Development of G9a-targeted BioPROTACs based on peptides and nanobodies

This project arose motivated by the high incidence that neoplastic pathologies have nowadays, and therefore by the need that our society has to find a solution against them. The main objective of this research was to identify specific peptides and nanobodies against G9a methyltransferase, whose role in cancer is to introduce epigenetic modifications to silence tumor suppressor genes. The strategy followed focuses on degrading this enzyme through the Biological Proteolysis Targeting Chimera (BioPROTAC) system, made up of either the aforementioned peptides or nanobodies. A search for these specific molecules was carried out using phage display, to find the most specific candidates against the previously produced and purified G9a. Later, the binding affinity of the selected clones was evaluated, the BioPROTAC construct was designed, and immunocytochemistry (ICQ) was performed to check their cellular internalization. In short, it was possible to select 3 peptide clones and 16 nanobodies with a good affinity for G9a, in addition to designing a system capable of being introduced into the tumor cell. This represents a project of Targeted Protein Degradation (TPD) for drug discovery with its main objectives met. Furthermore, this study will facilitate future investigations aimed at confirming the efficacy of the system to degrade the enzyme, in order to conduct additional testing of its therapeutic effect

A translational approach to assess the metabolomic impact of stabilized gold nanoparticles by NMR spectroscopy

