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The lack of a precise noninvasive, clinical evaluation method for cardiac fibrosis hinders the development of successful treatments that can effectively work in physiological settings, where tissues and organs are interconnected and moderating drug responses. To address this challenge and advance personalized medicine, researchers have turned to human-induced pluripotent stem (iPS) cells, which can be differentiated to resemble the human heart in terms of structure, function and cellular composition. In this chapter, we present an assay protocol that uses these iPS cells to generate heart organoids for the in vitro evaluation of cardiac fibrosis. By establishing this biological platform, we pave the way for conducting phenotype evaluation and treatment screening in a multiscale approach, aiming to discover effective interventions for the treatment of cardiac fibrosis.
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Diferenciação Celular , Fibrose , Células-Tronco Pluripotentes Induzidas , Organoides , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Organoides/patologia , Organoides/citologia , Miocárdio/patologia , Miocárdio/citologia , Técnicas de Cultura de Células/métodos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/patologia , Células CultivadasRESUMO
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-ß1 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.
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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.
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For regenerative cell therapies using pluripotent stem cell (PSC)-derived cells, large quantities of purified cells are required. Magnetic-activated cell sorting (MACS) is a powerful approach to collect target antigen-positive cells; however, it remains a challenge to purify various cell types efficiently at large scale without using antibodies specific to the desired cell type. Here we develop a technology that combines microRNA (miRNA)-responsive mRNA switch (miR-switch) with MACS (miR-switch-MACS) to purify large amounts of PSC-derived cells rapidly and effectively. We designed miR-switches that detect specific miRNAs expressed in target cells and controlled the translation of a CD4-coding transgene as a selection marker for MACS. For the large-scale purification of induced PSC-derived cardiomyocytes (iPSC-CMs), we transferred miR-208a-CD4 switch-MACS and obtained purified iPSC-CMs efficiently. Moreover, miR-375-CD4 switch-MACS highly purified pancreatic insulin-producing cells and their progenitors expressing Chromogranin A. Overall, the miR-switch-MACS method can efficiently purify target PSC-derived cells for cell replacement therapy.
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Células-Tronco Pluripotentes Induzidas , MicroRNAs , Diferenciação Celular/genética , Separação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fenômenos Magnéticos , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
Differentiation protocols to direct cell fate decision from pluripotent stem cells to cardiac myocytes normally achieve high purity and quality of cells. Nonetheless, the highly specialized anatomy of the heart enables the possibility that acquisition of terminal somatic differentiation from pluripotency might imply heterogeneity of non-desire cell lineages. Directed cardiac differentiation empowers differentiation of pool of cells commonly reported to contain different proportions of ventricular, atrial, and nodal-like cells. RNA sequencing (RNA-Seq) allows a precise transcriptional profiling, ensuring a quality checking of the cell identity our protocol has derived as a main outcome. Here we describe a workflow methodology on how to adapt RNA sequencing analysis for integration into the R analysis pipeline in order to characterize chamber-specific gene signatures of the major cardiac lineages of myocytes in the heart.
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Perfilação da Expressão Gênica , Átrios do Coração/citologia , Ventrículos do Coração/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/química , RNA-Seq/métodos , Transcriptoma , Diferenciação Celular/genética , Células Cultivadas , Análise por Conglomerados , Ontologia Genética , Átrios do Coração/química , Ventrículos do Coração/química , Humanos , Análise de Componente Principal , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Fluxo de TrabalhoRESUMO
The EMX (Empty Spiracles Homeobox) genes EMX1 and EMX2 are two homeodomain gene members of the EMX family of transcription factors involved in the regulation of various biological processes, such as cell proliferation, migration, and differentiation, during brain development and neural crest migration. They play a role in the specification of positional identity, the proliferation of neural stem cells, and the differentiation of certain neuronal cell phenotypes. In general, they act as transcription factors in early embryogenesis and neuroembryogenesis from metazoans to higher vertebrates. The EMX1 and EMX2's potential as tumor suppressor genes has been suggested in some cancers. Our work showed that EMX1/EMX2 act as tumor suppressors in sarcomas by repressing the activity of stem cell regulatory genes (OCT4, SOX2, KLF4, MYC, NANOG, NES, and PROM1). EMX protein downregulation, therefore, induced the malignance and stemness of cells both in vitro and in vivo. In murine knockout (KO) models lacking Emx genes, 3MC-induced sarcomas were more aggressive and infiltrative, had a greater capacity for tumor self-renewal, and had higher stem cell gene expression and nestin expression than those in wild-type models. These results showing that EMX genes acted as stemness regulators were reproduced in different subtypes of sarcoma. Therefore, it is possible that the EMX genes could have a generalized behavior regulating proliferation of neural crest-derived progenitors. Together, these results indicate that the EMX1 and EMX2 genes negatively regulate these tumor-altering populations or cancer stem cells, acting as tumor suppressors in sarcoma.
