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Pulmonary arteriovenous malformations (PAVMs) are vascular anomalies resulting in abnormal connections between pulmonary arteries and veins. In 80% of cases, PAVMs are present from birth, but clinical manifestations are rarely seen in childhood. These congenital malformations are typically associated with Hereditary Hemorrhagic Telangiectasia (HHT), a rare disease that affects 1 in 5000/8000 individuals. HHT disease is frequently caused by mutations in genes involved in the TGF-ß pathway. However, approximately 15% of patients do not have a genetic diagnosis and, among the genetically diagnosed, more than 33% do not meet the Curaçao criteria. This makes clinical diagnosis even more challenging in the pediatric age group. Here, we introduce an 8-year-old patient bearing a severe phenotype of multiple diffuse PAVMs caused by an unknown mutation which ended in lung transplantation. Phenotypically, the case under study follows a molecular pattern which is HHT-like. Therefore, molecular- biological and cellular-functional analyses have been performed in primary endothelial cells (ECs) isolated from the explanted lung. The findings revealed a loss of functionality in lung endothelial tissue and a stimulation of endothelial-to-mesenchymal transition. Understanding the molecular basis of this transition could potentially offer new therapeutic strategies to delay lung transplantation in severe cases.
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Células Endoteliais , Artéria Pulmonar , Veias Pulmonares , Telangiectasia Hemorrágica Hereditária , Humanos , Telangiectasia Hemorrágica Hereditária/genética , Telangiectasia Hemorrágica Hereditária/patologia , Criança , Artéria Pulmonar/anormalidades , Artéria Pulmonar/patologia , Veias Pulmonares/anormalidades , Veias Pulmonares/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Masculino , Mutação , Malformações Arteriovenosas/genética , Malformações Arteriovenosas/patologia , Malformações Arteriovenosas/metabolismo , Transição Epitelial-Mesenquimal/genética , Transplante de Pulmão , Fístula Arteriovenosa/patologia , Fístula Arteriovenosa/genética , Pulmão/patologia , Pulmão/irrigação sanguínea , FemininoRESUMO
Cerebral cavernous malformation (CCM) or familial cavernomatosis is a rare, autosomal dominant, inherited disease characterized by the presence of vascular malformations consisting of blood vessels with an abnormal structure in the form of clusters. Based on the altered gene (CCM1/Krit1, CCM2, CCM3) and its origin (spontaneous or familial), different types of this disease can be found. In this work we have isolated and cultivated primary endothelial cells (ECs) from peripheral blood of a type 1 CCM patient. Differential functional and gene expression profiles of these cells were analyzed and compared to primary ECs from a healthy donor. The mutation of the familial index case consisted of a heterozygous point mutation in the position +1 splicing consensus between exons 15 and 16, causing failure in RNA processing and in the final protein. Furthermore, gene expression analysis by quantitative PCR revealed a decreased expression of genes involved in intercellular junction formation, angiogenesis, and vascular homeostasis. Cell biology analysis showed that CCM1 ECs were impaired in angiogenesis and cell migration. Taken together, the results obtained suggest that the alterations found in CCM1 ECs are already present in the heterozygous condition, suffering from vascular impairment and somewhat predisposed to vascular damage.
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Células Endoteliais , Junções Intercelulares , Humanos , Movimento Celular/genética , Éxons , ConsensoRESUMO
Due to the limited effectiveness of current treatments, the survival rate of patients with metastatic castration-resistant prostate cancer (mCRPC) is significantly reduced. Consequently, it is imperative to identify novel therapeutic targets for managing these patients. Since the invasive ability of cells is crucial for establishing and maintaining metastasis, the aim of this study was to identify the essential regulators of invasive abilities of mCRPC cells by conducting two independent high-throughput CRISPR/Cas9 screenings. Furthermore, some of the top hits were validated using siRNA technology, with protein arginine methyltransferase 7 (PRMT7) emerging as the most promising candidate. We demonstrated that its inhibition or depletion via genetic or pharmacological approaches significantly reduces invasive, migratory and proliferative abilities of mCRPC cells in vitro. Moreover, we confirmed that PRMT7 ablation reduces cell dissemination in chicken chorioallantoic membrane and mouse xenograft assays. Molecularly, PRMT7 reprograms the expression of several adhesion molecules by methylating various transcription factors, such as FoxK1, resulting in the loss of adhesion from the primary tumor and increased motility of mCRPC cells. Furthermore, PRMT7 higher expression correlates with tumor aggressivity and poor overall survival in prostate cancer patients. Thus, this study demonstrates that PRMT7 is a potential therapeutic target and potential biomarker for mPCa.