[EN] Gold nanoparticles have high potential in the biomedical area, especially in disease diagnosis and treatment. The application of these nanoparticles requires the presence of stabilizers to avoid their agglomeration. Nowadays, there is a lack of reliable methods for characterising the effect of stabilised nanoparticles on biological systems. To this end, in this study, we apply an experimental approach based on nuclear magnetic resonance spectroscopy to study the effect of gold nanoparticles, stabilised with cerium oxide or chitosan, on a human cancer cell model. The results showed that both systems have a significant effect, even at non-toxic levels, on the cellular antioxidant system. However, although particles functionalised with chitosan exerted a strong effect on the aerobic respiration, nanoparticles stabilised with cerium oxide had a higher impact on the mechanisms associated with anaerobic energy production. Therefore, even though both systems contained similar gold nanoparticles, the presence of different stabilizers strongly influenced their mode of action and potential applications in biomedicine.This work was supported by the Carlos III Health Institute, the European Regional Development Fund (PI16/02064 and CP13/00252) and the Spanish Ministerio de Economia y Competitividad (SAF2014-53977-R, SAF2017-89229-R and RD12/0036/0025). In addition, JRH is a recipient of a contract from the Ministry of Health of the Carlos III Health Institute.Herance, JR.; García Gómez, H.; Guitierrez Carcedo, P.; Navalón Oltra, S.; Pineda-Lucena, A.; Palomino-Schätzlein, M. (2019). A translational approach to assess the metabolomic impact of stabilized gold nanoparticles by NMR spectroscopy. The Analyst. 144(4):1265-1274. https://doi.org/10.1039/c8an01827hS126512741444Shi, J., Kantoff, P. W., Wooster, R., & Farokhzad, O. C. (2016). Cancer nanomedicine: progress, challenges and opportunities. Nature Reviews Cancer, 17(1), 20-37. doi:10.1038/nrc.2016.108Gioria, S., Lobo Vicente, J., Barboro, P., La Spina, R., Tomasi, G., Urbán, P., … Chassaigne, H. (2016). A combined proteomics and metabolomics approach to assess the effects of gold nanoparticlesin vitro. Nanotoxicology, 10(6), 736-748. doi:10.3109/17435390.2015.1121412Beik, J., Khademi, S., Attaran, N., Sarkar, S., Shakeri-Zadeh, A., Ghaznavi, H., & Ghadiri, H. (2017). A Nanotechnology-based Strategy to Increase the Efficiency of Cancer Diagnosis and Therapy: Folate-conjugated Gold Nanoparticles. Current Medicinal Chemistry, 24(39). doi:10.2174/0929867324666170810154917Nagi, N. M. S., Khair, Y. A. M., & Abdalla, A. M. E. (2017). Capacity of gold nanoparticles in cancer radiotherapy. Japanese Journal of Radiology, 35(10), 555-561. doi:10.1007/s11604-017-0671-6Gharatape, A., & Salehi, R. (2017). Recent progress in theranostic applications of hybrid gold nanoparticles. European Journal of Medicinal Chemistry, 138, 221-233. doi:10.1016/j.ejmech.2017.06.034Fang, J., Nakamura, H., & Maeda, H. (2011). The EPR effect: Unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect. Advanced Drug Delivery Reviews, 63(3), 136-151. doi:10.1016/j.addr.2010.04.009Haume, K., Rosa, S., Grellet, S., Śmiałek, M. A., Butterworth, K. T., Solov’yov, A. V., … Mason, N. J. (2016). Gold nanoparticles for cancer radiotherapy: a review. Cancer Nanotechnology, 7(1). doi:10.1186/s12645-016-0021-xAzevedo, R. S. S., de Sousa, J. R., Araujo, M. T. F., Martins Filho, A. J., de Alcantara, B. N., Araujo, F. M. C., … Vasconcelos, P. F. C. (2018). In situ immune response and mechanisms of cell damage in central nervous system of fatal cases microcephaly by Zika virus. Scientific Reports, 8(1). doi:10.1038/s41598-017-17765-5Lin, Y., Wu, Z., Wen, J., Ding, K., Yang, X., Poeppelmeier, K. R., & Marks, L. D. (2015). Adhesion and Atomic Structures of Gold on Ceria Nanostructures: The Role of Surface Structure and Oxidation State of Ceria Supports. Nano Letters, 15(8), 5375-5381. doi:10.1021/acs.nanolett.5b02694Menchón, C., Martín, R., Apostolova, N., Victor, V. M., Álvaro, M., Herance, J. R., & García, H. (2012). Gold Nanoparticles Supported on Nanoparticulate Ceria as a Powerful Agent against Intracellular Oxidative Stress. Small, 8(12), 1895-1903. doi:10.1002/smll.201102255Tiwari, P., Vig, K., Dennis, V., & Singh, S. (2011). Functionalized Gold Nanoparticles and Their Biomedical Applications. Nanomaterials, 1(1), 31-63. doi:10.3390/nano1010031Fathy, M. M., Mohamed, F. S., Elbialy, N., & Elshemey, W. M. (2018). Multifunctional Chitosan-Capped Gold Nanoparticles for enhanced cancer chemo-radiotherapy: An invitro study. Physica Medica, 48, 76-83. doi:10.1016/j.ejmp.2018.04.002Guo, X., Zhuang, Q., Ji, T., Zhang, Y., Li, C., Wang, Y., … Du, L. (2018). Multi-functionalized chitosan nanoparticles for enhanced chemotherapy in lung cancer. Carbohydrate Polymers, 195, 311-320. doi:10.1016/j.carbpol.2018.04.087Lee, Y.-H., Kim, J.-S., Kim, J.-E., Lee, M.-H., Jeon, J.-G., Park, I.-S., & Yi, H.-K. (2017). Nanoparticle mediated PPARγ gene delivery on dental implants improves osseointegration via mitochondrial biogenesis in diabetes mellitus rat model. Nanomedicine: Nanotechnology, Biology and Medicine, 13(5), 1821-1832. doi:10.1016/j.nano.2017.02.020Sun, I.-C., Na, J. H., Jeong, S. Y., Kim, D.