Role of c-Src in Human MCF7 Breast Cancer Cell Tumorigenesis

Peer Reviewe

MicroRNA-dependent regulation of transcription in non-small cell lung cancer

Squamous cell lung cancer (SCC) and adenocarcinoma are the most common histological subtypes of non-small cell lung cancer (NSCLC), and have been traditionally managed in the clinic as a single entity. Increasing evidence, however, illustrates the biological diversity of these two histological subgroups of lung cancer, and supports the need to improve our understanding of the molecular basis beyond the different phenotypes if we aim to develop more specific and individualized targeted therapy. The purpose of this study was to identify microRNA (miRNA)-dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. In this work, paired miRNA (667 miRNAs by TaqMan Low Density Arrays (TLDA)) and mRNA profiling (Whole Genome 44 K array G112A, Agilent) was performed in tumor samples of 44 NSCLC patients. Nine miRNAs and 56 mRNAs were found to be differentially expressed in SCC versus adenocarcinoma samples. Eleven of these 56 mRNA were predicted as targets of the miRNAs identified to be differently expressed in these two histological conditions. Of them, 6 miRNAs (miR-149, miR-205, miR-375, miR-378, miR- 422a and miR-708) and 9 target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1) were validated by quantitative PCR in an independent cohort of 41 lung cancer patients. Furthermore, the inverse correlation between mRNAs and microRNAs expression was also validated. These results suggest miRNA-dependent transcriptional regulation differences play an important role in determining key hallmarks of NSCLC, and may provide new biomarkers for personalized treatment strategies

PTRF/Cavin-1 and MIF Proteins Are Identified as Non-Small Cell Lung Cancer Biomarkers by Label-Free Proteomics

With the completion of the human genome sequence, biomedical sciences have entered in the “omics” era, mainly due to high-throughput genomics techniques and the recent application of mass spectrometry to proteomics analyses. However, there is still a time lag between these technological advances and their application in the clinical setting. Our work is designed to build bridges between high-performance proteomics and clinical routine. Protein extracts were obtained from fresh frozen normal lung and non-small cell lung cancer samples. We applied a phosphopeptide enrichment followed by LC-MS/MS. Subsequent label-free quantification and bioinformatics analyses were performed. We assessed protein patterns on these samples, showing dozens of differential markers between normal and tumor tissue. Gene ontology and interactome analyses identified signaling pathways altered on tumor tissue. We have identified two proteins, PTRF/cavin-1 and MIF, which are differentially expressed between normal lung and non-small cell lung cancer. These potential biomarkers were validated using western blot and immunohistochemistry. The application of discovery-based proteomics analyses in clinical samples allowed us to identify new potential biomarkers and therapeutic targets in non-small cell lung cancer

Structure-dependent cytotoxic effects of eremophilanolide sesquiterpenes

The aim of this research was to determine the cytotoxic action of sixteen structurally-related eremophilane-type sesquiterpenes, isolated from several species of Senecio, against a panel of cancer cell lines. The cytotoxic activities were evaluated by WST-1 test and the IC50 values calculated. The investigated compounds exerted dose-dependent cytotoxic actions against selected cancer cell lines and no-tumoral HS5 cell line. The comparative structure-activity relationships demonstrated the importance of C-1, C-6, and C-8 substituents in the molecule. Our results show that eremophilane-type sesquiterpenes may represent an important source of novel potential antitumor agents due to their pronounced cytotoxic actions towards malignant cells.This work has been supported by grants CTQ2012-38219-C03-01 and CTQ2015-64049-C3-1-R, MINECO, Spain.Peer Reviewe

A non-catalytic function of the Src family tyrosine kinases controls prolactin-induced Jak2 signaling

The cytokine prolactin (PRL) plays important roles in the proliferation and differentiation of the mammary gland and it has been implicated in tumorigenesis. The prolactin receptor (PRLR) is devoid of catalytic activity and its mitogenic response is controlled by cytoplasmic tyrosine kinases of the Src (SFK) and Jak families. How PRLR uses these kinases for signaling is not well understood. Previous studies indicated that PRLR-induced Jak2 activation does not require SFK catalytic activity in favor of separate signaling operating on this cellular response. Here we show that, nevertheless, PRLR requires Src-SH2 and -SH3 domains for Jak2 signaling. In W53 lymphoid cells, conditional expression of two c-Src non-catalytic mutants, either SrcK295M/Y527F or Src{increment}K, whose SH3 and SH2 domains are exposed, controls Jak2/Stat5 activation by recruiting Jak2, avoiding its activation by endogenous active SFK. In contrast, the kinase inactive SrcK295M mutant, with inaccessible SH3 and SH2 domains, does not. Furthermore, all three mutants attenuate PRLR-induced Akt and p70S6K activation. Accordingly, PRLR-induced Jak2/Stat5 signaling is inhibited in MCF7 breast cancer cells by Src depletion, expression of SrcK295M/Y527F or active Src harboring an inactive SH2 (SrcR175L) or SH3 domain (SrcW118A). Finally, Jak2/Stat5 pathway is also reduced in Src-/- mice mammary glands. We thus conclude that, in addition to Akt and p70S6K, SFK regulate PRLR-induced Jak2 signaling through a kinase-independent mechanism. © 2009 Elsevier Inc. All rights reserved.This work was supported by grants from MCyT (SAF2003-02188/SAF2006-00371/SAF2009-09254), FIS (01-1316/04-0682/03C03-10), Fundación de Investigación Médica Mutua Madrileña and from MECHF2007-0107 to JM-P and from Public Health Service: R01CA085736–P01HD38129 to SMA. JMG-M was supported by a FIS fellowship and SAF2003-02188/SAF2006-00371, LG by a fellowship from Fundación Carolina and LG and MTA-O by FIS 03C03/10. HW had a fellowship from Cancer League of Colorado and University of Colorado Cancer Center. JMP is member of GEICAM (Spanish Research Group on Breast Cancer).Peer Reviewe