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Neoplasias de Próstata Resistentes à Castração , Proteína-Arginina N-Metiltransferases , Masculino , Animais , Camundongos , Humanos , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Sistemas CRISPR-Cas , Genes Essenciais , Detecção Precoce de CâncerRESUMO
Liver cancer represents a major health problem worldwide with growing incidence and high mortality, hepatocellular carcinoma (HCC) being the most frequent. Hepatocytes are likely the cellular origin of most HCCs through the accumulation of genetic alterations, although hepatic progenitor cells (HPCs) might also be candidates in specific cases, as discussed here. HCC usually develops in a context of chronic inflammation, fibrosis, and cirrhosis, although the role of fibrosis is controversial. The interplay between hepatocytes, immune cells and hepatic stellate cells is a key issue. This review summarizes critical aspects of the liver tumor microenvironment paying special attention to platelets as new key players, which exert both pro- and anti-tumor effects, determined by specific contexts and a tight regulation of platelet signaling. Additionally, the relevance of specific signaling pathways, mainly HGF/MET, EGFR and TGF-ß is discussed. HGF and TGF-ß are produced by different liver cells and platelets and regulate not only tumor cell fate but also HPCs, inflammation and fibrosis, these being key players in these processes. The role of C3G/RAPGEF1, required for the proper function of HGF/MET signaling in HCC and HPCs, is highlighted, due to its ability to promote HCC growth and, regulate HPC fate and platelet-mediated actions on liver cancer.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Neoplasias Hepáticas/metabolismo , Carcinoma Hepatocelular/metabolismo , Fígado/metabolismo , Hepatócitos/metabolismo , Cirrose Hepática/metabolismo , Fibrose , Fator de Crescimento Transformador beta/metabolismo , Inflamação/metabolismo , Microambiente TumoralRESUMO
PURPOSE: Von Hippel-Lindau (VHL) is a rare inherited disease mainly characterized by the growth of tumours, predominantly hemangioblastomas (Hbs) in the CNS and retina, and renal carcinomas. The natural history of VHL disease is variable, differing in the age of onset and its penetrance, even among relatives. Unfortunately, sometimes VHL shows more severe than average: the onset starts in adolescence, and surgeries are required almost every year. In these cases, the factor that triggers the appearance and growth of Hbs usually remains unknown, although additional mutations are suspected. METHODS: We present the case of a VHL patient whose first surgery was at 13 years of age. Then, along his next 8 years, he has undergone 5 surgeries for resection of 10 CNS Hbs. To clarify this severe VHL condition, DNA from a CNS Hb and white blood cells (WBC) was sequenced using next-generation sequencing technology. RESULTS: Massive DNA sequencing of the WBC (germ line) revealed a pathogenic mutation in CHEK2 and the complete loss of a VHL allele (both tumour suppressors). Moreover, in the tumour sample, several mutations, in BRAF1 and PTPN11 were found. Familiar segregation studies showed that CHEK2 mutation was in the maternal lineage, while VHL was inherited by paternal lineage. CONCLUSIONS: Finally, clinical history correlated to the different genotypes in the family, concluding that the severity of these VHL manifestations are due to both, VHL-and-CHEK2 mutations. This case report aims to notice the importance of deeper genetic analyses, in inherited rare diseases, to uncover non-expected mutations.
