Caracterización del gen Nampt en el proceso de patogénesis tumoral, valoración diagnóstica, pronóstica y como diana terapeútica.

El cáncer es una enfermedad genética compleja donde el metabolismo juega un papel fundamental. Las reacciones de reducción-oxidación son fundamentales para generar energía en forma de ATP desde los nutrientes que adquirimos en la dieta. La oxidación de dichos nutrientes genera cofactores reducidos, donde el NADH es uno de los más importantes al ser un cofactor esencial en la degradación no oxidativa de la glucosa. Esta degradación no oxidativa se conoce como glicólisis anaerobia. El proceso de patogénesis o iniciación tumoral que da lugar al cáncer es un proceso multifásico. Todas las características del fenotipo tumoral tienen en común la dependencia del reciclaje de NAD+ como principal intercambiador de electrones en las reacciones de reducción-oxidación de las que dependen. La nicotinamida fosforribosil transferasa (NAMPT) es el enzima limitante de la vía de rescate de generación del NAD+. Esta vía es el centro de actividad mayoritaria de síntesis de NAD+ celular. Por ello este enzima es el mayor contribuyente al mantenimiento, reciclaje y homeostasis de NAD+. NAMPT se encuentra expresado en todos los tejidos, con mayores niveles en médula ósea, hígado y células de fibras musculares, donde el aporte energético es mayor. NAMPT se encuentra sobreexpresado en muchos tipos de cáncer. Estos incluyen cáncer de mama, colon, próstata, tiroides, estómago y algunos tipos de cáncer hematopoyéticos. En algunos tipos de cáncer, como los sarcomas y los carcinomas gástricos, tiroideos y prostáticos, NAMPT correlaciona con una mayor capacidad invasiva del tumor. Además, la expresión de NAMPT también correlaciona con un mayor potencial metastásico y mayor resistencia a la quimioterapia. En este trabajo decidimos analizar en profundidad el papel de NAMPT en los procesos de proliferación cellular, “stemness” y evasión de la apoptosis así como estudiar el mecanismo molecular a través del cual NAMPT puede intervenir en estos procesos. Para ellos nos centramos en vías donde la desacetilación, ADP ribosilación y la regulación del metabolismo dependientes de NAD+ juegan un papel importante. Elegimos como modelo dos tumores de origen diferente, uno epithelial (cáncer colorectal) y otro de origen mesenquimal glioblastoma). Encontramos que tanto en glioma como en cáncer de colon, la obreexpresión de NAMPT es un factor de mal pronóstico en la supervivencia del paciente. Hemos encontrado que en todos los modelos analizados, tanto in vivo (tumores humanos y xenoinjertos) como in vitro, la sobreexpresión de NAMPT dió lugar a un incremento de las propiedades tumorigénicas tales como aumento de la velocidad de crecimiento, incremeneto de la capacidad de invasión, incremento a la resistencia a muerte celular y a un incremento de las propiedades de célula madre del cáncer. Hemos encontrado que, además, la sobreexpresión de NAMPT provoca un incremento del fenotipo de célula madre del cáncer, induciendo la transcripción del núcleo de genes que mantienen la pluripotencia e inducen desdiferenciación celular, promoviendo además la transición epitelio-mesénquima, paso crucial en el desarrollo de metástasis. Para ello, la expresión de NAMPT asocia con la activación de principales vías de señalización que median el fenotipo de célula mádre del cáncer: Notch, Hippo, Sonic Hedgehog y Wnt. La quimioresistencia a fármacos se debe, en parte, al aporte metabólico producido por NAMPT. son capaces de adquirir una mayor quimioresistencia a fármacos. De hecho, muchos de los procesos de detoxificación celular y reparación del ADN son dependientes de NAD+. Esto refuerza la hipótesis de que NAMPT es un potente oncogén implicado en diversos procesos celulares que ayudan al desarrollo y mantenimiento del fenotipo tumoral. El fenotipo tumoral mediado por NAMPT tiene una principal contribución a través del restablecimiento metabólico de la reserva y reciclaje de NAD+, en particular gracias al restablecimiento mediado por su producto enzimático, la nicotinamida mononucleótido (NMN). En todos los casos hemos encontrado una mejor respuesta a los tratamientos estándar para Glioma y Cáncer de Colon en combinación con la inhibición de NAMPT, con importantes resultados sobre la toxicidad inducida directamente a las células madre del cáncer. Todos estos datos indican que NAMPT puede aplicarse en la clínica como un