-E., Kwon, I. C., Choi, K., … Kim, K. (2013). Biocompatible Glycol Chitosan-Coated Gold Nanoparticles for Tumor-Targeting CT Imaging. Pharmaceutical Research, 31(6), 1418-1425. doi:10.1007/s11095-013-1142-0Costa, P. M., & Fadeel, B. (2016). Emerging systems biology approaches in nanotoxicology: Towards a mechanism-based understanding of nanomaterial hazard and risk. Toxicology and Applied Pharmacology, 299, 101-111. doi:10.1016/j.taap.2015.12.014Lv, M., Huang, W., Chen, Z., Jiang, H., Chen, J., Tian, Y., … Xu, F. (2015). Metabolomics techniques for nanotoxicity investigations. Bioanalysis, 7(12), 1527-1544. doi:10.4155/bio.15.83Nagana Gowda, G. A., Barding, G. A., Dai, J., Gu, H., Margineantu, D. H., Hockenbery, D. M., & Raftery, D. (2018). A Metabolomics Study of BPTES Altered Metabolism in Human Breast Cancer Cell Lines. Frontiers in Molecular Biosciences, 5. doi:10.3389/fmolb.2018.00049Ali, M. R. K., Wu, Y., Han, T., Zang, X., Xiao, H., Tang, Y., … El-Sayed, M. A. (2016). Simultaneous Time-Dependent Surface-Enhanced Raman Spectroscopy, Metabolomics, and Proteomics Reveal Cancer Cell Death Mechanisms Associated with Gold Nanorod Photothermal Therapy. Journal of the American Chemical Society, 138(47), 15434-15442. doi:10.1021/jacs.6b08787Esumi, K., Takei, N., & Yoshimura, T. (2003). Antioxidant-potentiality of gold–chitosan nanocomposites. Colloids and Surfaces B: Biointerfaces, 32(2), 117-123. doi:10.1016/s0927-7765(03)00151-6Du, S., Kendall, K., Toloueinia, P., Mehrabadi, Y., Gupta, G., & Newton, J. (2012). Aggregation and adhesion of gold nanoparticles in phosphate buffered saline. Journal of Nanoparticle Research, 14(3). doi:10.1007/s11051-012-0758-zUlrich, E. L., Akutsu, H., Doreleijers, J. F., Harano, Y., Ioannidis, Y. E., Lin, J., … Markley, J. L. (2007). BioMagResBank. Nucleic Acids Research, 36(Database), D402-D408. doi:10.1093/nar/gkm957Wishart, D. S., Feunang, Y. D., Marcu, A., Guo, A. C., Liang, K., Vázquez-Fresno, R., … Scalbert, A. (2017). HMDB 4.0: the human metabolome database for 2018. Nucleic Acids Research, 46(D1), D608-D617. doi:10.1093/nar/gkx1089Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65(1-2), 55-63. doi:10.1016/0022-1759(83)90303-4Yang, L., Shang, L., & Nienhaus, G. U. (2013). Mechanistic aspects of fluorescent gold nanocluster internalization by live HeLa cells. Nanoscale, 5(4), 1537. doi:10.1039/c2nr33147kZhitomirsky, B., Farber, H., & Assaraf, Y. G. (2018). LysoTracker and MitoTracker Red are transport substrates of P‐glycoprotein: implications for anticancer drug design evading multidrug resistance. Journal of Cellular and Molecular Medicine, 22(4), 2131-2141. doi:10.1111/jcmm.13485Chong, J., Soufan, O., Li, C., Caraus, I., Li, S., Bourque, G., … Xia, J. (2018). MetaboAnalyst 4.0: towards more transparent and integrative metabolomics analysis. Nucleic Acids Research, 46(W1), W486-W494. doi:10.1093/nar/gky310Stepanenko, A. A., & Dmitrenko, V. V. (2015). Pitfalls of the MTT assay: Direct and off-target effects of inhibitors can result in over/underestimation of cell viability. Gene, 574(2), 193-203. doi:10.1016/j.gene.2015.08.009Choi, S. Y., Jang, S. H., Park, J., Jeong, S., Park, J. H., Ock, K. S., … Lee, S. Y. (2012). Cellular uptake and cytotoxicity of positively charged chitosan gold nanoparticles in human lung adenocarcinoma cells. Journal of Nanoparticle Research, 14(12). doi:10.1007/s11051-012-1234-5De Carvalho, T. G., Garcia, V. B., de Araújo, A. A., da Silva Gasparotto, L. H., Silva, H., Guerra, G. C. B., … de Araújo Júnior, R. F. (2018). Spherical neutral gold nanoparticles improve anti-inflammatory response, oxidative stress and fibrosis in alcohol-methamphetamine-induced liver injury in rats. International Journal of Pharmaceutics, 548(1), 1-14. doi:10.1016/j.ijpharm.2018.06.008Carrola, J., Bastos, V., Ferreira de Oliveira, J. M. P., Oliveira, H., Santos, C., Gil, A. M., & Duarte, I. F. (2016). Insights into the impact of silver nanoparticles on human keratinocytes metabolism through NMR metabolomics. Archives of Biochemistry and Biophysics, 589, 53-61. doi:10.1016/j.abb.2015.08.022Fröhlich, E. (2012). The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles. International Journal of Nanomedicine, 5577. doi:10.2147/ijn.s36111Gürer, H., Özgünes, H., Saygin, E., & Ercal, N. (2001). Antioxidant Effect of Taurine Against Lead-Induced Oxidative Stress. Archives of Environmental Contamination and Toxicology, 41(4), 397-402. doi:10.1007/s002440010265Lee, S.-H., Wang, T.-Y., Hong, J.-H., Cheng, T.-J., & Lin, C.-Y. (2016). NMR-based metabolomics to determine acute inhalation effects of nano- and fine-sized ZnO particles in the rat lung. Nanotoxicology, 10(7), 924-934. doi:10.3109/17435390.2016.1144825Saborano, R., Wongpinyochit, T., Totten, J. D., Johnston, B. F., Seib, F. P., & Duarte, I. F. (2017). Metabolic Reprogramming of Macrophages Exposed to Silk, Poly(lactic-co-glycolic acid), and Silica Nanoparticles. Advanced Healthcare Materials, 6(14), 1601240. doi:10.1002/adhm.201601240Bo, Y., Jin, C., Liu, Y., Yu, W., & Kang, H. (2014). Metabolomic analysis on the toxicological effects of TiO2nanoparticles in mouse fibroblast cells: from the perspective of perturbations in amino acid metabolism. Toxicology Mechanisms and Methods, 24(7), 461-469. doi:10.3109/15376516.2014.939321Shea, T. B., Ekinci, F. J., Ortiz, D., Dawn-Linsley, M., Wilson, T. O., & Nicolosi, R. J. (2002). Efficacy of vitamin E, phosphatidyl choline, and pyruvate on buffering neuronal degeneration and oxidative stress in cultured cortical neurons and in central nervous tissue of apolipoprotein E-deficient mice. Free Radical Biology and Medicine, 33(2), 276-282. doi:10.1016/s0891-5849(02)00872-9Schätzlein, M. P., Becker, J., Schulze-Sünninghausen, D., Pineda-Lucena, A., Herance, J. R., & Luy, B. (2018). Rapid two-dimensional ALSOFAST-HSQC experiment for metabolomics and fluxomics studies: application to a 13C-enriched cancer cell model treated with gold nanoparticles. Analytical and Bioanalytical Chemistry, 410(11), 2793-2804. doi:10.1007/s00216-018-0961-6HUNTER, A. (2006). Molecular hurdles in polyfectin design and mechanistic background to polycation induced cytotoxicity☆. Advanced Drug Delivery Reviews, 58(14), 1523-1531. doi:10.1016/j.addr.2006.09.008Ratnasekhar, C., Sonane, M., Satish, A., & Mudiam, M. K. R. (2015). Metabolomics reveals the perturbations in the metabolome ofCaenorhabditis elegansexposed to titanium dioxide nanoparticles. Nanotoxicology, 9(8), 994-1004. doi:10.3109/17435390.2014.99334