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Genes Homeobox , Proteínas de Homeodomínio/genética , Sarcoma/genética , Fatores de Transcrição/genética , Animais , Carcinogênese , Linhagem Celular Tumoral , Xenoenxertos , Proteínas de Homeodomínio/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Sarcoma/metabolismo , Sarcoma/patologia , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Sarcomas are a very heterogeneous group of tumors with intrinsic developmental programs derived from the cell of origin. This implies a functional hierarchy inside tumors governed by sarcoma stem cells. Therefore, genetic and/or epigenetic changes profoundly affect the biology of sarcoma tumor stem cells. EMX genes are proposed to be transcription factors that are involved in the sarcomagenesis process, regardless of the neural or mesodermal embryological sarcoma origin. It has been shown that EMX1 or EMX2 overexpression reduces tumorigenic properties, while reducing the levels of these genes enhances these properties. Furthermore, it has been shown that EMX genes decrease the expression of stem cell regulatory genes and the stem cell phenotype. Taken together, these results indicate that the EMX1 and EMX2 genes negatively regulate these tumor-remodeling populations or sarcoma stem cells, acting as tumor suppressors in sarcoma. METHODS: Bioinformatic analysis, quantitative mRNA and protein expression analysis, cell models of sarcoma by ectopic expression of EMX genes. By cell biology methods we measured tumorigenesis and populations enriched on stem cell phenotypes, either in vitro or in vivo. RESULTS: In this work, we showed that the canonical Wnt pathway is one of the mechanisms that explains the relationships of EMX1/EMX2 and stem cell genes in sarcoma. The Wnt-EMX1/EMX2 relationship was validated in silico with sarcoma patient datasets, in vitro in primary derived sarcoma cell lines, and in vivo. EMX expression was found to negatively regulate the Wnt pathway. In addition, the constitutive activation of the Wnt pathway revers to a more aggressive phenotype with stem cell properties, and stemness gene transcription increased even in the presence of EMX1 and/or EMX2 overexpression, establishing the relationship among the Wnt pathway, stem cell genes and the EMX transcription factors. CONCLUSIONS: Our data showed that Empty Spiracles Homeobox Genes EMX1 and EMX2 represses WNT signalling and activation of WNT pathway bypass EMX-dependent stemness repression and induces sarcomagenesis. These results also suggest the relevance of the Wnt/b-catenin/stemness axis as a therapeutic target in sarcoma.
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Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Sarcoma/genética , Sarcoma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt , Carcinogênese , Humanos , Sarcoma/patologia , TransfecçãoRESUMO
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.
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Biomarcadores/metabolismo , Separação Celular , Terapia Baseada em Transplante de Células e Tecidos , Células-Tronco Pluripotentes Induzidas/citologia , Desenvolvimento Muscular , Músculo Esquelético/citologia , Células-Tronco/citologia , Animais , Sequência de Bases , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Genes Reporter , Camundongos Transgênicos , Fator Regulador Miogênico 5 , Fator de Transcrição PAX7/metabolismo , RNA-Seq , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Regeneração , Transcrição Gênica , Transcriptoma/genéticaRESUMO
One of the earliest maturation steps in cardiomyocytes (CMs) is the sarcomere protein isoform switch between TNNI1 and TNNI3 (fetal and neonatal/adult troponin I). Here, we generate human induced pluripotent stem cells (hiPSCs) carrying a TNNI1EmGFP and TNNI3mCherry double reporter to monitor and isolate mature sub-populations during cardiac differentiation. Extensive drug screening identifies two compounds, an estrogen-related receptor gamma (ERRγ) agonist and an S-phase kinase-associated protein 2 inhibitor, that enhances cardiac maturation and a significant change to TNNI3 expression. Expression, morphological, functional, and molecular analyses indicate that hiPSC-CMs treated with the ERRγ agonist show a larger cell size, longer sarcomere length, the presence of transverse tubules, and enhanced metabolic function and contractile and electrical properties. Here, we show that ERRγ-treated hiPSC-CMs have a mature cellular property consistent with neonatal CMs and are useful for disease modeling and regenerative medicine.
