c-Src signaling in triple negative breast cancer cells: role of Cyr61

Póster presentado al CNIO Frontiers Meeting: "Metastasis Initiation: Mechanistic Insights and Therapeutic Opportunities", celebrado en el Spanish National Cancer Research Centre (CNIO) Auditorium del 28 al 30 de septiembre de 2015.The SFKs (Src Family Kinases) control cellular pathways involved in division, motility, adhesion, angiogenesis, and survival. Therefore, their deregulation is associated with tumorigenesis, and metastasis. c-Src is overexpressed and/or aberrantly activated in epithelial tumors: pancreatic, colorectal, prostatic, ovarian, breast, etc. We previously showed that SFKs catalytic inhibitors (Dasatinib, PP2, and SU6656) reduce proliferation, migration, and invasiveness of MDA-MB-231. Here, we analyzed c-Src contribution to initial steps of metastasis by Tet-On conditional expression of a specific shRNA-c-Src, which suppressed c-Src mRNA and protein levels in MDA-MB-231. c-Src suppression did not alter cell proliferation or survival, but it significantly reduced anchorage-independent growth. Concomitantly with diminished tyrosine-phosphorylation/activation of Fak, caveolin-1, paxillin and p130CAS, c-Src depletion inhibited migration, invasion, transendothelial migration, and reduced MMP2, MMP7 and MMP9 in secretome. Quantitative proteomic analyses of secretome showed that Cyr61 levels, detected in exosomal fraction, were diminished upon shRNA-c-Src expression. However, Cyr61 expression was unaltered inside cells. Cyr61 partially colocalized with cis-Golgi gp74 marker, and with exosomal marker CD63, but c-Src depletion did not alter their distribution. In SUM159PT, transient c-Src suppression also reduced secreted exosomal Cyr61. Furthermore, conditional expression of c-Src dominant negative mutant (c-Src-K295M/Y527F) in MDA-MB-231 and in SUM159PT diminished secreted Cyr61 as well. Cyr61 transient suppression in MDA-MB-231 inhibited invasion and transendothelial migration. Finally, in both MDA-MB-231 and SUM159PT, a neutralizing Cyr61 antibody restrained migration. Collectively, these results suggest that c-Src regulates secreted proteins, including exosomal Cyr61, which are involved in modulating the metastatic potential of triple negative breast cancer cells.Peer Reviewe

Screening health-promoting compounds for their capacity to induce the activity of FOXO3

Several chemical compounds including natural products have been suggested as being effective against age-related diseases or as beneficial for a healthy life. On the other hand, forkhead box O (FOXO) proteins are emerging as key cellular components associated with extreme human longevity. FOXO proteins are mainly regulated by posttranslational modifications and as these modifications are reversible, activation and inactivation of FOXO are attainable through pharmacological treatment. Here, we questioned whether a panel of compounds with known health-beneficial properties has the capacity to induce the activity of FOXO factors. We show that resveratrol, a phytoalexin present in grapes and other food products, the amide alkaloid piperlongumine found in the fruit of the long pepper, and the plant-derived β-carboline compound harmine induced nuclear translocation of FOXO3. We also show that piperlongumine and harmine but not resveratrol activate FOXO-dependent transcription. We determined the half maximal effective concentration (EC50) values for resveratrol, piperlongumine, and harmine for FOXO translocation, and analyzed their inhibitory impact on chromosomal maintenance 1 (CRM1)-mediated nuclear export and the production of reactive oxygen species (ROS). We also used chemical biology approach and Western blot analysis to explore the underlying molecular mechanisms. We show that harmine, piperlongumine, and resveratrol activate FOXO3 independently of phosphoinositide 3-kinase (PI3K)/AKT signaling and the CRM1-mediated nuclear export. The effect of harmine on FOXO3 activity is at least partially mediated through the inhibition of dual-specificity tyrosine (Y) phosphorylationregulated kinase 1A (DYRK1A) and can be reverted by the inhibition of sirtuins (SIRTs).This work was supported by Fundação para a Ciência e a Tecnologia (FCT) Research Center Grant UID/BIM/04773/2013 Centre for Biomedical Research (CBMR) and by the Spanish Ministry of Science, Innovation and Universities through grant RTI2018-094629-B-I00 to W.L. B.I.F. was supported by FCTSFRH/BPD/100434/2014 and Marie Curie Individual Fellowship project TRIBBLES (#748585). This work was also supported by 2 LPCC-NRS/Terry Fox grants (2016/2017 and 2017/2018). F.B. was supported by Deutsche Forschungsgemeinschaft (grant BR1034/6-1)

c-Src functionality controls self-renewal and glucose metabolism in MCF7 breast cancer stem cells