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Carcinoma de Células Renais , Hemangioblastoma , Neoplasias Renais , Doença de von Hippel-Lindau , Masculino , Adolescente , Humanos , Hemangioblastoma/genética , Hemangioblastoma/cirurgia , Hemangioblastoma/patologia , Mutação/genética , Doença de von Hippel-Lindau/diagnóstico , Doença de von Hippel-Lindau/genética , Doença de von Hippel-Lindau/patologiaRESUMO
Previous data indicate that C3G (RapGEF1) main isoform is highly expressed in liver progenitor cells (or oval cells) compared to adult mature hepatocytes, suggesting it may play an important role in oval cell biology. Hence, we have explored C3G function in the regulation of oval cell properties by permanent gene silencing using shRNAs. We found that C3G knock-down enhanced migratory and invasive ability of oval cells by promoting a partial epithelial to mesenchymal transition (EMT). This is likely mediated by upregulation of mRNA expression of the EMT-inducing transcription factors, Snail1, Zeb1 and Zeb2, induced in C3G-silenced oval cells. This EMT is associated to a higher expression of the stemness markers, CD133 and CD44. Moreover, C3G down-regulation increased oval cells clonogenic capacity by enhancing cell scattering. However, C3G knock-down did not impair oval cell differentiation into hepatocyte lineage. Mechanistic studies revealed that HGF/MET signaling and its pro-invasive activity was impaired in oval cells with low levels of C3G, while TGF-ß signaling was increased. Altogether, these data suggest that C3G might be tightly regulated to ensure liver repair in chronic liver diseases such as non-alcoholic steatohepatitis. Hence, reduced C3G levels could facilitate oval cell expansion, after the proliferation peak, by enhancing migration.
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Transição Epitelial-Mesenquimal , Células-Tronco , Transição Epitelial-Mesenquimal/genética , Regulação para Baixo/genética , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , RNA Mensageiro/metabolismoRESUMO
Research on cancer therapies focuses on processes such as angiogenesis, cell signaling, stemness, metastasis, and drug resistance and inflammation, all of which are influenced by the cellular and molecular microenvironment of the tumor. Different strategies, such as antibodies, small chemicals, hormones, cytokines, and, recently, gene editing techniques, have been tested to reduce the malignancy and generate a harmful microenvironment for the tumor. Few therapeutic agents have shown benefits when administered alone, but a few more have demonstrated clear improvement when administered in combination with other therapeutic molecules. In 2008 (and for the first time in the clinic), the therapeutic benefits of the ß-adrenergic receptor antagonist, propranolol, were described in benign tumors, such as infantile hemangioma. Propranolol, initially prescribed for high blood pressure, irregular heart rate, essential tremor, and anxiety, has shown, in the last decade, increasing evidence of its antitumoral properties in more than a dozen different types of cancer. Moreover, the use of propranolol in combination therapies with other drugs has shown synergistic antitumor effects. This review highlights the clinical trials in which propranolol is taking part as adjuvant therapy at single administration or in combinatorial human trials, arising as a good pick and roll partner in anticancer strategies.
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Hereditary Hemorrhagic Telangiectasia (HHT) is a rare disorder of vascular development. Common manifestations include epistaxis, telangiectasias and arteriovenous malformations in multiple organs. Different deletions or nonsense mutations have been described in the ENG (HHT1) or ACVRL1/ALK1 (HHT2) genes, all affecting endothelial homeostasis. A novel mutation in ACVRL1/ALK1 has been identified in a Peruvian family with a clinical history compatible to HHT. Subsequently, 23 DNA samples from oral exchanges (buccal swaps) of the immediate family members were analyzed together with their clinical histories. A routine cDNA PCR followed by comparative DNA sequencing between the founder and another healthy family member showed the presence of the aforementioned specific mutation. The single mutation detected (c.525 + 1G > T) affects the consensus splice junction immediately after exon 4, provokes anomalous splicing and leads to the inclusion of intron IV between exons 4 and 5 in the ACVRL1/ALK1 mRNA and, therefore, to ALK1 haploinsufficiency. Complete sequencing determined that 10 of the 25 family members analyzed were affected by the same mutation. Notably, the approach described in this report could be used as a diagnostic technique, easily incorporated in clinical practice in developing countries and easily extrapolated to other patients carrying such a mutation.
