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

Opposite effects of protein kinase C beta1 (PKCβ1) and PKCε in the metastatic potential of a breast cancer murine model

In this paper we investigated whether protein kinase C (PKC) b1 and PKCe, members of the classical and novel PKC family respectively, induce phenotypic alterations that could be associated with tumor progression and metastatic dissemination in a murine model of breast cancer. Stable overexpression of PKCb1 in LM3 cells altered their ability to proliferate, adhere, and survive, and impaired their tumorigenicity and metastatic capacity. Moreover, PKCb1 induced the re-expression of fibronectin, an extracellular matrix glycoprotein which loss has been associated with the acquisition of a transformed phenotype in different cell models, and exerted an important inhibition on proteases production, effects that probably impact on LM3 invasiveness and dissemination. Conversely, PKCe overexpression enhanced LM3 survival, anchorage-independent growth, and caused a significant increase in spontaneous lung metastasis. Our results suggest PKCb1 functions as an inhibitory protein for tumor growth and metastasis dissemination whereas PKCe drives metastatic dissemination without affecting primary tumor growth.Fil: Grossoni, Valeria Carla. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Todaro, Laura Beatriz. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Kazanietz, Marcelo Gabriel. University of Pennsylvania; Estados UnidosFil: Bal, Elisa Dora. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Urtreger, Alejandro Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentin

PKCε Is an Essential Mediator of Prostate Cancer Bone Metastasis.

UNLABELLED: The bone is a preferred site for metastatic homing of prostate cancer cells. Once prostate cancer patients develop skeletal metastases, they eventually succumb to the disease; therefore, it is imperative to identify key molecular drivers of this process. This study examines the involvement of protein kinase C epsilon (PKCε), an oncogenic protein that is abnormally overexpressed in human tumor specimens and cell lines, on prostate cancer cell bone metastasis. PC3-ML cells, a highly invasive prostate cancer PC3 derivative with bone metastatic colonization properties, failed to induce skeletal metastatic foci upon inoculation into nude mice when PKCε expression was silenced using shRNA. Interestingly, while PKCε depletion had only marginal effects on the proliferative, adhesive, and migratory capacities of PC3-ML cells in vitro or in the growth of xenografts upon s.c. inoculation, it caused a significant reduction in cell invasiveness. Notably, PKCε was required for transendothelial cell migration (TEM) as well as for the growth of PC3-ML cells in a bone biomimetic environment. At a mechanistic level, PKCε depletion abrogates the expression of IL1β, a cytokine implicated in skeletal metastasis. Taken together, PKCε is a key factor for driving the formation of bone metastasis by prostate cancer cells and is a potential therapeutic target for advanced stages of the disease. IMPLICATIONS: This study uncovers an important new function of PKCε in the dissemination of cancer cells to the bone; thus, highlighting the promising potential of this oncogenic kinase as a therapeutic target for skeletal metastasis

ΔNp63α suppresses cells invasion by downregulating PKCγ/Rac1 signaling through miR-320a

ΔNp63α, a member of the p53 family of transcription factors, is overexpressed in a number of cancers and plays a role in proliferation, differentiation, migration, and invasion. ΔNp63α has been shown to regulate several microRNAs that are involved in development and cancer. We identified miRNA miR-320a as a positively regulated target of ΔNp63α. Previous studies have shown that miR-320a is downregulated in colorectal cancer and targets the small GTPase Rac1, leading to a reduction in noncanonical WNT signaling and EMT, thereby inhibiting tumor metastasis and invasion. We showed that miR-320a is a direct target of ΔNp63α. Knockdown of ΔNp63α in HaCaT and A431 cells downregulates miR-320a levels and leads to a corresponding elevation in PKCγ transcript and protein levels. Rac1 phosphorylation at Ser71 was increased in the absence of ΔNp63α, whereas overexpression of ΔNp63α reversed S71 phosphorylation of Rac1. Moreover, increased PKCγ levels, Rac1 phosphorylation and cell invasion observed upon knockdown of ΔNp63α was reversed by either overexpressing miR-320a mimic or Rac1 silencing. Finally, silencing PKCγ or treatment with the PKC inhibitor Gö6976 reversed increased Rac1 phosphorylation and cell invasion observed upon silencing ΔNp63α. Taken together, our data suggest that ΔNp63α positively regulates miR-320a, thereby inhibiting PKCγ expression, Rac1 phosphorylation, and cancer invasion.Fil: Aljagthmi, Amjad A.. Wright State University; Estados UnidosFil: Hill, Natasha T.. Wright State University; Estados UnidosFil: Cooke, Mariana. University of Pennsylvania; Estados UnidosFil: Kazanietz, Marcelo Gabriel. 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: Long, Weiwen. Wright State University; Estados UnidosFil: Kadakia, Madhavi P.. Wright State University; Estados Unido

P-REX1-Independent, Calcium-Dependent RAC1 Hyperactivation in Prostate Cancer

The GTPase Rac1 is a well-established master regulator of cell motility and invasiveness contributing to cancer metastasis. Dysregulation of the Rac1 signaling pathway, resulting in elevated motile and invasive potential, has been reported in multiple cancers. However, there are limited studies on the regulation of Rac1 in prostate cancer. Here, we demonstrate that aggressive androgen-independent prostate cancer cells display marked hyperactivation of Rac1. This hyperactivation is independent of P-Rex1 activity or its direct activators, the PI3K product PIP3 and Gβγ subunits. Furthermore, we demonstrate that the motility and invasiveness of PC3 prostate cancer cells is independent of P-Rex1, supporting the analysis of publicly available datasets indicating no correlation between high P-Rex1 expression and cancer progression in patients. Rac1 hyperactivation was not related to the presence of activating Rac1 mutations and was insensitive to overexpression of a Rac-GAP or the silencing of specific Rac-GEFs expressed in prostate cancer cells. Interestingly, active Rac1 levels in these cells were markedly reduced by elevations in intracellular calcium or by serum stimulation, suggesting the presence of an alternative means of Rac1 regulation in prostate cancer that does not involve previously established paradigms.Centro 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