diana terapéutica en estos tipos de cáncer, en tratamientos personalizados tanto en monoterapia como en combinación, habiendo demostrado un potencial efecto terapéutico sobre las células madre del cancer in vitro.Cancer is a genetically complex disease where cellular metabolism plays a key role on its establishment. In order to generate ATP-derived energy from our dietary intake, oxidation-reduction reactions are ultimately essential. The oxidation of such nutrients generate reduced cellular cofactors where NADH is listed as one of the most important due to its essential role within non-oxidative glucose degradation. This non-oxidative degradation is known as anaerobic glycolysis. Tumor initiation process that eventually sets the basis to Cancer disease is a multistep process. Every single feature involving the biology of the tumor phenotype have in common NAD+ pool recycling dependence as a main electron exchanger within oxidation-reduction reactions that they rely on. Nicotinamide phosphoribosyl transferase (NAMPT) is the rate-limiting enzyme of the Salvage pathway in cellular NAD+ biosynthesis. This pathway is the main source for NAD+ synthesis. As a consequence, this enzyme plays a key role in NAD+ pool maintenance, recycling and homeostasis. NAMPT is expressed in all tissues, with higher levels in bone marrow, liver and muscle myocytes, where the metabolic requirements are higher. NAMPT is also over-expressed in many cancer types. These include breast, colon, prostate, thyroid, stomach and some hematological cancers. In some cancer types like sarcomas and gastric, thyroid and prostatic carcinomas, NAMPT correlates with enhanced tissue invasion capability. Besides, NAMPT expression also correlates with enhanced metastatic potential and greater chemo resistance capability. In this work we decide to further analyze NAMPT role in cellular proliferation rate processes and apoptosis evasion as well as studying the molecular mechanism underlying NAMPT, which may be ultimately linked to the aforementioned processes. To this end, we explore pathways where NAD+ dependent deacetylation, ADP-ribosylation and metabolic regulation may play an important role in Cancer. For this, we choose as a model two tumors with different origins, an epithelial one (colon cancer) and a mesenchymal one (glioblastoma). We find that in both cancer types, glioma and colon cancer, NAMPT overexpression is a poor prognosis factor regarding patient survival. We find in every single analyzed model, either in vivo (human tumors and xenografts) or in vitro (cell lines), that NAMPT over expression results in a increase of tumorigenic properties such: increased rate of growth, increased invasiveness, increased resistance to cell death, and an increase in cancer stem cell properties. Furthermore, we have found that NAMPT overexpression results in an increase of the cancer stem cell phenotype, leading to the transcription of the main core of genes driving pluripotency and inducing cell dedifferentiation. Besides, NAMPT over expression promotes epithelial-mesenchymal transition, crucial step in metastases development. For this, NAMPT associates with the activation of main signaling pathways controlling cancer stem cell like properties: Notch, Hippo, Sonic and Wnt. This phenotype is partially achieved as a consequence to the metabolic support given by NAMPT, ultimately acquiring a higher chemo resistance to treatments. In fact, many cellular detoxifying processes and DNA repair systems rely on NAD+ pool recycling. Altogether, these data reinforce the hypothesis that NAMPT may be a potent oncogene involved in different cellular processes helping to develop and maintain the tumor phenotype. Indeed, the tumor phenotype mediated by NAMPT has a major contribution through metabolic reestablishment of NAD+ pool recycling, in particular through its metabolic product Nicotinamide Mononucleotide (NMN). In all cases we have found a better treatment response to standard treatment for glioma and colon cancer, using the NAMPT inhibitor, with great results over toxicity induced directly to cancer stem cells, recovering chemo sensibilization. Altogether, these data indicate that NAMPT can be applied on the clinic as a therapeutic marker in these cancer types, using personalized treatments in either combination or monotherapy, being demonstrated a potential therapeutic effect over cancer stem cells.Premio Extraordinario de Doctorado U