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Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Receptores de Estrogênio/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Miócitos Cardíacos/metabolismo , Receptores de Estrogênio/química , Proteínas Quinases Associadas a Fase S/antagonistas & inibidores , Sarcolema/efeitos dos fármacos , Sarcolema/metabolismo , Sarcômeros/efeitos dos fármacos , Sarcômeros/metabolismo , Transcriptoma/efeitos dos fármacos , Troponina I/genética , Troponina I/metabolismoRESUMO
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.
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Cancer cells are in continuous communication with the surrounding microenvironment and this communication can affect tumor evolution. In this work, we show that phospholipase D2 (PLD2) was overexpressed in colon tumors and is secreted by cancer cells, inducing senescence in neighboring fibroblasts. This occurs through its lipase domain. Senescence induced by its product, phosphatidic acid, leads to a senescence-associated secretory phenotype (SASP) able to increase the stem properties of cancer cells. This increase in stemness occurs by Wnt pathway activacion. This closes a feedback loop in which senescence acts as a crosspoint for the generation of CSCs mediated by phospholipid metabolism. We also demonstrate the connexion of both phenomena in mouse models in vivo showing that a high PLD2 expression increased stemness and tumorigenesis. Thus, the patients with colon cancer show high levels of PLD2 and SASP factor genes expression correlating with Wnt pathway activation. Therefore, we demonstrate that tumor cell-secreted PLD2 contributes to tumor development by modifying the microenvironment, making it a possible therapeutic target for cancer treatment. This mechanism may also explain the high levels of Wnt pathway activation in colon cancer.
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Carcinogênese/genética , Neoplasias do Colo/genética , Fosfolipase D/genética , Microambiente Tumoral/genética , Animais , Proliferação de Células/genética , Senescência Celular , Neoplasias do Colo/epidemiologia , Neoplasias do Colo/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Masculino , Camundongos , Células-Tronco Neoplásicas/patologia , Via de Sinalização Wnt/genéticaRESUMO
Purpose: Colorectal cancer is the second most common cancer in women and the third most common in men worldwide. However, despite current progress, many patients with advanced and metastatic tumors still die from the malignancy. Refractory disease often relies on nicotinamide adenine dinucleotide (NAD)-dependent mechanisms. NAD metabolism and a stable NAD regeneration circuit are required to maintain tissue homeostasis and metabolism. However, high levels of NAD confer therapy resistance to tumors.Experimental Design: Ectopic overexpression of nicotinamide phosphoribosil transferase (NAMPT) and shRNAs in colorectal cancer cell lines, tumorigenic and stemness properties and transcription measurement in culture and in vivo Transcriptional analysis in public databases. Therapeutic approaches.Results: NAMPT, the rate-limiting enzyme responsible for the highest source of physiologic NAD biosynthesis, increases tumorigenic properties and induces cancer stem cell-like properties through pathways that control stem cell signaling, thus enriching the cancer-initiating cell (CIC) population. Furthermore, NAMPT expression correlated with high levels of CIC-like cells in colon tumors directly extracted from patients, and transcription meta-analysis revealed that NAMPT is also a key factor that induces cancer stem pathways in colorectal cancer tumors. This effect is mediated by PARP and SIRT1. In addition, we report a novel NAMPT-driven signature that stratifies prognosis from high to low expression groups. The NAMPT signature contained SIRT1 and PARP1 levels as well as other cancer stem cell-related genes. Finally, NAMPT inhibition increased the sensitivity to apoptosis in both NAMPT-expressing cells and tumorspheres.Conclusions: NAMPT represents a novel therapeutic target in colon cancer progression and relapse, particularly the CIC subset of human colon cancers. Clin Cancer Res; 24(5); 1202-15. ©2017 AACR.
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Neoplasias do Colo/patologia , Citocinas/metabolismo , Células-Tronco Neoplásicas/patologia , Nicotinamida Fosforribosiltransferase/metabolismo , Animais , Linhagem Celular Tumoral , Colo/patologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/mortalidade , Citocinas/genética , Conjuntos de Dados como Assunto , Resistencia a Medicamentos Antineoplásicos , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos Nus , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Oncogenes , Poli(ADP-Ribose) Polimerase-1/metabolismo , Prognóstico , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Sirtuína 1/metabolismo , Análise de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Gliomas are the most prevalent primary malignant brain tumors associated with poor prognosis. NAMPT, a rate-limiting enzyme that boosts the nicotinamide adenine dinucleotide (NAD) regeneration in the salvage pathway, is commonly expressed in these tumors. NAD metabolism is required to maintain tissue homeostasis. To maintain metabolism, cancer cells require a stable NAD regeneration circuit. However, high levels of NAD confer resistance to therapy to these tumors, usually treated with Temozolomide (TMZ). We report that NAMPT overexpression in glioma cell lines increases tumorigenic properties controlling stem cell pathways and enriching the cancer-initiating cell (CIC) population. Furthermore, NAMPT expression correlated with high levels of Nanog, CD133 and CIC-like cells in glioblastoma directly extracted from patients. Meta-analysis reveals that NAMPT is also a key factor inducing cancer stem pathways in glioma cells. Furthermore, we report a novel NAMPT-driven signature which stratify prognosis within tumor staging. NAMPT signature also correlates directly with EGFR positive and IDH negative tumors. Finally, NAMPT inhibition increases sensitivity to apoptosis in both NAMPT-expressing cells and tumorspheres. Therefore, NAMPT represents a novel therapeutic target in Glioma progression and relapse.