Mild Muscle Mitochondrial Fusion Distress Extends Drosophila Lifespan through an Early and Systemic Metabolome Reorganization

[EN] In a global aging population, it is important to understand the factors affecting systemic aging and lifespan. Mitohormesis, an adaptive response caused by different insults affecting the mitochondrial network, triggers a response from the nuclear genome inducing several pathways that promote longevity and metabolic health. Understanding the role of mitochondrial function during the aging process could help biomarker identification and the development of novel strategies for healthy aging. Herein, we interfered the muscle expression of the Drosophila genes Marf and Opa1, two genes that encode for proteins promoting mitochondrial fusion, orthologues of human MFN2 and OPA1. Silencing of Marf and Opa1 in muscle increases lifespan, improves locomotor capacities in the long term, and maintains muscular integrity. A metabolomic analysis revealed that muscle down-regulation of Marf and Opa1 promotes a non-autonomous systemic metabolome reorganization, mainly affecting metabolites involved in the energetic homeostasis: carbohydrates, lipids and aminoacids. Interestingly, the differences are consistently more evident in younger flies, implying that there may exist an anticipative adaptation mediating the protective changes at the older age. We demonstrate that mild mitochondrial muscle disturbance plays an important role in Drosophila fitness and reveals metabolic connections between tissues. This study opens new avenues to explore the link of mitochondrial dynamics and inter-organ communication, as well as their relationship with muscle-related pathologies, or in which muscle aging is a risk factor for their appearance. Our results suggest that early intervention in muscle may prevent sarcopenia and promote healthy aging.Work in the laboratory of M.I.G. was funded by PROMETEU/2018/135 from "Conselleria, de Sanitat de la Generalitat Valenciana". Part of the equipment employed in this work has been funded by Generalitat Valenciana and co-financed with ERDF funds (OP ERDF of Comunitat Valenciana 2014-2020).Tapia, A.; Palomino-Schätzlein, M.; Roca, M.; Lahoz, A.; Pineda-Lucena, A.; López Del Amo, V.; Galindo, MI. (2021). Mild Muscle Mitochondrial Fusion Distress Extends Drosophila Lifespan through an Early and Systemic Metabolome Reorganization. International Journal of Molecular Sciences. 22(22):1-20. https://doi.org/10.3390/ijms222212133120222