Pyrrolizidine alkaloids from Canarian endemic plants and their biological effects

14 pages, 2 figures, 5 tables.-- Available online Oct 25, 2007.Pyrrolizidine alkaloid (PA) producing plants belonging to the Boraginaceae (Echium wildpretti) and Asteraceae (Canariothamnus palmensis, Kleinia neriifolia, Pericallis appendiculata, Pericallis echinata, Pericallis hansenii, Pericallis multiflora, Pericallis steetzii and Senecio bollei) were selected to study their alkaloidal composition and the defensive properties (antifeedant and phytotoxic effects) of their ethanolic and alkaloidal extracts plus their isolated PAs against insects (Spodoptera littoralis, Leptinotarsa decemlineata, the aphids Myzus persicae and Rhopalosiphum padi) and Lactuca sativa seeds. We also tested the selective cytotoxic effects of these PAs on insect-derived Sf9 and mammalian CHO cells. Most of the insect antifeedant effects were found in the ethanolic extracts. The isolated PAs had species- and structure-dependent antifeedant effects and all of them decreased L. sativa radicle growth, suggesting a specific mode of action against insects and a generalized one against plants. Given the relatively low alkaloid content of these species, we assume that their herbivore defenses are not only alkaloid-based.This work was supported by grants CTQ2006-15597-C02-01/PPQ and an I3P-CSIC fellowship to D.M. Domínguez. Plant collection was possible thanks to the "Cabildos" of Tenerife, Gomera and Lanzarote

Prevalence of Hypoproteinemia and Hypoalbuminemia in Pregnant Women from Three Different Socioeconomic Populations

Protein requirements of pregnant women are increased due to anatomical and physiological changes. However, optimal levels of plasma proteins do not receive adequate attention from health professionals and researchers. We aimed to evaluate the plasma protein status in pregnant women receiving care at health centers, with the intention of identifying potential deficiency states and their relationship with quality of life during pregnancy. This is a population-based, prospective, and observational study among a cohort of 215 pregnant women from three different socioeconomic areas (urban, semi-urban, and rural). Blood samples in the first (T1), second (T2), and third (T3) trimester of pregnancy were obtained to quantify the proteins and albumin levels. Statically significant differences regarding the age of pregnant women (p = 0.002), education status (p = 0.034), and socioeconomic level (p = 0.000), were found among groups. Prevalence of protein and albumin deficits was much higher in women from rural and semi-urban areas than in women from urban areas (p = 0.001). Moreover, these deficits were associated with the appearance of edema. Plasma total protein deficit could be an undervalued public health problem in pregnant women receiving prenatal care that could affect the quality of life in the gestational period. It would be important to establish reference intervals for plasma protein monitoring in each trimester of pregnancy, and protein levels should be measured routinely throughout pregnancy

Pyrrolizidine alkaloids from Canarian endemic plants and their biological effects

14 pages, 2 figures, 5 tables.-- Available online Oct 25, 2007.Pyrrolizidine alkaloid (PA) producing plants belonging to the Boraginaceae (Echium wildpretti) and Asteraceae (Canariothamnus palmensis, Kleinia neriifolia, Pericallis appendiculata, Pericallis echinata, Pericallis hansenii, Pericallis multiflora, Pericallis steetzii and Senecio bollei) were selected to study their alkaloidal composition and the defensive properties (antifeedant and phytotoxic effects) of their ethanolic and alkaloidal extracts plus their isolated PAs against insects (Spodoptera littoralis, Leptinotarsa decemlineata, the aphids Myzus persicae and Rhopalosiphum padi) and Lactuca sativa seeds. We also tested the selective cytotoxic effects of these PAs on insect-derived Sf9 and mammalian CHO cells. Most of the insect antifeedant effects were found in the ethanolic extracts. The isolated PAs had species- and structure-dependent antifeedant effects and all of them decreased L. sativa radicle growth, suggesting a specific mode of action against insects and a generalized one against plants. Given the relatively low alkaloid content of these species, we assume that their herbivore defenses are not only alkaloid-based.This work was supported by grants CTQ2006-15597-C02-01/PPQ and an I3P-CSIC fellowship to D.M. Domínguez. Plant collection was possible thanks to the "Cabildos" of Tenerife, Gomera and Lanzarote