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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. Clin Cancer Res; 23(14); 3871-83. ©2017 AACR.
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Proteínas de Membrana/genética , Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Proteínas do Tecido Nervoso/genética , Animais , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células HeLa , Humanos , Camundongos , Neoplasias/patologia , Células-Tronco Neoplásicas/patologia , Receptores Notch/genética , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The PIM family of Ser/Thr kinase proteins has been implicated in tumorigenesis at different levels. PIM proteins are overexpressed in several tumor types and have been associated with chemoresistance. However, their role in hormone-dependent female tissues has not been explored, especially in the uterus, breast and ovary. We generated conditional transgenic mice with confined expression of human PIM1 or PIM2 genes in these tissues. We characterized the tumoral response to these genetic alterations corroborating their role as oncogenes since they induce hyperproliferation in all tissues and tumors in mammary gland and uterus. Furthermore, we observed a high degree of inflammatory infiltration in these tissues of transgenic mice accompanied by NFAT and mTOR activation and IL6 expression. Moreover, PIM1/2 were overexpressed in human breast, uterine and ovarian tumors, correlating with inflammatory features and stem cell markers. Our data suggest that PIM1/2 kinase overexpression provoke tissue alterations and a large IL6-dependent inflammatory response that may act synergistically during the process of tumorigenesis. The possible end-point is an increased percentage of cancer stem cells, which may be partly responsible for the therapy resistance found in tumors overexpressing PIM kinases.
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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.
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Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células-Tronco Neoplásicas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Carcinogênese , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Endocitose , Transição Epitelial-Mesenquimal , Feminino , Células HeLa , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Camundongos , Células-Tronco Neoplásicas/citologia , Receptores Notch/metabolismo , Sarcoma/metabolismoRESUMO
The PIM family of serine/threonine kinases has three highly conserved isoforms (PIM1, PIM2 and PIM3). PIM proteins are regulated through transcription and stability by JAK/STAT pathways and are overexpressed in hematological malignancies and solid tumors. The PIM kinases possess weak oncogenic abilities, but enhance other genes or chemical carcinogens to induce tumors. We generated conditional transgenic mice that overexpress PIM1 or PIM2 in male reproductive organs and analyzed their contribution to tumorigenesis. We found an increase in alterations of sexual organs and hyperplasia in the transgenic mice correlating with inflammation. We also found that PIM1/2 are overexpressed in a subset of human male germ cells and prostate tumors correlating with inflammatory features and stem cell markers. Our data suggest that PIM1/2 kinase overexpression is a common feature of male reproductive organs tumors, which provoke tissue alterations and a large inflammatory response that may act synergistically during the process of tumorigenesis. There is also a correlation with markers of cancer stem cells, which may contribute to the therapy resistance found in tumors overexpressing PIM kinases.
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Biomarcadores Tumorais , Neoplasias da Próstata , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-pim-1 , Proteínas Proto-Oncogênicas , Animais , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-pim-1/biossíntese , Proteínas Proto-Oncogênicas c-pim-1/genéticaRESUMO
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.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Dasatinibe/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Compostos Organoplatínicos/farmacologia , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Dasatinibe/administração & dosagem , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Camundongos Nus , Metástase Neoplásica , Compostos Organoplatínicos/administração & dosagem , Oxaliplatina , Transfecção , Quinases da Família src/metabolismoRESUMO
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.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias do Colo/metabolismo , Expressão Gênica , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Idoso , Neoplasias do Colo/mortalidade , Neoplasias do Colo/patologia , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Proteínas dos Microfilamentos/genética , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Proteínas do Tecido Nervoso/genética , PrognósticoRESUMO
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.