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Rare Diseases (RD) are defined by their prevalence in less than 5 in 10,000 of the general population. Considered individually, each RD may seem insignificant, but together they add up to more than 7000 different diseases. Research in RD is not attractive for pharmaceutical companies since it is unlikely to recover development costs for medicines aimed to small numbers of patients. Since most of these diseases are life threatening, this fact underscores the urgent need for treatments. Drug repurposing consists of identifying new uses for approved drugs outside the scope of the original medical indication. It is an alternative option in drug development and represents a viable and risk-managed strategy to develop for RDs. In 2008, the "off label" therapeutic benefits of propranolol were described in the benign tumor Infantile Hemangioma. Propranolol, initially prescribed for high blood pressure, irregular heart rate, essential tremor, and anxiety, has, in the last decade, shown increasing evidence of its antiangiogenic, pro-apoptotic, vasoconstrictor and anti-inflammatory properties in different RDs, including vascular or oncological pathologies. This review highlights the finished and ongoing trials in which propranolol has arisen as a good repurposing drug for improving the health condition in RDs.
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Propranolol , Doenças Vasculares , Antagonistas Adrenérgicos beta/uso terapêutico , Reposicionamento de Medicamentos , Humanos , Propranolol/uso terapêutico , Doenças Raras/tratamento farmacológico , Doenças Vasculares/tratamento farmacológicoRESUMO
Von Hippel-Lindau (VHL) syndrome is a rare inherited cancer disease where the lack of VHL protein triggers the development of multisystemic tumors such us retinal hemangioblastomas (HBs), CNS-HBs, and clear cell renal cell carcinoma (ccRCC). Since standard therapies in VHL have shown limited response, leaving surgery as the only possible treatment, targeting of the ß2-adrenergic receptor (ADRB2) has shown therapeutic antitumor benefits on VHL-retinal HBs (clinical trial), VHL-CNS HBs, and VHL-ccRCC (in vitro and in vivo). In the present study, we wanted to look deep into the effects of the ADRB2 blockers propranolol and ICI-118,551 on two main aspects of cancer progression: (i) the changes on the inflammatory response of ccRCC cells; and (ii) the modulation on the Warburg effect (glycolytic metabolism), concretely, on the expression of genes involved in the cell reactive oxygen species (ROS) balance and levels. Accordingly, in vitro studies with primary VHL-ccRCC and 786-O cells measuring ROS levels, ROS-expression of detoxifying enzymes, and the expression of p65/NF-κB targets by RT-PCR were carried out. Furthermore, histological analyses of ccRCC samples from heterotopic mouse xenografts were performed. The obtained results show that ADRB2 blockade in ccRCC cells reduces the level of oxidative stress and stabilizes the inflammatory response. Thus, these data further support the idea of targeting ADRB2 as a promising strategy for the treatment of VHL and other non-VHL tumors.
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Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Inflamação/tratamento farmacológico , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Receptores Adrenérgicos beta 2/metabolismo , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hemangioblastoma/tratamento farmacológico , Hemangioblastoma/metabolismo , Humanos , Inflamação/metabolismo , Masculino , Camundongos , Propanolaminas/farmacologia , Propranolol/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Doença de von Hippel-Lindau/tratamento farmacológico , Doença de von Hippel-Lindau/metabolismoRESUMO
Von Hippel-Lindau disease (VHL) is a rare hereditary disease characterized by the predisposal to develop different types of highly vascularized tumors. VHL patients carry a VHL mutation that causes partial lack of functional VHL protein (pVHL) in all cells, and a total lack thereof in cells harboring a second hit mutation. Absence of pVHL generates a prolonged state of pseudo-hypoxia in the cell due to accumulation of hypoxia inducible factor, an important transcription factor regulating pro-tumorigenic genes. The work here presented focuses on characterizing the endothelium of VHL patients, by means of blood outgrowth endothelial cells (BOECs). Transcriptome analysis of VHL-derived BOECs, further supported by in vitro assays, shows that these cells are at a disadvantage, as evidenced by loss of cell adhesion capacity, angiogenesis defects, and immune response and oxidative metabolic gene downregulation, which induce oxidative stress. These results suggest that the endothelium of VHL patients is functionally compromised and more susceptible to tumor development. These findings contribute to shedding light on the vascular landscape of VHL patients preceding the second hit mutation in the VHL gene. This knowledge could be useful in searching for new therapies for these patients and other vascular diseases.
