Synergistic effect of antimetabolic and chemotherapy drugs in triple-negative breast cancer

The triple-negative breast cancer (TNBC) subtype comprises approximately 15% of all breast cancers and is associated with poor long-term outcomes. Classical chemotherapy remains the standard of treatment, with toxicity and resistance being major limitations. TNBC is a high metabolic group, and antimetabolic drugs are effective in inhibiting TNBC cell growth. We analyzed the combined effect of chemotherapy and antimetabolic drug combinations in MDA-MB-231, MDA-MB-468 and HCC1143 human TNBC cell lines. Cells were treated with each drug or with drug combinations at a range of concentrations to establish the half-maximal inhibitory concentrations (IC50). The dose-effects of each drug or drug combination were calculated, and the synergistic or antagonistic effects of drug combinations were defined. Chemotherapy and antimetabolic drugs exhibited growth inhibitory effects on TNBC cell lines. Antimetabolic drugs targeting the glycolysis pathway had a synergistic effect with chemotherapy drugs, and antiglycolysis drug combinations also had a synergistic effect. The use of these drug combinations could lead to new therapeutic strategies that reduce chemotherapy drug doses, decreasing their toxic effect, or that maintain the doses but enhance their efficacy by their synergistic effect with other drugsMaría I. Lumbreras-Herrera and Andrea Zapater-Moros are supported by Consejería de Educación e Investigación de la Comunidad de Madrid (IND2018/BMD-9262). Elena López-Camacho is supported by the Spanish Economy and Competitiveness Ministry (PTQ2018–009760). This work is supported by an unrestricted grant from Roch

An 8-gene qRT-PCR-based gene expression score that has prognostic value in early breast cancer

<p>Abstract</p> <p>Background</p> <p>Gene expression profiling may improve prognostic accuracy in patients with early breast cancer. Our objective was to demonstrate that it is possible to develop a simple molecular signature to predict distant relapse.</p> <p>Methods</p> <p>We included 153 patients with stage I-II hormonal receptor-positive breast cancer. RNA was isolated from formalin-fixed paraffin-embedded samples and qRT-PCR amplification of 83 genes was performed with gene expression assays. The genes we analyzed were those included in the 70-Gene Signature, the Recurrence Score and the Two-Gene Index. The association among gene expression, clinical variables and distant metastasis-free survival was analyzed using Cox regression models.</p> <p>Results</p> <p>An 8-gene prognostic score was defined. Distant metastasis-free survival at 5 years was 97% for patients defined as low-risk by the prognostic score versus 60% for patients defined as high-risk. The 8-gene score remained a significant factor in multivariate analysis and its performance was similar to that of two validated gene profiles: the 70-Gene Signature and the Recurrence Score. The validity of the signature was verified in independent cohorts obtained from the GEO database.</p> <p>Conclusions</p> <p>This study identifies a simple gene expression score that complements histopathological prognostic factors in breast cancer, and can be determined in paraffin-embedded samples.</p

Utility of CYP2D6 copy number variants as prognostic biomarker in localized anal squamous cell carcinoma

Background: Anal squamous cell carcinoma (ASCC) is an infrequent tumor whose treatment has not changed since the 1970s. The aim of this study is the identification of biomarkers allowing personalized treatments and improvement of therapeutic outcomes. Methods: Forty-six paraffin tumor samples from ASCC patients were analyzed by whole-exome sequencing. Copy number variants (CNVs) were identified and their relation to disease-free survival (DFS) was studied and validated in an independent retrospective cohort of 101 ASCC patients from the Multidisciplinary Spanish Digestive Cancer Group (GEMCAD). GEMCAD cohort proteomics allowed assessing the biological features of these tumors. Results: On the discovery cohort, the median age was 61 years old, 50% were males, stages I/II/III: 3 (7%)/16 (35%)/27 (58%), respectively, median DFS was 33 months, and overall survival was 45 months. Twenty-nine genes whose duplication was related to DFS were identified. The most representative was duplications of the CYP2D locus, including CYP2D6, CYP2D7P, and CYP2D8P genes. Patients with CYP2D6 CNV had worse DFS at 5 years than those with two CYP2D6 copies (21% vs. 84%; p <.0002, hazard ratio [HR], 5.8; 95% confidence interval [CI], 2.7–24.9). In the GEMCAD validation cohort, patients with CYP2D6 CNV also had worse DFS at 5 years (56% vs. 87%; p =.02, HR = 3.6; 95% CI, 1.1–5.7). Mitochondria and mitochondrial cell-cycle proteins were overexpressed in patients with CYP2D6 CNV. Conclusions: Tumor CYP2D6 CNV identified patients with a significantly worse DFS at 5 years among localized ASCC patients treated with 5-fluorouracil, mitomycin C, and radiotherapy. Proteomics pointed out mitochondria and mitochondrial cell-cycle genes as possible therapeutic targets for these high-risk patients. Plain Language Summary: Anal squamous cell carcinoma is an infrequent tumor whose treatment has not been changed since the 1970s. However, disease-free survival in late staged tumors is between 40% and 70%. The presence of an alteration in the number of copies of CYP2D6 gene is a biomarker of worse disease-free survival. The analysis of the proteins in these high-risk patients pointed out mitochondria and mitochondrial cell-cycle genes as possible therapeutic targets. Therefore, the determination of the number of copies of CYP2D6 allows the identification of anal squamous carcinoma patients with a high-risk of relapse that could be redirected to a clinical trial. Additionally, this study may be useful to suggest new treatment strategies to increase current therapy efficacyIdiPAZ, Grant/Award Number: Jesús Antolín Garciarena Fellowship; European Proteomics Infrastructure Consortium, Grant/Award Number: 823839, Horizon 2020 Programm