Identification of Bioactive Compounds in Polar and Nonpolar Extracts of Araujia sericifera

Araujia sericifera is a native perennial, climbing laticiferous shrub from South America that is currently naturalized in many other countries. Previous data describe promising properties for A. sericifera, but no systematic study of its bioactive compounds and possible medicinal applications has been conducted to date. In the present study, aerial parts of A. sericifera (leaves, stems, and fruits) were explored by combining GC-MS and NMR spectroscopy analysis for both nonpolar (hexane) and polar (methanol) extracts. The hexanic extracts contained high amounts of pentacyclic triterpenes including two new metabolites, 3-tigloyl germanicol (18) and 3-tigloyl lupeol (19). The methanolic extracts revealed the presence of luteolin-7-glucoside (24), trigonelline (22), and conduritol F (23) as the main constituents. A multivariate study of a meaningful number of extracts allowed us to determine the distribution of compounds inside the plant. A cytotoxic evaluation in vitro showed that both leaf and fruit hexanic extracts presented a moderate activity against human breast carcinoma cell lines (MDA-MB-453 and MCF-7) and human colon carcinoma cell line (HCT-116) by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay

In Vivo Detection of Perinatal Brain Metabolite Changes in a Rabbit Model of Intrauterine Growth Restriction (IUGR)

Background Intrauterine growth restriction (IUGR) is a risk factor for abnormal neurodevelopment.We studied a rabbit model of IUGR by magnetic resonance imaging (MRI) and spectroscopy (MRS), to assess in vivo brain structural and metabolic consequences, and identify potential metabolic biomarkers for clinical translation. Methods IUGR was induced in 3 pregnant rabbits at gestational day 25, by 40-50% uteroplacental vessel ligation in one horn; the contralateral horn was used as control. Fetuses were delivered at day 30 and weighted. A total of 6 controls and 5 IUGR pups underwent T2-w MRI and localized proton MRS within the first 8 hours of life, at 7T. Changes in brain tissue volumes and respective contributions to each MRS voxel were estimated by semi-automated registration of MRI images with a digital atlas of the rabbit brain. MRS data were used for: (i) absolute metabolite quantifications, using linear fitting; (ii) local temperature estimations, based on the water chemical shift; and (iii) classification, using spectral pattern analysis. Results Lower birth weight was associated with (i) smaller brain sizes, (ii) slightly lower brain temperatures, and (iii) differential metabolite profile changes in specific regions of the brain parenchyma. Specifically, we found estimated lower levels of aspartate and N-acetylaspartate (NAA) in the cerebral cortex and hippocampus (suggesting neuronal impairment), and higher glycine levels in the striatum (possible marker of brain injury). Our results also suggest that the metabolic changes in cortical regions are more prevalent than those detected in hippocampus and striatum. Conclusions IUGR was associated with brain metabolic changes in vivo, which correlate well with the neurostructural changes and neurodevelopment problems described in IUGR. Metabolic parameters could constitute non invasive biomarkers for the diagnosis and abnormal neurodevelopment of perinatal origin