Deregulation of Src kinases is associated with cancer. We previously showed that SrcDN conditional expression in MCF7 cells reduces tumorigenesis and causes tumor regression in mice. However, it remained unclear whether SrcDN affected breast cancer stem cell functionality or it reduced tumor mass. Here, we address this question by isolating an enriched population of Breast Cancer Stem Cells (BCSCs) from MCF7 cells with inducible expression of SrcDN. Induction of SrcDN inhibited self-renewal, and stem-cell marker expression (Nanog, Oct3-4, ALDH1, CD44). Quantitative proteomic analyses of mammospheres from MCF7-Tet-On-SrcDN cells (data are available via ProteomeXchange with identifier PXD017789, project DOI: 10.6019/PXD017789) and subsequent GSEA showed that SrcDN expression inhibited glycolysis. Indeed, induction of SrcDN inhibited expression and activity of hexokinase, pyruvate kinase and lactate dehydrogenase, resulting in diminished glucose consumption and lactate production, which restricted Warburg effect. Thus, c-Src functionality is important for breast cancer stem cell maintenance and renewal, and stem cell transcription factor expression, effects linked to glucose metabolism reduction.This work has been supported by grand SAF2016–75991-R (MINECO, AEI/FEDER, UE) to Jorge Martín-Pérez and ISCIII [grand PI 16/00789] to Miguel Ángel Fernández-Moreno. Víctor Mayoral-Varo was supported by the grand SAF2016–75991-R (MINECO, AEI/FEDER, UE). We acknowledge support for publication fee by the CSIC Open Access Publication Support Initiative through its Unit for Information Resources for Research (URICI)

The critical role of trib2 in cancer and therapy resistance

© 2021 by the authors.The Tribbles pseudokinases family consists of TRIB1, TRIB2, TRIB3 and STK40 and, although evolutionarily conserved, they have distinctive characteristics. Tribbles members are expressed in a context and cell compartment-dependent manner. For example, TRIB1 and TRIB2 have potent oncogenic activities in vertebrate cells. Since the identification of Tribbles proteins as modulators of multiple signalling pathways, recent studies have linked their expression with several pathologies, including cancer. Tribbles proteins act as protein adaptors involved in the ubiquitin-proteasome degradation system, as they bridge the gap between substrates and E3 ligases. Between TRIB family members, TRIB2 is the most ancestral member of the family. TRIB2 is involved in protein homeostasis regulation of C/EBPα, β-catenin and TCF4. On the other hand, TRIB2 interacts with MAPKK, AKT and NFkB proteins, involved in cell survival, proliferation and immune response. Here, we review the characteristic features of TRIB2 structure and signalling and its role in many cancer subtypes with an emphasis on TRIB2 function in therapy resistance in melanoma, leukemia and glioblastoma. The strong evidence between TRIB2 expression and chemoresistance provides an attractive opportunity for targeting TRIB2.This work was supported by the Spanish Ministry of Science, Innovation and Universities through Grant RTI2018-094629-B-I00 to W.L

L'Auto-vélo : automobilisme, cyclisme, athlétisme, yachting, aérostation, escrime, hippisme / dir. Henri Desgranges

04 août 19101910/08/04 (A11,N3580)

Role of Src Family Kinases in Prolactin Signaling

Prolactin (PRL) is a polypeptide hormone/cytokine mainly synthesized by the lactotrophic cells of the adenohypophysis. In addition to the best-known role in mammary gland development and the functional differentiation of its epithelium, PRL is involved in regulation of multiple physiological processes in higher organisms contributing to their homeostasis. PRL has been also associated with pathology, including breast cancer. Therefore, it is relevant to determine the molecular mechanisms by which PRL controls cellular functions. Here, we analyze the role of Src family kinases (SFKs) in the intracellular signaling pathways controlled by PRL in several model systems. The data show that SFKs are essential components in transmitting signals upon PRL receptor stimulation, as they control activation of Jak2/Stat5 and other routes that regulate PRL cellular responses.The authors acknowledge the support of the SAF2009-09254/SAF2012-38048 grant.Peer Reviewe