The cargo protein MAP17 (PDZK1IP1) regulates the cancer stem cell pool activating the Notch pathway by abducting NUMB

Purpose: Cancer stem cells (CSC) are self-renewing tumor cells, with the ability to generate diverse differentiated tumor cell subpopulations. They differ from normal stem cells in the deregulation of the mechanisms that normally control stem cell physiology. CSCs are the origin of metastasis and highly resistant to therapy. Therefore, the understanding of the CSC origin and deregulated pathways is important for tumor control. Experimental Design: We have included experiments in vitro, in cell lines and tumors of different origins. We have used patient-derived xenografts (PDX) and public transcriptomic databases of human tumors. Results: MAP17 (PDZKIP1), a small cargo protein overexpressed in tumors, interacts with NUMB through the PDZ-binding domain activating the Notch pathway, leading to an increase in stem cell factors and cancer-initiating–like cells. Identical behavior was mimicked by inhibiting NUMB. Conversely, MAP17 downregulation in a tumor cell line constitutively expressing this gene led to Notch pathway inactivation and a marked reduction of stemness. In PDX models, MAP17 levels directly correlated with tumorsphere formation capability. Finally, in human colon, breast, or lung there is a strong correlation of MAP17 expression with a signature of Notch and stem cell genes. Conclusions: MAP17 overexpression activates Notch pathway by sequestering NUMB. High levels of MAP17 correlated with tumorsphere formation and Notch and Stem gene transcription. Its direct modification causes direct alteration of tumorsphere number and Notch and Stem pathway transcription. This defines a new mechanism of Notch pathway activation and Stem cell pool increase that may be active in a large percentage of tumors.Ministerio de Economía y Competitividad PI15/00045, CTS-1848Junta de Andalucía PI-00-96-2014, PI-0306-201

Immunosuppressants Tacrolimus and Sirolimus revert the cardiac antifibrotic properties of p38-MAPK inhibition in 3D-multicellular human iPSC-heart organoids

Immunosuppressive regimens after a heart transplant can hamper good therapeutic progressions in fibrosis. 京都大学プレスリリース. 2022-11-15.より本物の心臓に近い心臓オルガノイドによる薬剤の効果を評価するシステム --細胞間コミュニケーションを研究できるツールの開発--. 京都大学プレスリリース. 2022-11-21.Cardiac reactive fibrosis is a fibroblast-derived maladaptive process to tissue injury that exacerbates an uncontrolled deposition of large amounts of extracellular matrix (ECM) around cardiomyocytes and vascular cells, being recognized as a pathological entity of morbidity and mortality. Cardiac fibrosis is partially controlled through the sustained activation of TGF-β1 through IL-11 in fibroblasts. Yet, preclinical studies on fibrosis treatment require human physiological approaches due to the multicellular crosstalk between cells and tissues in the heart. Here, we leveraged an iPSC-derived multi-lineage human heart organoid (hHO) platform composed of different cardiac cell types to set the basis of a preclinical model for evaluating drug cardiotoxicity and assessing cardiac fibrosis phenotypes. We found that the inhibition of the p38-MAPK pathway significantly reduces COL1A1 depositions. Yet, concomitant treatment with organ-rejection immunosuppressant drugs Tacrolimus or Sirolimus reverts this effect, opening new questions on the clinical considerations of combined therapies in reducing fibrosis after organ transplantation

Numb-like (NumbL) downregulation increases tumorigenicity, cancer stem cell-like properties and resistance to chemotherapy