A Drosophila model of GDAP1 function reveals the involvement of insulin signalling in the mitochondria-dependent neuromuscular degeneration

[EN] Charcot-Marie-Tooth disease is a rare peripheral neuropathy for which there is no specific treatment. Some forms of Charcot-Marie-Tooth are due to mutations in the GDAP1 gene. A striking feature of mutations in GDAP1 is that they have a variable clinical manifestation, according to disease onset and progression, histology and mode of inheritance. Studies in cellular and animal models have revealed a role of GDAP1 in mitochondrial morphology and distribution, calcium homeostasis and oxidative stress. To get a better understanding of the disease mechanism we have generated models of over-expression and RNA interference of the Drosophila Gdapl gene. In order to get an overview about the changes that Gdapl mutations cause in our disease model, we have combined a comprehensive determination of the metabolic profile in the flies by nuclear magnetic resonance spectroscopy with gene expression analyses and biophysical tests. Our results revealed that both up- and down-regulation of Gdapl results in an early systemic inactivation of the insulin pathway before the onset of neuromuscular degeneration, followed by an accumulation of carbohydrates and an increase in the (3-oxidation of lipids. Our findings are in line with emerging reports of energy metabolism impairments linked to different types of neural pathologies caused by defective mitochondrial function, which is not surprising given the central role of mitochondria in the control of energy metabolism. The relationship of mitochondrial dynamics with metabolism during neurodegeneration opens new avenues to understand the cause of the disease, and for the discovery of new biomarkers and treatments.This work was supported by a project grant from the Association Francaise contre les Myopathies [AFM 18540 to M.I.G]; a collaborative grant from International Rare Diseases Research consortium (IRDiRC) and Institute de Salud Carlos III [IR11/TREAT-CMT to M.I.G. (partner 12) and F.V.P. (partner 8)]; funding from Institute de Salud Carlos III through Biomedical Network Research Center for Rare Diseases and the INGENIO 2010 program to F.V.P.; and a project grant from the Spanish Government (Secretaria de Estado de Investigacion, Desarollo e Innovacion, Ministerio de Economia y Competitividad) [SAF2014-53977-R to A.P.].Lopez Del Amo, V.; Palomino-Schätzlein, M.; Seco-Cervera, M.; Garcia-Gimenez, JL.; Pallardó-Calatayud, FV.; Pineda-Lucena, A.; Galindo-Orozco, MI. (2017). A Drosophila model of GDAP1 function reveals the involvement of insulin signalling in the mitochondria-dependent neuromuscular degeneration. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 1863(3):801-809. https://doi.org/10.1016/j.bbadis.2017.01.003S8018091863

Vaginal metabolome: towards a minimally invasive diagnosis of microbial invasion of the amniotic cavity in women with preterm labor