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Células Endoteliais/patologia , Neovascularização Patológica , Doença de von Hippel-Lindau/patologia , Estudos de Casos e Controles , Adesão Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Mutação , Neovascularização Patológica/genética , Estresse Oxidativo , Fenótipo , Transdução de Sinais , Transcriptoma , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Doença de von Hippel-Lindau/genética , Doença de von Hippel-Lindau/imunologia , Doença de von Hippel-Lindau/metabolismoRESUMO
C3G (RAPGEF1) is a guanine nucleotide exchange factor (GEF) for GTPases from the Ras superfamily, mainly Rap1, although it also acts through GEF-independent mechanisms. C3G regulates several cellular functions. It is expressed at relatively high levels in specific brain areas, playing important roles during embryonic development. Recent studies have uncovered different roles for C3G in cancer that are likely to depend on cell context, tumour type, and stage. However, its role in brain tumours remained unknown until very recently. We found that C3G expression is downregulated in GBM, which promotes the acquisition of a more mesenchymal phenotype, enhancing migration and invasion, but not proliferation. ERKs hyperactivation, likely induced by FGFR1, is responsible for this pro-invasive effect detected in C3G silenced cells. Other RTKs (Receptor Tyrosine Kinases) are also dysregulated and could also contribute to C3G effects. However, it remains undetermined whether Rap1 is a mediator of C3G actions in GBM. Various Rap1 isoforms can promote proliferation and invasion in GBM cells, while C3G inhibits migration/invasion. Therefore, other RapGEFs could play a major role regulating Rap1 activity in these tumours. Based on the information available, C3G could represent a new biomarker for GBM diagnosis, prognosis, and personalised treatment of patients in combination with other GBM molecular markers. The quantification of C3G levels in circulating tumour cells (CTCs) in the cerebrospinal liquid and/or circulating fluids might be a useful tool to improve GBM patient treatment and survival.
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Glioblastoma/metabolismo , Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Animais , Glioblastoma/genética , Fator 2 de Liberação do Nucleotídeo Guanina/genética , Humanos , Células Neoplásicas Circulantes/metabolismo , Proteínas rap1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
Von Hippel-Lindau (VHL), is a rare autosomal dominant inherited cancer in which the lack of VHL protein triggers the development of multisystemic tumors such us retinal hemangioblastomas (HB), CNS-HB, and clear cell renal cell carcinoma (ccRCC). ccRCC ranks third in terms of incidence and first in cause of death. Standard systemic therapies for VHL-ccRCC have shown limited response, with recurrent surgeries being the only effective treatment. Targeting of ß2-adrenergic receptor (ADRB) has shown therapeutic antitumor benefits on VHL-retinal HB (clinical trial) and VHL-CNS HB (in vitro). Therefore, the in vitro and in vivo antitumor benefits of propranolol (ADRB-1,2 antagonist) and ICI-118,551 (ADRB-2 antagonist) on VHL-/- ccRCC primary cultures and 786-O tumor cell lines have been addressed. Propranolol and ICI-118,551 activated apoptosis inhibited gene and protein expression of HIF-2α, CAIX, and VEGF, and impaired partially the nuclear internalization of HIF-2α and NFĸB/p65. Moreover, propranolol and ICI-118,551 reduced tumor growth on two in vivo xenografts. Finally, ccRCC patients receiving propranolol as off-label treatment have shown a positive therapeutic response for two years on average. In summary, propranolol and ICI-118,551 have shown antitumor benefits in VHL-derived ccRCC, and since ccRCCs comprise 63% of the total RCCs, targeting ADRB2 becomes a promising drug for VHL and other non-VHL tumors.