The RacGAP β2-Chimaerin Selectively Mediates Axonal Pruning in the Hippocampus

SummaryAxon pruning and synapse elimination promote neural connectivity and synaptic plasticity. Stereotyped pruning of axons that originate in the hippocampal dentate gyrus (DG) and extend along the infrapyramidal tract (IPT) occurs during postnatal murine development by neurite retraction and resembles axon repulsion. The chemorepellent Sema3F is required for IPT axon pruning, dendritic spine remodeling, and repulsion of DG axons. The signaling events that regulate IPT axon pruning are not known. We find that inhibition of the small G protein Rac1 by the Rac GTPase-activating protein (GAP) β2-Chimaerin (β2Chn) mediates Sema3F-dependent pruning. The Sema3F receptor neuropilin-2 selectively binds β2Chn, and ligand engagement activates this GAP to ultimately restrain Rac1-dependent effects on cytoskeletal reorganization. β2Chn is necessary for axon pruning both in vitro and in vivo, but it is dispensable for axon repulsion and spine remodeling. Therefore, a Npn2/β2Chn/Rac1 signaling axis distinguishes DG axon pruning from the effects of Sema3F on repulsion and dendritic spine remodeling

A regulatory axis connecting PKCα and ZEB1 modulates epithelial-mesenchymal transition and invasiveness of breast cancer cells

The Epithelial-Mesenchymal Transition (EMT) is an essential program of embryogenesis and tumor progression, ZEB1 is amaster regulator of the EMT. While extensive evidence confirmed the importance of ZEB1 as an EMT transcription factor thatpromotes tumor invasiveness and metastasis, little is known about its regulation. The aim of this work was to explore thesignaling pathways that regulate ZEB1 levels and functionality, and how this regulation impacts on the dynamics of the EMT incancer cells. We screened for potential regulatory links between ZEB1 and multiple cellular kinases. Our preliminary in silicostudies revealed a plethora of potential phosphorylation sites for several kinases. Due to this level of complexity, we decided tofollow up this analysis using ZEB1 deletion mutants (ZD1-HD and NZEB1), these constructs represent 60% and 10% of the full-length protein, respectively, and both retain the capacity to repress the E-cadherin promoter in cells, as determined with aluciferase reporter assay in cells. Intriguingly, we found that NZEB1 is enriched in PKC-specific sites and a substrate of p-PKCantibodies in cell extracts, thus suggesting an unforeseen regulatory role of PKC kinases on ZEB1 biology. Our initialexperiments showed that NZEB1 and full length ZEB1 (ZEB1-FL) levels were actively reduced when NMuMMG-NZEB1 orMDA-MB-231cells were treated with the pharmacological inhibitors of PKCs GF109203X and Gö69761. To study thepenetrance of this phenotype with ZEB1-FL, we investigated the levels of three well-known PKCs paralogs (α, δ and ε), ZEB1and EMT makers in a group of 9 breast cancer cell lines. Strikingly, we found that PKCα and ZEB1 had a significant positivecorrelation, being both proteins overexpressed in cell lines with more aggressive phenotypes. Subsequent validation experimentsusing siRNAs against PKCα in MDA-MB231 cells 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 theinhibition of mesenchymal phenotypes, including inhibition in cell migration and invasiveness. These findings were extended toan in vivo model, by demonstrating that the stable knockdown of PKCα using lentiviral shRNAs markedly impaired themetastatic potential of MDA-MB-231 breast cancer cells. Conclusion: We demonstrated for the first time that the PKCα signaltransduction pathway regulates the biological function of ZEB1, defining a novel regulatory axis of the EMT program in breastcancer cell lines, which might stimulate the evaluation of PKC inhibitors for metastatic breast cancer therapy.Fil: Llorens de Los Ríos, María Candelaria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Rossi, Fabiana Alejandra. 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 de Investigación y Desarrollo en Inmunología y Enfermedades Infecciosas. Universidad Católica de Córdoba. Centro de Investigación y Desarrollo en Inmunología y Enfermedades Infecciosas; ArgentinaFil: Cooke, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina. University of Pennsylvania; Estados UnidosFil: Rossi, Mari. 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; 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; ArgentinaThe LV Annual SAIB Meeting and XIV PABMB CongressSaltaArgentinaSociedad Argentina de Investigación Bioquímica y Biología MolecularPanamerican Association of Biochemestry and Molecular Biolog

Protein Kinase C Epsilon Cooperates with PTEN Loss for Prostate Tumorigenesis through the CXCL13-CXCR5 Pathway

PKCε, an oncogenic member of the PKC family, is aberrantly overexpressed in epithelial cancers. To date, little is known about functional interactions of PKCε with other genetic alterations, as well as the effectors contributing to its tumorigenic and metastatic phenotype. Here, we demonstrate that PKCε cooperates with the loss of the tumor suppressor Pten for the development of prostate cancer in a mouse model. Mechanistic analysis revealed that PKCε overexpression and Pten loss individually and synergistically upregulate the production of the chemokine CXCL13, which involves the transcriptional activation of the CXCL13 gene via the non-canonical nuclear factor κB (NF-κB) pathway. Notably, targeted disruption of CXCL13 or its receptor, CXCR5, in prostate cancer cells impaired their migratory and tumorigenic properties. In addition to providing evidence for an autonomous vicious cycle driven by PKCε, our studies identified a compelling rationale for targeting the CXCL13-CXCR5 axis for prostate cancer treatment.Centro 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