In-depth proteomics characterization of ∆Np73 effectors identifies key proteins with diagnostic potential implicated in lymphangiogenesis, vasculogenesis and metastasis in colorectal cancer

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death worldwide. Alterations in proteins of the p53-family are a common event in CRC. ΔNp73, a p53-family member, shows oncogenic properties and its effectors are largely unknown. We performed an in-depth proteomics characterization of transcriptional control by ∆Np73 of the secretome of human colon cancer cells and validated its clinical potential. The secretome was analyzed using high-density antibody microarrays and stable isotopic metabolic labeling. Validation was performed by semiquantitative PCR, ELISA, dot-blot and western blot analysis. Evaluation of selected effectors was carried out using 60 plasma samples from CRC patients, individuals carrying premalignant colorectal lesions and colonoscopy-negative controls. In total, 51 dysregulated proteins were observed showing at least 1.5-foldchange in expression. We found an important association between the overexpression of ∆Np73 and effectors related to lymphangiogenesis, vasculogenesis and metastasis, such as brain-derived neurotrophic factor (BDNF) and the putative aminoacyl tRNA synthase complex-interacting multifunctional protein 1 (EMAP-II)–vascular endothelial growth factor C–vascular endothelial growth factor receptor 3 axis. We further demonstrated the usefulness of BDNF as a potential CRC biomarker able to discriminate between CRC patients and premalignant individuals from controls with high sensitivity and specificity.This study has been funded by Instituto de Salud Carlos III (ISCIII) through the project “PI18/00473” and co-funded by the European Union (FEDER funds) and Catedra UAM-Roche en Medicina de Innovacion to GD, and the Ramon y Cajal Programme of the MINECO, PI17CIII/00045 and PI20CIII/00019 research projects from AES-ISCIII to RB. MG-A and JR-C were supported by contracts of the Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejerıa de Educacion, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo. AM-C FPU predoctoral contract is supported by the MECD. GSF is a recipient of a predoctoral contract (grant number 1193818N) supported by The Flanders Research Foundation (FWO).Peer reviewe

Effectiveness of a novel gene nanotherapy based on putrescine for cancer treatment

Gene therapy has long been proposed for cancer treatment. However, the use of therapeutic nucleic acids presents several limitations such as enzymatic degradation, rapid clearance, and poor cellular uptake and efficiency. In this work we propose the use of putrescine, a precursor for higher polyamine biosynthesis for the preparation of cationic nanosystems for cancer gene therapy. We have formulated and characterized putrescine-sphingomyelin nanosystems (PSN) and studied their endocytic pathway and intracellular trafficking in cancer cells. After loading a plasmid DNA (pDNA) encoding the apoptotic Fas Ligand (FasL), we proved their therapeutic activity by measuring the cell death rate after treatment of MDA-MB-231 cells. We have also used xenografted zebrafish embryos as a first in vivo approach to demonstrate the efficacy of the proposed PSN-pDNA formulation in a more complex model. Finally, intratumoral and intraperitoneal administration to mice-bearing MDA-MB-231 xenografts resulted in a significant decrease in tumour cell growth, highlighting the potential of the developed gene therapy nanoformulation for the treatment of triple negative breast cancer.This research was funded by Instituto de Salud Carlos III (ISCIII) and the European Regional Development Fund (FEDER) (grant numbers: CB16/12/00328, PI18/00176; AC18/00045; CIBERONC; CB16/12/00295), by ERA-NET EURONANOMED III project METASTARG (grant number JTC2018-045), by Roche-Chus Joint Unit, Axencia Galega de Innovación (GAIN), Consellería de Economía, Emprego e Industria (IN853B 2018/03), by the Spanish Ministry of Economy and Innovation (PID2019-104644RB-I00), and by the AECC Scientific Foundation (FC_AECC PROYE19036MOR). S. L. and P. H. also acknowledge the funding given by GAIN (IN606A-2019/003 and IN606A-2018/019). G. M. B. thanks the financial support given. We gratefully thank Raquel Antón Segurado for her technical support in scanning transmission electron microscopy, Montserrat García Lavandeira and Marta Picado for their technical support with confocal imaging, Lidia Martínez and Saleta Morales for their technical support cell culture maintenance and treatment, and all the co-workers in our laboratories for the help and discussion day by day. Funding for open access charge: Universidade de Santiago de Compostela/CISUG

Intracellular delivery of an antibody targeting gasdermin-b reduces her2 breast cancer aggressiveness

© 2019 American Association for Cancer Research.[Purpose]: Gasdermin B (GSDMB) overexpression/amplification occurs in about 60% of HER2 breast cancers, where it promotes cell migration, resistance to anti-HER2 therapies, and poor clinical outcome. Thus, we tackle GSDMB cytoplasmic overexpression as a new therapeutic target in HER2 breast cancers. Experimental Design: We have developed a new targeted nanomedicine based on hyaluronic acid–biocompatible nanocapsules, which allow the intracellular delivery of a specific anti-GSDMB antibody into HER2 breast cancer cells both in vitro and in vivo.[Results]: Using different models of HER2 breast cancer cells, we show that anti-GSDMB antibody loaded to nanocapsules has significant and specific effects on GSDMB-overexpressing cancer cells' behavior in ways such as (i) lowering the in vitro cell migration induced by GSDMB; (ii) enhancing the sensitivity to trastuzumab; (iii) reducing tumor growth by increasing apoptotic rate in orthotopic breast cancer xenografts; and (iv) diminishing lung metastasis in MDA-MB-231-HER2 cells in vivo. Moreover, at a mechanistic level, we have shown that AbGB increases GSDMB binding to sulfatides and consequently decreases migratory cell behavior and may upregulate the potential intrinsic procell death activity of GSDMB.[Conclusions]: Our findings portray the first evidence of the effectiveness and specificity of an antibody-based nanomedicine that targets an intracellular oncoprotein. We have proved that intracellular-delivered anti-GSDMB reduces diverse protumor GSDMB functions (migration, metastasis, and resistance to therapy) in an efficient and specific way, thus providing a new targeted therapeutic strategy in aggressive HER2 cancers with poor prognosis.This work was supported by grants from the Instituto de Salud Carlos III (ISCIII) and FEDER (PI16/00134), CIBERONC (CB16/12/00295), and the AECC (Grupos Estables de Investigacion 2018-AECC) to G. Moreno-Bueno. D. Torres and M.J. Alonso received support from the Xunta de Galicia (Competitive Reference Groups: ED431C 2017/09). J. Palacios received support from CIBERONC (CB16/12/00316). A. Molina-Crespo and D. Sarrio are funded by CIBERONC (CB16/12/00295). A. Cadete was funded in the Nanofar program by the European Commission (EACEA), an Erasmus Mundus Joint Doctorate (EMJD). A. Olivera was funded by grant BES-2015-071236 (Spanish Ministry of Science, Innovation and Universities). A. Gonella was funded by Marie Sklodowska - Curie Grant 642028 (NABBA)