Protein content of blood-derived extracellular vesicles: An approach to the pathophysiology of cerebral hemorrhage

Introduction: Extracellular vesicles (EVs) participate in cell-to-cell paracrine signaling and can be biomarkers of the pathophysiological processes underlying disease. In intracerebral hemorrhage, the study of the number and molecular content of circulating EVs may help elucidate the biological mechanisms involved in damage and repair, contributing valuable information to the identification of new therapeutic targets.Methods: The objective of this study was to describe the number and protein content of blood-derived EVs following an intracerebral hemorrhage (ICH). For this purpose, an experimental ICH was induced in the striatum of Sprague-Dawley rats and EVs were isolated and characterized from blood at baseline, 24 h and 28 days. The protein content in the EVs was analyzed by mass spectrometric data-dependent acquisition; protein quantification was obtained by sequential window acquisition of all theoretical mass spectra data and compared at pre-defined time points.Results: Although no differences were found in the number of EVs, the proteomic study revealed that proteins related to the response to cellular damage such as deubiquitination, regulation of MAP kinase activity (UCHL1) and signal transduction (NDGR3), were up-expressed at 24 h compared to baseline; and that at 28 days, the protein expression profile was characterized by a higher content of the proteins involved in healing and repair processes such as cytoskeleton organization and response to growth factors (COR1B) and the regulation of autophagy (PI42B).Discussion: The protein content of circulating EVs at different time points following an ICH may reflect evolutionary changes in the pathophysiology of the disease

MALDI Profiling of Human Lung Cancer Subtypes

Proteomics is expected to play a key role in cancer biomarker discovery. Although it has become feasible to rapidly analyze proteins from crude cell extracts using mass spectrometry, complex sample composition hampers this type of measurement. Therefore, for effective proteome analysis, it becomes critical to enrich samples for the analytes of interest. Despite that one-third of the proteins in eukaryotic cells are thought to be phosphorylated at some point in their life cycle, only a low percentage of intracellular proteins is phosphorylated at a given time.In this work, we have applied chromatographic phosphopeptide enrichment techniques to reduce the complexity of human clinical samples. A novel method for high-throughput peptide profiling of human tumor samples, using Parallel IMAC and MALDI-TOF MS, is described. We have applied this methodology to analyze human normal and cancer lung samples in the search for new biomarkers. Using a highly reproducible spectral processing algorithm to produce peptide mass profiles with minimal variability across the samples, lineal discriminant-based and decision tree–based classification models were generated. These models can distinguish normal from tumor samples, as well as differentiate the various non–small cell lung cancer histological subtypes.A novel, optimized sample preparation method and a careful data acquisition strategy is described for high-throughput peptide profiling of small amounts of human normal lung and lung cancer samples. We show that the appropriate combination of peptide expression values is able to discriminate normal lung from non-small cell lung cancer samples and among different histological subtypes. Our study does emphasize the great potential of proteomics in the molecular characterization of cancer

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

Estudio de la asociación y activación de Src por el receptor de prolactina y del papel de Jak2 en la fosforilación del receptor