Microbial invasion of the amniotic cavity (MIAC) is only identified by amniocentesis, an invasive procedure that limits its clinical translation. Here, we aimed to evaluate whether the vaginal metabolome discriminates the presence/absence of MIAC in women with preterm labor (PTL) and intact membranes. We conducted a case-control study in women with symptoms of PTL below 34 weeks who underwent amniocentesis to discard MIAC. MIAC was defined as amniotic fluid positive for microorganisms identified by specific culture media. The cohort included 16 women with MIAC and 16 control (no MIAC). Both groups were matched for age and gestational age at admission. Vaginal fluid samples were collected shortly after amniocentesis. Metabolic profiles were analyzed by nuclear magnetic resonance (NMR) spectroscopy and compared using multivariate and univariate statistical analyses to identify significant differences between the two groups. The vaginal metabolomics profile of MIAC showed higher concentrations of hypoxanthine, proline, choline and acetylcholine and decreased concentrations of phenylalanine, glutamine, isoleucine, leucine and glycerophosphocholine. In conclusion, metabolic changes in the NMR-based vaginal metabolic profile are able to discriminate the presence/absence of MIAC in women with PTL and intact membranes. These metabolic changes might be indicative of enhanced glycolysis triggered by hypoxia conditions as a consequence of bacterial infection, thus explaining the utilization of alternative energy sources in an attempt to replenish glucose

Characterization of triple‐negative breast cancer preclinical models provides functional evidence of metastatic progression

Triple‐negative breast cancer (TNBC), an aggressive, metastatic and recurrent breast cancer (BC) subtype, currently suffers from a lack of adequately described spontaneously metastatic preclinical models that faithfully reproduce the clinical scenario. We describe two preclinical spontaneously metastatic TNBC orthotopic murine models for the development of advanced therapeutics: an immunodeficient human MDA‐MB‐231‐Luc model and an immunocompetent mouse 4T1 model. Furthermore, we provide a broad range of multifactorial analysis for both models that could provide relevant information for the development of new therapies and diagnostic tools. Our comparisons uncovered differential growth rates, stromal arrangements and metabolic profiles in primary tumors, and the presence of cancer‐associated adipocyte infiltration in the MDA‐MB‐231‐Luc model. Histopathological studies highlighted the more rapid metastatic spread to the lungs in the 4T1 model following a lymphatic route, while we observed both homogeneous (MDA‐MB‐231‐Luc) and heterogeneous (4T1) metastatic spread to axillary lymph nodes. We encountered unique metabolomic signatures in each model, including crucial amino acids and cell membrane components. Hematological analysis demonstrated severe leukemoid and lymphoid reactions in the 4T1 model with the partial reestablishment of immune responses in the immunocompromised MDA‐MB‐231‐Luc model. Additionally, we discovered β‐immunoglobulinemia and increased basal levels of G‐CSF correlating with a metastatic switch, with G‐CSF also promoting extramedullary hematopoiesis (both models) and causing hepatosplenomegaly (4T1 model). Overall, we believe that the characterization of these preclinical models will foster the development of advanced therapeutic strategies for TNBC treatment, especially for the treatment of patients presenting both, primary tumors and metastatic spread

Splicing Factor SLU7 Prevents Oxidative Stress-Mediated Hepatocyte Nuclear Factor 4α Degradation, Preserving Hepatic Differentiation and Protecting From Liver Damage