NumbL, or Numb-like, is a close homologue of Numb, and is part of an evolutionary conserved protein family implicated in some important cellular processes. Numb is a protein involved in cell development, in cell adhesion and migration, in asymmetric cell division, and in targeting proteins for endocytosis and ubiquitination. NumbL exhibits some overlapping functions with Numb, but its role in tumorigenesis is not fully known. Here we showed that the downregulation of NumbL alone is sufficient to increase NICD nuclear translocation and induce Notch pathway activation. Furthermore, NumbL downregulation increases epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC)-related gene transcripts and CSC-like phenotypes, including an increase in the CSC-like pool. These data suggest that NumbL can act independently as a tumor suppressor gene. Furthermore, an absence of NumbL induces chemoresistance in tumor cells. An analysis of human tumors indicates that NumbL is downregulated in a variable percentage of human tumors, with lower levels of this gene correlated with worse prognosis in colon, breast and lung tumors. Therefore, NumbL can act as an independent tumor suppressor inhibiting the Notch pathway and regulating the cancer stem cell pool.España, Ministerio de Economía y Competitividad PI12/00137, PI15/00045España, Consejería de Ciencia e Innovación CTS-6844España, Consejería de Ciencia e Innovación CTS-1848España, Junta de Andalucía, Consejería de Salud PI-0306-2012España, Junta de Andalucía, Consejería de Salud PI-0096-201

CDH18 is a fetal epicardial biomarker regulating differentiation towards vascular smooth muscle cells

CDH18は胎児期の心外膜細胞の指標であり胎児心外膜から平滑筋細胞の分化を制御している. 京都大学プレスリリース. 2022-02-09.Using stem cells to regenerate the heart. 京都大学プレスリリース. 2022-02-09.The epicardium is a mesothelial layer covering the myocardium serving as a progenitor source during cardiac development. The epicardium reactivates upon cardiac injury supporting cardiac repair and regeneration. Fine-tuned balanced signaling regulates cell plasticity and cell-fate decisions of epicardial-derived cells (EPCDs) via epicardial-to-mesenchymal transition (EMT). However, powerful tools to investigate epicardial function, including markers with pivotal roles in developmental signaling, are still lacking. Here, we recapitulated epicardiogenesis using human induced pluripotent stem cells (hiPSCs) and identified type II classical cadherin CDH18 as a biomarker defining lineage specification in human active epicardium. The loss of CDH18 led to the onset of EMT and specific differentiation towards cardiac smooth muscle cells. Furthermore, GATA4 regulated epicardial CDH18 expression. These results highlight the importance of tracing CDH18 expression in hiPSC-derived epicardial cells, providing a model for investigating epicardial function in human development and disease and enabling new possibilities for regenerative medicine

Characterization of hiPSC-Derived Muscle Progenitors Reveals Distinctive Markers for Myogenic Cell Purification Toward Cell Therapy

骨格筋幹細胞を純化する方法を確立 --筋肉の細胞移植治療の実現に向けて--. 京都大学プレスリリース. 2021-04-02.Enhanced muscle regeneration using stem cells. 京都大学プレスリリース. 2021-04-02.The transplantation of muscle progenitor cells (MuPCs) differentiated from human induced pluripotent stem cells (hiPSCs) is a promising approach for treating skeletal muscle diseases such as Duchenne muscular dystrophy (DMD). However, proper purification of the MuPCs before transplantation is essential for clinical application. Here, by using MYF5 hiPSC reporter lines, we identified two markers for myogenic cell purification: CDH13, which purified most of the myogenic cells, and FGFR4, which purified a subset of MuPCs. Cells purified with each of the markers showed high efficiency for regeneration after transplantation and contributed to the restoration of dystrophin expression in DMD-immunodeficient model mice. Moreover, we found that MYF5 regulates CDH13 expression by binding to the promoter regions. These findings suggest that FGFR4 and CDH13 are strong candidates for the purification of hiPSC-derived MuPCs for therapeutical application