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 09-07-1997RESUMEN La transducción de señales por prolactina (PRL) requiere la unión de una molécula de PRL con dos moléculas de receptor. El receptor de prolactina es una proteína con un único dominio transmembrana que pertenece a la superfamilia de receptores de citoquinas. La familia de proteína tirosina quinasas Jak desempeña un papel crítico en la transducción de las señales que parten de los receptores de citoquinas activados por la unión de sus ligandos. Estas quinasas interaccionan con los receptores en la porción citoplasmática próxima a la membrana, donde se encuentran las secuencias conservadas de la Caja 1 y la Caja 2. La activación de todos los receptores de citoquinas conocidos induce la fosforilación/activación de las Jaks asociadas con ellos y la fosforilación del receptor. La activación de las Jaks es imprescindible para la mayoría, si no lo es para todas, de las funciones de estos receptores. La Jak que se asocia al receptor de prolactina (PRLR) es Jak2. Los receptores de citoquinas inducen diversas rutas de señalización mediante la asociación y la activación de varias moléculas señalizadoras, además de las Jaks. Se ha descrito que ciertos receptores de citoquinas pueden asociar y activar la familia de tirosina quinasas Src. La estimulación del PRLR induce la activación de las moléculas de Src o Fyn que interaccionan con él. Los objetivos fundamentales del presente trabajo han sido estudiar los mecanismos de esta asociación y activación, y analizar el papel de la actividad quinasa de Jak2 en la fosforilación del PRLR. Nuestros resultados demuestran que la estimulación de Src por el PRLR es independiente de la actividad de Jak2, que la asociación de Src con el receptor puede estar mediada por el dominio único de Src, pero no por sus dominios SH2 y SH3, y que Src no fosforila el PRLR. La fosforilación del receptor está mediada por la actividad quinasa de Jak2 y no por otra quinasa asociada al receptor. Estos datos sugieren que Src puede funcionar de manera complementaria, pero independiente, a Jak2 para transducir alguna de las señales que parten del PRLR.SUMMARY Prolactin (PRL) signal transduction requires binding of one PRL molecule to two receptor molecules. The prolactin receptor is a single transmembrane protein that belongs to the cytokine receptor superfamily. The Jak family of tyrosine kinases play critical roles in transducing the signals emanating from ligand-activated cytokine receptor complexes. They bind to receptors at the membrane-proximal cytoplasmic portion that contains the conserved Box 1 and Box 2 sequences. Activation of all known cytokine receptors induces the phosphorylation/activation of the associated Jaks and phosphorylation of the receptor. Jaks activation is required for most, if not all, receptor functions. The Jak associated with the prolactin receptor (PRLR) is Jak2. Cytokine receptors normally induce a number of signalling pathways by recruiting and activating signalling molecules others than Jaks. It has been described that certain cytokine receptors can associate and activate the Src family of tyrosine kinases. Stimulation of PRLR activates the associated Src or Fyn molecules. The aim of the present work has been to study the mechanisms of that association and activation, and to analyse the role of Jak2 kinase activity in the phosphorylation of the PRLR. Our results demonstrate that stimulation of Src by PRLR is independent of the Jak2 activity, that Src association with the receptor may be mediated by the unique domain of Src, but not by its SH2, SH3 domains, and that Src does not phosphorylate the PRLR. The receptor phosphorylation is mediated by Jak2 kinase activity and not by any other receptor associated kinase. These findings suggest that Src may function in conjunction, but independently, with Jak2 to elicit one or more of the signals emanating from the PRLR

Prolactin receptor is associated with c-src kinase in rat liver

The mechanism of action of the pituitary hormone PRL was studied in hepatocytes of lactating rats. PRL receptor immune complexes obtained from liver lysates have an associated tyrosine kinase activity. The tyrosine kinase has been identified in isolated hepatocytes as pp60c-src. Incubation of hepatocytes with PRL induces the association of PRL receptor with pp60c-src and the resultant stimulation of its tyrosine kinase activity. Furthermore, PRL stimulates the gene expression of c-fos, c-jun, and c-src. All of these findings support the idea that the pp60c-src tyrosine kinase participates in the early steps of the PRL intracellular signaling that promotes cell growth in liver cells.This work was supported by grants from the DGICYT (PM91-0223 and PB93-0136), CAM (C137/91and C263/91A), and the Ramón Areces Foundation.Peer Reviewe

Src Family Kinases Are Required for Prolactin Induction of Cell Proliferation

Prolactin (PRL) is a pleiotropic cytokine promoting cellular proliferation and differentiation. Because PRL activates the Src family of tyrosine kinases (SFK), we have studied the role of these kinases in PRL cell proliferation signaling. PRL induced [(3)H]thymidine incorporation upon transient transfection of BaF-3 cells with the PRL receptor. This effect was inhibited by cotransfection with the dominant negative mutant of c-Src (K>A295/Y>F527, SrcDM). The role of SFK in PRL-induced proliferation was confirmed in the BaF-3 PRL receptor-stable transfectant, W53 cells, where PRL induced Fyn and Lyn activation. The SFK-selective inhibitors PP1/PP2 and herbimycin A blocked PRL-dependent cell proliferation by arresting the W53 cells in G1, with no evident apoptosis. In parallel, PP1/PP2 inhibited PRL induction of cell growth-related genes c-fos, c-jun, c-myc, and odc. These inhibitors have no effect on PRL-mediated activation of Ras/Mapk and Jak/Start pathways. In contrast, they inhibited the PRL-dependent stimulation of the SFKs substrate Sam68, the phosphorylation of the tyrosine phosphatase Shp2, and the PI3K-dependent Akt and p70S6k serine kinases. Consistently, transient expression of SrcDM in W53 cells also blocked PRL activation of Akt. These results demonstrate that activation of SFKs is required for cell proliferation induced by PRL