Background and Aims: Hepatocellular dedifferentiation is emerging as an important determinant in liver disease progression. Preservation of mature hepatocyte identity relies on a set of key genes, predominantly the transcription factor hepatocyte nuclear factor 4α (HNF4α) but also splicing factors like SLU7. How these factors interact and become dysregulated and the impact of their impairment in driving liver disease are not fully understood. Approach and Results: Expression of SLU7 and that of the adult and oncofetal isoforms of HNF4α, driven by its promoter 1 (P1) and P2, respectively, was studied in diseased human and mouse livers. Hepatic function and damage response were analyzed in wild-type and Slu7-haploinsufficient/heterozygous (Slu7) mice undergoing chronic (CCl) and acute (acetaminophen) injury. SLU7 expression was restored in CCl-injured mice using SLU7-expressing adeno-associated viruses (AAV-SLU7). The hepatocellular SLU7 interactome was characterized by mass spectrometry. Reduced SLU7 expression in human and mouse diseased livers correlated with a switch in HNF4α P1 to P2 usage. This response was reproduced in Slu7 mice, which displayed increased sensitivity to chronic and acute liver injury, enhanced oxidative stress, and marked impairment of hepatic functions. AAV-SLU7 infection prevented liver injury and hepatocellular dedifferentiation. Mechanistically we demonstrate a unique role for SLU7 in the preservation of HNF4α1 protein stability through its capacity to protect the liver against oxidative stress. SLU7 is herein identified as a key component of the stress granule proteome, an essential part of the cell’s antioxidant machinery. Conclusions: Our results place SLU7 at the highest level of hepatocellular identity control, identifying SLU7 as a link between stress-protective mechanisms and liver differentiation. These findings emphasize the importance of the preservation of hepatic functions in the protection from liver injury.Supported by MINECO/AEI/FEDER (UE SAF2016-75972-R, PID2019-104265RB-I00/AEI/10.13039/501100011033, and PID2019-104878RB-100/AEI/10.13039/501100011033), CIBERehd, Fundación La Caixa (HEPACARE), an AECC postdoctoral fellowship (POSTD18014AREC, to M.A.), a Ministerio de Educación FPU fellowship (FPU18/01461, to M.G.R.), a Ministerio de Educación FPI fellowship (BES-2017-079883, to M.R.); a Ramón y Cajal Program contract (RYC2018-024475-1, to M.G.F.B.), the Fundación Eugenio Rodríguez Pascual, the Fundación Mario Losantos, the Fundación M. Torres, and a generous donation from Mr. Eduardo Avila

Splicing factor SLU7 prevents oxidative stress-mediated hepatocyte nuclear factor 4α degradation, preserving hepatic differentiation and protecting from liver damage

Background and Aims: Hepatocellular dedifferentiation is emerging as an important determinant in liver disease progression. Preservation of mature hepatocyte identity relies on a set of key genes, predominantly the transcription factor hepatocyte nuclear factor 4α (HNF4α) but also splicing factors like SLU7. How these factors interact and become dysregulated and the impact of their impairment in driving liver disease are not fully understood. Approach and Results: Expression of SLU7 and that of the adult and oncofetal isoforms of HNF4α, driven by its promoter 1 (P1) and P2, respectively, was studied in diseased human and mouse livers. Hepatic function and damage response were analyzed in wild-type and Slu7-haploinsufficient/heterozygous (Slu7+/−) mice undergoing chronic (CCl4) and acute (acetaminophen) injury. SLU7 expression was restored in CCl4-injured mice using SLU7-expressing adeno-associated viruses (AAV-SLU7). The hepatocellular SLU7 interactome was characterized by mass spectrometry. Reduced SLU7 expression in human and mouse diseased livers correlated with a switch in HNF4α P1 to P2 usage. This response was reproduced in Slu7+/− mice, which displayed increased sensitivity to chronic and acute liver injury, enhanced oxidative stress, and marked impairment of hepatic functions. AAV-SLU7 infection prevented liver injury and hepatocellular dedifferentiation. Mechanistically we demonstrate a unique role for SLU7 in the preservation of HNF4α1 protein stability through its capacity to protect the liver against oxidative stress. SLU7 is herein identified as a key component of the stress granule proteome, an essential part of the cell’s antioxidant machinery. Conclusions: Our results place SLU7 at the highest level of hepatocellular identity control, identifying SLU7 as a link between stress-protective mechanisms and liver differentiation. These findings emphasize the importance of the preservation of hepatic functions in the protection from liver injury.Supported by MINECO/AEI/FEDER (UE SAF2016‐75972‐R, PID2019‐104265RB‐I00/AEI/10.13039/501100011033, and PID2019‐104878RB‐100/AEI/10.13039/501100011033), CIBERehd, Fundación La Caixa (HEPACARE), an AECC postdoctoral fellowship (POSTD18014AREC, to M.A.), a Ministerio de Educación FPU fellowship (FPU18/01461, to M.G.R.), a Ministerio de Educación FPI fellowship (BES‐2017‐079883, to M.R.); a Ramón y Cajal Program contract (RYC2018‐024475‐1, to M.G.F.B.), the Fundación Eugenio Rodríguez Pascual, the Fundación Mario Losantos, the Fundación M. Torres, and a generous donation from Mr. Eduardo Avila