NAMPT as a Dedifferentiation-Inducer Gene: NAD+ as Core Axis for Glioma Cancer Stem-Like Cells Maintenance

Glioma Cancer Stem-Like Cells (GSCs) are a small subset of CD133+ cells with self-renewal properties and capable of initiating new tumors contributing to Glioma progression, maintenance, hierarchy, and complexity. GSCs are highly resistant to chemo and radiotherapy. These cells are believed to be responsible for tumor relapses and patients' fatal outcome after developing a recurrent Glioblastoma (GBM) or High Grade Glioma (HGG). GSCs are cells under replicative stress with high demands on NAD+ supply to repair DNA, maintain self-renewal capacity and to induce tumor plasticity. NAD+ feeds Poly-ADP polymerases (PARP) and NAD+-dependent deacetylases (SIRTUINS) contributing to GSC phenotype. This energetic core axis is mainly controlled by the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT), an important oncogene contributing to tumor dedifferentiation. Targeting GSCs depicts a new frontier in Glioma therapy; hence NAMPT could represent a key regulator for GSCs maintenance. Its inhibition may attenuate GSCs properties by decreasing NAD+ supply, consequently contributing to a better outcome together with current therapies for Glioma control

Dasatinib, a Src inhibitor, sensitizes liver metastatic colorectal carcinoma to oxaliplatin in tumors with high levels of phospho-Src.

Despite the development of new antineoplastic agents for the treatment of colorectal cancer (CRC), oxaliplatin and fluoropyrimidines remain the most commonly employed drugs for the treatment of both early and advanced disease. Intrinsic or acquired resistance is, however, an important limitation to pharmacological therapy, and the development of chemosensitization strategies constitute a major goal with important clinical implications. In the present work, we determined that high levels of activated Src kinase, measured as phospho-Src at the Tyr419 residue in CRC cell lines, can promote colorectal carcinoma cell resistance to oxaliplatin, but not to 5-fluorouracil (5FU), and that inhibition of this protein restores sensitivity to oxaliplatin. Similar results were observed with in vivo patient-derived xenograft (PDX) models that were orthotopically grown in murine livers. In PDX tumor lines derived from human CRC liver metastasis, dasatinib, a Src inhibitor, increases sensitivity to oxaliplatin only in tumors with high p-Src. However, dasatinib did not modify sensitivity to 5FU in any of the models. Our data suggest that chemoresistance induced by p-Src is specific to oxaliplatin, and that p-Src levels can be used to identify patients who may benefit from this combination therapy. These results are relevant for clinicians as they identify a novel biomarker of drug resistance that is suitable to pharmacological manipulation.HUVR-IBiS Biobanco del Sistema Sanitario Público de Andalucía y ISCIII-Red de Biobancos [PT13/0010/0056].FEDER from Regional Development European Funds (European Union), Ministerio de Economía y Competitividad, Plan Nacional de I+D+I 2008-2011, Plan Estatal de I+D+i 2013-2016, ISCIII [Fis: PI12/00137, PI13/02295, PI15/00045, RTICC: RD12/0036/0028].Consejería de Ciencia e Innovación [CTS-1848] y Consejería de Salud de la Junta de Andalucía [PI-0306-2012, PI-0096-2014]

MAP17 and SGLT1 Protein Expression Levels as Prognostic Markers for Cervical Tumor Patient Survival