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Von Hippel-Lindau syndrome (VHL) is a rare disease of dominant inheritance that increases susceptibility to tumor development, with a complete penetrance at the age of 60. In this report, we present the unprecedented case of a VHL carrier who remains healthy at 72. Under the course of this study, it was discovered that this patient carries a mutation for a second rare disease, Neuronal Ceroid Lipofuscinosis (NCL or CNL). We hypothesize that the CLN mutation she carries offers a protective effect, preventing tumor development in the cells potentially suffering a VHL second hit mutation. To test this hypothesis, we ran a series of molecular experiments and confirmed that cell viability of primary endothelial cells decreases upon CLN5 silencing. Our results further elucidate the cell biology implications of two rare diseases interacting.
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Hemangioblastoma , Neoplasias , Doença de von Hippel-Lindau , Células Endoteliais , Feminino , Humanos , Proteínas de Membrana Lisossomal , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Doença de von Hippel-Lindau/genéticaRESUMO
The diagnosis of hereditary hemorrhagic telangiectasia (HHT) is based on the Curaçao criteria: epistaxis, telangiectases, arteriovenous malformations in internal organs, and family history. Genetically speaking, more than 90% of HHT patients show mutations in ENG or ACVRL1/ALK1 genes, both belonging to the TGF-ß/BMP9 signaling pathway. Despite clear knowledge of the symptoms and genes of the disease, we still lack a definite cure for HHT, having just palliative measures and pharmacological trials. Among the former, two strategies are: intervention at "ground zero" to minimize by iron and blood transfusions in order to counteract anemia. Among the later, along the last 15 years, three different strategies have been tested: (1) To favor coagulation with antifibrinolytic agents (tranexamic acid); (2) to increase transcription of ENG and ALK1 with specific estrogen-receptor modulators (bazedoxifene or raloxifene), antioxidants (N-acetylcysteine, resveratrol), or immunosuppressants (tacrolimus); and (3) to impair the abnormal angiogenic process with antibodies (bevacizumab) or blocking drugs like etamsylate, and propranolol. This manuscript reviews the main strategies and sums up the clinical trials developed with drugs alleviating HHT.
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Reliable detection of disseminated tumor cells and of the biodistribution of tumor-targeting therapeutic antibodies within the entire body has long been needed to better understand and treat cancer metastasis. Here, we developed an integrated pipeline for automated quantification of cancer metastases and therapeutic antibody targeting, named DeepMACT. First, we enhanced the fluorescent signal of cancer cells more than 100-fold by applying the vDISCO method to image metastasis in transparent mice. Second, we developed deep learning algorithms for automated quantification of metastases with an accuracy matching human expert manual annotation. Deep learning-based quantification in 5 different metastatic cancer models including breast, lung, and pancreatic cancer with distinct organotropisms allowed us to systematically analyze features such as size, shape, spatial distribution, and the degree to which metastases are targeted by a therapeutic monoclonal antibody in entire mice. DeepMACT can thus considerably improve the discovery of effective antibody-based therapeutics at the pre-clinical stage. VIDEO ABSTRACT.
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Anticorpos/uso terapêutico , Aprendizado Profundo , Diagnóstico por Computador/métodos , Quimioterapia Assistida por Computador/métodos , Neoplasias/patologia , Animais , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos SCID , Metástase Neoplásica , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Software , Microambiente TumoralRESUMO
Phenotypic drug discovery must take advantage of the large amount of clinical data currently available. In this sense, the impact of microRNAs (miRs) on human disease and clinical therapeutic responses is becoming increasingly well documented. Accordingly, it might be possible to use miR-based signatures as phenotypic read-outs of pathological status, for example in cancer. Here, we propose to use the information accumulating regarding the biology of miRs from clinical research in the preclinical arena, adapting it to the use of miR biosensors in the earliest steps of drug screening. Thus, we have used an amperometric dual magnetosensor capable of monitoring a miR-21/miR-205 signature to screen for new drugs that restore these miRs to non-tumorigenic levels in cell models of breast cancer and glioblastoma. In this way we have been able to identify a new chemical entity, 11PS04 ((3aR,7aS)-2-(3-propoxyphenyl)-7,7a-dihydro-3aH-pyrano[3,4-d]oxazol-6(4H)-one), the therapeutic potential of which was suggested in mechanistic assays of disease models, including 3D cell culture (oncospheres) and xenografts. These assays highlighted the potential of this compound to attack cancer stem cells, reducing the growth of breast and glioblastoma tumors in vivo. These data demonstrate the enhanced chain of translatability of this strategy, opening up new perspectives for drug-discovery pipelines and highlighting the potential of miR-based electro-analytical sensors as efficient tools in modern drug discovery.