Role of c-Src in human MCF7 breast cancer cell tumorigenesis

To study the role of c-Src in breast cancer tumorigenesis, we generated a cell line derived from MCF7 carrying an inducible dominant negative c-Src (c-SrcDN: K295M/Y527F) under tetracycline control (Tet-On system). c-SrcDN expression caused phenotypic changes, relocation of c-Src, Fak, and paxillin, and loss of correct actin fiber assembly. These alterations were coupled to increased Fak-Tyr397 autophosphorylation and to inhibition of Fak-Tyr925, p130CAS, and paxillin phosphorylation. An increased association of total Src with Fak and a decreased interaction of p130CAS and p85-PI3K with Fak were also observed. SrcDN inhibited cell attachment, spreading, and migration. Serum and EGF-induced stimulation of cell proliferation and Akt phosphorylation were also significantly reduced by SrcDN, whereas p27Kip1 expression was increased. Consistently, silencing c-Src expression by siRNA in MCF7 cells significantly reduced cell migration, attachment, spreading and proliferation. Inoculation of MCF7 cells carrying inducible SrcDN to nude mice generated tumors. However, doxycycline administration to mice significantly reduced tumorigenesis, and when doxycycline treatment was installed after tumor development, a significant tumor regression was observed. In both situations, inhibition of tumorigenesis was associated with decreased Ki67 staining and increased apoptosis in tumors. These data undoubtedly demonstrate the relevance of the Src/Fak complex in breast cancer tumorigenesis. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.This work was supported by Grants from Ministerio de Educación y Ciencia (SAF2003-02188) and Fondo de Investigaciones Sanitarias (01/1316, 04/0682, and 03C03/10).Peer Reviewe

MicroRNA-Dependent Regulation of Transcription in Non-Small Cell Lung Cancer

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.Squamous cell lung cancer (SCC) and adenocarcinoma are the most common histological subtypes of non-small cell lung cancer (NSCLC), and have been traditionally managed in the clinic as a single entity. Increasing evidence, however, illustrates the biological diversity of these two histological subgroups of lung cancer, and supports the need to improve our understanding of the molecular basis beyond the different phenotypes if we aim to develop more specific and individualized targeted therapy. The purpose of this study was to identify microRNA (miRNA)-dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. In this work, paired miRNA (667 miRNAs by TaqMan Low Density Arrays (TLDA)) and mRNA profiling (Whole Genome 44 K array G112A, Agilent) was performed in tumor samples of 44 NSCLC patients. Nine miRNAs and 56 mRNAs were found to be differentially expressed in SCC versus adenocarcinoma samples. Eleven of these 56 mRNA were predicted as targets of the miRNAs identified to be differently expressed in these two histological conditions. Of them, 6 miRNAs (miR-149, miR-205, miR-375, miR-378, miR- 422a and miR-708) and 9 target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1) were validated by quantitative PCR in an independent cohort of 41 lung cancer patients. Furthermore, the inverse correlation between mRNAs and microRNAs expression was also validated. These results suggest miRNA-dependent transcriptional regulation differences play an important role in determining key hallmarks of NSCLC, and may provide new biomarkers for personalized treatment strategies.LPA is funded by Fondo de Investigación Sanitaria (PI081156, PI1102688 and R12/0036/0028), Proyecto de Excelencia de la Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía (P08-CVI-04090), and the Roche Fellowship in 75th Anniversary, Spain. SMP is funded by Fondo de Investigación Sanitaria (CD1100153), Fundación Científica de la Asociación Española Contra el Cáncer, Consejería de Salud, Servicio Andaluz de Salud (PI-0224/2009 and PI-0046/2012), and Fundación Mutua Madrileña (2009). MDP is funded by Fondo de Investigación Sanitaria (CD0900148). RGC is funded by Fondo de Investigación Sanitaria (PI10/02164). GMB is funded by SAF2010-20175 and Madrid Regional Government (S2010/BMD-2303). AC lab was supported by grants to from the Spanish Ministry of Economy and Competitivity, ISCIII (PI12/00137, RTICC: RD12/0036/0028), Consejeria de Ciencia e Innovacion (CTS-6844) and Consejeria de Salud of the Junta de Andalucia (PI-0135-2010 and PI-0306-2012).Peer reviewe