MAP17 is a membrane-associated protein that is overexpressed in human tumors. Because the expression of MAP17 increases reactive oxygen species (ROS) generation through SGLT1 in cancer cells, in the present work, we investigated whether MAP17 and/or SGLT1 might be markers for the activity of treatments involving oxidative stress, such as cisplatin or radiotherapy. First, we confirmed transcriptional alterations in genes involved in the oxidative stress induced by MAP17 expression in HeLa cervical tumor cells and found that Hela cells expressing MAP17 were more sensitive to therapies that induce ROS than were parental cells. Furthermore, MAP17 increased glucose uptake through SGLT receptors. We then analyzed MAP17 and SGLT1 expression levels in cervical tumors treated with cisplatin plus radiotherapy and correlated the expression levels with patient survival. MAP17 and SGLT1 were expressed in approximately 70% and 50% of cervical tumors of different types, respectively, but they were not expressed in adenoma tumors. Furthermore, there was a significant correlation between MAP17 and SGLT1 expression levels. High levels of either MAP17 or SGLT1 correlated with improved patient survival after treatment. However, the patients with high levels of both MAP17 and SGLT1 survived through the end of this study. Therefore, the combination of high MAP17 and SGLT1 levels is a marker for good prognosis in patients with cervical tumors after cisplatin plus radiotherapy treatment. These results also suggest that the use of MAP17 and SGLT1 markers may identify patients who are likely to exhibit a better response to treatments that boost oxidative stress in other cancer types. © 2013 Perez et al.This work was supported by grants from the Spanish Ministry of Science and Innovation and FEDER funds (SAF2009-08605), Consejeria de Ciencia e Innovacion and Consejeria de Salud of the Junta de Andalucia (CTS-6844 and PI-0142) and FIS (PI12/00137). AC’s laboratory is also funded by a fellowship from the Fundacion Oncologica FERO, supported by Fundació Josep Botet.Peer Reviewe

Spinophilin loss correlates with poor patient prognosis in advanced stages of colon carcinoma

[Purpose+ The genomic region 17q21 is frequently associated with microsatellite instability and LOH in cancer, including gastric and colorectal carcinomas. This region contains several putative tumor suppressor genes, including Brca1, NM23, prohibitin, and spinophilin (Spn, PPP1R9B, neurabin II). The scaffold protein Spn is one of the regulatory subunits of phosphatase-1 (PP1) that targets PP1 to distinct subcellular locations and couples PP1 to its target. Thus, Spn may alter cell-cycle progression via the regulation of the phosphorylation status of the retinoblastoma protein, a direct target of PP1. Therefore, we analyzed whether Spn levels were reduced in colorectal carcinomas and whether Spn levels correlated with prognosis or response to therapy.[Experimental Design] By means of immunohistochemistry or quantitative PCR, we studied the levels of Spn in stages II, III, and IV colorectal carcinoma tumors and correlated to other clinicopathologic features as well as prognosis or response to therapy.[Results] Spn was lost in a percentage of human gastric, small intestine, and colorectal carcinomas. In patients with colorectal carcinoma, tumoral Spn downregulation correlated with a more aggressive histologic phenotype (poorer tumor differentiation and higher proliferative Ki67 index). Consistent with this observation, lower Spn protein expression levels were associated with faster relapse and poorer survival in patients with stage III colorectal carcinoma, particularly among those receiving adjuvant fluoropyrimidine therapy. We validated this result in an independent cohort of patients with metastatic colorectal carcinoma treated with standard chemotherapy. Although patients that achieved an objective tumor response exhibited Spn levels similar to nontumoral tissue, nonresponding patients showed a significant reduction in Spn mRNA levels.[Conclusions] Our data suggest that Spn downregulation contributes to a more aggressive biologic behavior, induces chemoresistance, and is associated with a poorer survival in patients with advanced stages of colorectal carcinoma. © 2013 American Association for Cancer Research.This work was supported by grants from the Spanish Ministry of Science and Innovation (SAF2009-08605), Fondo de Investigacion Sanitaria (PI12/00137), Consejeria de Ciencia e Innovacion, and Consejeria de Salud of the Junta de Andalucia (CTS-6844 and PI-0142). A. Carnero's laboratory is also funded by a fellowship from Fundacion Oncologica FERO. P. Estevez-Garcia and I. Lopez-Calderero are supported by Rio Ortega Fellowships and S. Molina-Pinelo is supported by a Sara Borrell fellowship. R. Garcia-Carbonero is funded by the Instituto de Salud Carlos III, Ministerio de Sanidad, Spain (PI 10.02164).Peer Reviewe