Cyr61 as mediator of Src signaling in triple negative breast cancer cells

This work is licensed under a Creative Commons Attribution 3.0 License.-- et al.SFKs are involved in tumorigenesis and metastasis. Here we analyzed c-Src contribution to initial steps of metastasis by tetracycline-dependent expression of a specific shRNA-c-Src, which suppressed c-Src mRNA and protein levels in metastatic MDA-MB-231 cells. c-Src suppression did not alter cell proliferation or survival, but it significantly reduced anchorage-independent growth. Concomitantly with diminished tyrosine-phosphorylation/activation of Fak, caveolin-1, paxillin and p130CAS, c-Src depletion also inhibited cellular migration, invasion and transendothelial migration. Quantitative proteomic analyses of the secretome showed that Cyr61 levels, which were detected in the exosomal fraction, were diminished upon shRNA-c-Src expression. In contrast, Cyr61 expression was unaltered inside cells. Cyr61 partially colocalized with cis-Golgi gp74 marker and with exosomal marker CD63, but c-Src depletion did not alter their cellular distribution. In SUM159PT cells, transient c-Src suppression also reduced secreted exosomal Cyr61 levels. Furthermore, conditional expression of a c-Src dominant negative mutant (SrcDN, c-Src-K295M/Y527F) in MDA-MB-231 and in SUM159PT diminished secreted Cyr61 as well. Cyr61 transient suppression in MDA-MB-231 inhibited invasion and transendothelial migration. Finally, in both MDA-MB-231 and SUM159PT, a neutralizing Cyr61 antibody restrained migration. Collectively, these results suggest that c-Src regulates secreted proteins, including the exosomal Cyr61, which are involved in modulating the metastatic potential of triple negative breast cancer cells.MP. S-B. was supported by a FPI fellowship from Ministerio de Economía y Competitividad. JM-P is a member of GEICAM and of IdPAZ. This work was supported by SAF2009–09254 and SAF2012–38048 from Ministerio de Economía y Competitividad, Spain. KUW is supported, in part, by the Public Health Service grants CA117930.Peer Reviewe

Validation of miR205 targets.

<p>(A) miR205 expression determined by a TaqMan^TM assay in SUM159_Control (Ctrl), and SUM159_miR205 (miR) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188637#sec002" target="_blank">Materials and Methods</a>). Results are shown as mean ± SD of relative miR205 levels in three independent experiments in triplicate, considering arbitrarily the first sample of SUM159_Control (Ctrl) as 1 (**p<0.01). (B) EGFP-positive cells were isolated by FACS-Vantage SE from SUM159_miR205 infected with a bicistronic lentivirus containing Anti-miR205/EGFP to establish SUM159_miR205_Anti-miR205 cell line (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188637#sec002" target="_blank">Materials and Methods</a>). Result is shown as histogram from background (EGFP-) and positive populations (EGFP+) of sorted SUM159_miR205_Anti-miR205 cells. (C) Immunoblotting detection of VEGF-A, ErbB3, ZEB1, Fyn, and Lyn A/B from extracts of SUM159_Control (Ctrl), SUM159_miR205 (miR), SUM159_Control_Anti-miR205 (Ctrl_Anti-miR), and SUM159_miR205_Anti-miR205 (miR_Anti-miR) cells; GAPDH or β-actin were determined for loading controls. These are representative results from 3 independent experiments. The ratios referred to SUM159_Control (Ctrl) considered as 1.</p

Role of miR205 in mammospheres formation.

<p>(A) Cell extracts from exponentially growing SUM159_Control (Ctrl), _miR205 (miR), _Control_Anti-miR205 (Ctrl_Anti-miR), _miR205_Anti-miR205 (miR_Anti-miR) cells were used to determine expression of CD44, TAZ, E2A.E12, Twist, Snail1, and CK5 by immunoblotting, β-Actin and GAPDH were assessed as loading control. (B) Cells plated at 2x10³ cells/well were cultured in conditions for mammosphere growth, after 10 days, mammospheres were disaggregated and seeded again, and at the third passage, the Spheres Formation Efficiency (SFE) was determined (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188637#sec002" target="_blank">Material and Methods</a>). The results are represented as percentage of SFE ± SD from three independent experiments in triplicate. (***p<0.001). Immunoblotting detection in mammospheres extracts of CD44, TAZ, E2A, and ALDH1, GAPDH was used as loading control. They are representative results from three independent experiments. The ratios referred to SUM159_Control (Ctrl) considered as 1.</p

Role of miR205 in cell proliferation and anchorage-independent growth.

<p>(A) Cell proliferation was determined by performing Trypan blue exclusion assay in SUM159_Control (Ctrl), SUM159_miR205 (miR), SUM159_Control_Anti-miR205 (Ctrl_Anti-miR), SUM159_miR205_Anti-miR205 (miR_Anti-miR) cells. Cells were seeded at 3x10⁵ cells/60mm dishes, and 72h later both Trypan blue-negative (viable cells) and Trypan blue-positive cells (dead cells) were counted (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188637#sec002" target="_blank">Materials and Methods</a>). Results are shown as measurements of Trypan blue-negative cells, mean ± SD from four independent experiments in triplicate. (B) Immunoblotting detection of p27^Kip1, cyclin D1, Myc, and PARP in cell extracts. Membranes were reblotted with anti-β-actin for loading control. Results are representative of three independent experiments. The ratios referred to SUM159_Control (Ctrl) considered as 1. (C) Number of colonies/field obtained after cell growth in soft-agar for 10 days (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188637#sec002" target="_blank">Material and Methods</a>). Average of colony number/field ± SD from three independent experiments in triplicate. (***p<0.001).</p