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Técnicas Biossensoriais , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/patologia , Oxazóis/farmacologia , Animais , Antineoplásicos/farmacologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/patologia , Fenômenos Magnéticos , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/patologia , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Oxazóis/química , Reprodutibilidade dos Testes , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Temozolomida/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hereditary hemorrhagic telangiectasia (HHT) is a vascular dysplasia characterized by recurrent and spontaneous epistaxis (nose bleeds), telangiectases on skin and mucosa, internal organ arteriovenous malformations, and dominant autosomal inheritance. Mutations in Endoglin and ACVRL1 / ALK1 , genes mainly expressed in endothelium, are responsible in 90% of the cases for the pathology. These genes are involved in the transforming growth factor-ß(TGF-ß) signaling pathway. Epistaxis remains as one of the most common symptoms impairing the quality of life of patients, becoming life-threatening in some cases. Different strategies have been used to decrease nose bleeds, among them is antiangiogenesis. The two main angiogenic pathways in endothelial cells depend on vascular endothelial growth factor and fibroblast growth factor (FGF). The present work has used etamsylate, the diethylamine salt of the 2,5-dihydroxybenzene sulfonate anion, also known as dobesilate, as a FGF signaling inhibitor. In endothelial cells, in vitro experiments show that etamsylate acts as an antiangiogenic factor, inhibiting wound healing and matrigel tubulogenesis. Moreover, etamsylate decreases phosphorylation of Akt and ERK1/2. A pilot clinical trial (EudraCT: 2016-003982-24) was performed with 12 HHT patients using a topical spray of etamsylate twice a day for 4 weeks. The epistaxis severity score (HHT-ESS) and other pertinent parameters were registered in the clinical trial. The significant reduction in the ESS scale, together with the lack of significant side effects, allowed the designation of topical etamsylate as a new orphan drug for epistaxis in HHT (EMA/OD/135/18).
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BACKGROUND: von Hippel-Lindau disease (VHL) is a multisystem cancer syndrome caused by mutations in the VHL gene. Retinal hemangioblastoma is one of the most common tumours, and when it appears near the optic nerve, its treatment is challenging and risky. To date, no treatment has proven effective in changing the course of the disease. This study was designed to evaluate the safety and effectiveness of propranolol in controlling these tumours. METHODS: Seven patients were included. All patients took a daily dose of 120 mg of propranolol for 1 year. Clinical variables were assessed at baseline, and at 1, 3, 6, 9 and 12 months. The primary endpoint of the study was the number and size of retinal hemangioblastomas. On every visit, retinal outcomes and blood biomarkers (such as vascular endothelial growth factor (VEGF) and miR210) were analysed. RESULTS: Number and size of retinal hemangioblastomas remained stable in all patients. All of them had initially increased levels of VEGF and miR210. There was a gradual reabsorption of retinal exudation in two patients, correlating with a progressive decrease of both biomarkers. The only adverse effect reported was hypotension in one patient. CONCLUSIONS: Propranolol could be used to treat retinal hemangioblastomas in VHL patients, although more studies are needed to determine the ideal dose and long-term effect. VEGF and miR210 should be explored as biomarkers of disease activity. As far as we know, these are the first biomarkers proposed to monitor the VHL disease activity. TRIAL REGISTRATION NUMBER: 2014-003671-30.
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The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8N/CEGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy.