protein expression, as well as PRKCE promoter activity. Moreover, a strong correlation was found between PKCε and phospho-Ser-727 (active) STAT1 levels in breast cancer cells. Our results may have significant implications for the development of approaches to target PKCε and its effectors in cancer therapeutics.Centro de Investigaciones Inmunológicas Básicas y AplicadasFacultad de Ciencias Médica

PKCα Modulates Epithelial-to-Mesenchymal Transition and Invasiveness of Breast Cancer Cells Through ZEB1

ZEB1 is a master regulator of the Epithelial-to-Mesenchymal Transition (EMT) program. While extensive evidence confirmed the importance of ZEB1 as an EMT transcription factor that promotes tumor invasiveness and metastasis, little is known about its regulation. In this work, we screened for potential regulatory links between ZEB1 and multiple cellular kinases. Exploratory in silico analysis aided by phospho-substrate antibodies and ZEB1 deletion mutants led us to identify several potential phospho-sites for the family of PKC kinases in the N-terminus of ZEB1. The analysis of breast cancer cell lines panels with different degrees of aggressiveness, together with the evaluation of a battery of kinase inhibitors, allowed us to expose a robust correlation between ZEB1 and PKCα both at mRNA and protein levels. Subsequent validation experiments using siRNAs against PKCα revealed that its knockdown leads to a concomitant decrease in ZEB1 levels, while ZEB1 knockdown had no impact on PKCα levels. Remarkably, PKCα-mediated downregulation of ZEB1 recapitulates the inhibition of mesenchymal phenotypes, including inhibition in cell migration and invasiveness. These findings were extended to an in vivo model, by demonstrating that the stable knockdown of PKCα using lentiviral shRNAs markedly impaired the metastatic potential of MDA-MB-231 breast cancer cells. Taken together, our findings unveil an unforeseen regulatory pathway comprising PKCα and ZEB1 that promotes the activation of the EMT in breast cancer cells.Centro de Investigaciones Inmunológicas Básicas y Aplicada

Nuevas aportaciones a la caracterización molecular del gen CHN2

[EN]the overall objective of this study was to analyze the presence of alterations in the gene CHN2 that could be associated with the emergence and development of cancer. Searching for pathogenic gene mutations CHN2 in tumor samples different types of cancer. Analysis of gene transcript levels in cell lines cancerígenas.Estudio CHN2 mechanisms regulating CHN2 gene promoter: promoter methylation[ES]el objetivo general de este trabajo se centró en analizar la presencia de alteraciones en el gen CHN2 que pudieran asociarse con la aparición y desarrollo de cáncer. Búsqueda de mutaciones patogénicas del gen CHN2 en muestras de tumor de distintos tipos de cáncer. Análisis de los niveles de transcripción del gen CHN2 en líneas celulares cancerígenas.Estudio de los mecanismos de regulación del promotor del gen CHN2: metilación del promoto

Association of a novel polymorphism of the β2-chimaerin gene (CHN2) with smoking

[Objective]: The CHN2 gene encodes the β2-chimaerin, a Rac-specific guanosine-5′-triphosphatase activating protein with an important role in the establishment of functional brain circuitry by controlling axon pruning. Genetic studies suggest that the CHN2 gene harbors variants that contribute to addiction vulnerability and smoking behavior. To further evaluate the role of β2-chimaerin in nicotine addiction, we investigated the association of 3 individual polymorphisms of the CHN2 gene with smoking dependence. [Methods]:Three hundred sixty-one healthy volunteers, 173 smokers (mean ± SD age, 60.4 ± 1.4 years) and 188 control subjects (mean ± SD age, 45.9 ± 1.4 years) were genotyped for 3 single-nucleotide polymorphisms in the CHN2 gene (rs3750103, rs12112301, and rs186911567). The association of these polymorphisms with smoking habits was analyzed. [Results]:There was no significant association of polymorphisms rs12112301 and rs3750103 with smoking. However, there was a significant difference in the frequency of the rs186911567 polymorphism between the smokers and the controls (P = 0.003).[Conclusions]: We report for the first time a significant association of the novel rs186911567 polymorphism of the CHN2 gene with smoking.This work was supported by the Spanish Ministry of Health (Fondo de Investigaciones Sanitarias, PI052096 to MJC, PI10/00219 to RGS), the Spanish Ministry of Science and Innovation cosponsored by the plan E (BFU2009-08051 to MJC), and the Castilla y Leon Autonomous Government (BIO103/VA44/11 to MJC).Peer Reviewe

Rho GTPases and their regulators in addiction: A focus on the association of a ß2-chimaerin polymorphism with smoking

Tobacco addiction is a complex disorder that involves multiple molecular mechanisms and is influenced by genetic factors. Repeated exposure to nicotine induces the remodeling of synaptic connections, a process that contributes to the long-lasting nature of tobacco addiction. Rho GTPases are key regulators of synaptic structure and function and, therefore, these proteins have a potential role in nicotine addiction. In this chapter we will briefly describe some studies that identify genes encoding Rho GTPases and their regulators as candidate genes for smoking-related behaviors, with special focus on the CHN2 gene. The CHN2 gene encodes the β2-chimaerin, a member of the chimaerin family of GTPase activating proteins that selectively inactivates the GTPase Rac. Genetic studies suggest that nucleotide variants of the CHN2 gene may influence success in smoking cessation. In addition, work has identified a significant association of the rs186911567 polymorphism in the CHN2 gene with smoking in a Spanish population. We discuss how this polymorphism could alter β2-chimaerin function and the signaling pathways mediated by this protein that could influence smoking behavior.Peer Reviewe

A new role of the Rac-GAP ß2-chimaerin in cell adhesion reveals opposite functions in breast cancer initiation and tumor progression

β2-chimaerin is a Rac1-specific negative regulator and a candidate tumor suppressor in breast cancer but its precise function in mammary tumorigenesis in vivo is unknown. Here, we study for the first time the role of β2-chimaerin in breast cancer using a mouse model and describe an unforeseen role for this protein in epithelial cell-cell adhesion. We demonstrate that expression of β2-chimaerin in breast cancer epithelial cells reduces E-cadherin protein levels, thus loosening cell-cell contacts. In vivo, genetic ablation of β2-chimaerin in the MMTV-Neu/ErbB2 mice accelerates tumor onset, but delays tumor progression. Finally, analysis of clinical databases revealed an inverse correlation between β2-chimaerin and E-cadherin gene expressions in Her2+ breast tumors. Furthermore, breast cancer patients with low β2-chimaerin expression have reduced relapse free survival but develop metastasis at similar times. Overall, our data redefine the role of β2-chimaerin as tumor suppressor and provide the first in vivo evidence of a dual function in breast cancer, suppressing tumor initiation but favoring tumor progression.This work was initially supported by a grant from the Spanish Ministry of Economy and Competitiveness to MJC (BFU2009-08051), but could be finished only thanks to the support from the Castilla-León Autonomous Government to MJC (grants BIO103/VA44/11, BIO/VA22/14, CSI090U14 and BIO/VA34/15).Peer Reviewe