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BACKGROUND: Some genetic polymorphisms (SNPs) have been proposed as predictors for different colorectal cancer (CRC) outcomes. This work aims to assess their performance in our cohort and find new SNPs associated with them. METHODS: A total of 833 CRC cases were analyzed for seven outcomes, including the use of chemotherapy, and stratified by tumor location and stage. The performance of 63 SNPs was assessed using a generalized linear model and area under the receiver operating characteristic curve, and local SNPs were detected using logistic regressions. RESULTS: In total 26 of the SNPs showed an AUC > 0.6 and a significant association (p < 0.05) with one or more outcomes. However, clinical variables outperformed some of them, and the combination of genetic and clinical data showed better performance. In addition, 49 suggestive (p < 5 × 10-6) SNPs associated with one or more CRC outcomes were detected, and those SNPs were located at or near genes involved in biological mechanisms associated with CRC. CONCLUSIONS: Some SNPs with clinical data can be used in our population as predictors of some CRC outcomes, and the local SNPs detected in our study could be feasible markers that need further validation as predictors.
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Celiac disease (CD) is a chronic and autoimmune disease that develops in genetically predisposed individuals upon exposure to dietary gluten. The availability of the target tissue for research has made it possible to identify alterations in the transcriptome and methylome in the celiac gut. However, gene expression and methylation is highly variable among different cell types, and separation of cellular populations in target tissue must be considered for the understanding of the specific cellular and immune responses to gluten. In this context, a few studies have demonstrated that focusing on an isolated cell population, novel candidate genes involved in the pathogenesis of the disease can be identified. Here, we describe a method to separate epithelial and immune cells from biopsy samples for DNA and RNA isolation. With minor variations, the same technique can be applied to other tissues and cell types.
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Glutens , Processamento de Proteína Pós-Traducional , Humanos , Transcriptoma/genética , BiópsiaRESUMO
Colorectal cancer is a major health concern since it is a highly diagnosed cancer and the second cause of death among cancers. Thus, the most suitable biomarkers for its diagnosis, prognosis, and treatment have been studied to improve and personalize the prevention and clinical management of colorectal cancer. The emergence of omic techniques has provided a great opportunity to better study CRC and make personalized medicine feasible. In this review, we will try to summarize how the analysis of the omic layers can be useful for personalized medicine and the existing difficulties. We will discuss how single and multiple omic layer analyses have been used to improve the prediction of the risk of CRC and its outcomes and how to overcome the challenges in the use of omic layers in personalized medicine.
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Neoplasias Colorretais , Humanos , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/terapia , Medicina de Precisão/métodos , PrognósticoRESUMO
One billion people worldwide get flu every year, including patients with non-small cell lung cancer (NSCLC). However, the impact of acute influenza A virus (IAV) infection on the composition of the tumor microenvironment (TME) and the clinical outcome of patients with NSCLC is largely unknown. We set out to understand how IAV load impacts cancer growth and modifies cellular and molecular players in the TME. Herein, we report that IAV can infect both tumor and immune cells, resulting in a long-term protumoral effect in tumor-bearing mice. Mechanistically, IAV impaired tumor-specific T-cell responses, led to the exhaustion of memory CD8+ T cells and induced PD-L1 expression on tumor cells. IAV infection modulated the transcriptomic profile of the TME, fine-tuning it toward immunosuppression, carcinogenesis, and lipid and drug metabolism. Consistent with these data, the transcriptional module induced by IAV infection in tumor cells in tumor-bearing mice was also found in human patients with lung adenocarcinoma and correlated with poor overall survival. In conclusion, we found that IAV infection worsened lung tumor progression by reprogramming the TME toward a more aggressive state.
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Carcinoma Pulmonar de Células não Pequenas , Vírus da Influenza A , Influenza Humana , Neoplasias Pulmonares , Infecções por Orthomyxoviridae , Humanos , Animais , Camundongos , Neoplasias Pulmonares/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Microambiente Tumoral , Linfócitos T CD8-Positivos , Pulmão , Infecções por Orthomyxoviridae/patologiaRESUMO
The role of microbiota in colorectal cancer has been studied since alterations in its composition were observed. In addition, there are more and more pieces of evidence that microbiota could be implicated in colorectal cancer progression. Thus, the components of the microbiota could be biomarkers for the diagnosis and prognosis of colorectal cancer. In addition, it is important to address how the microbiota interacts with the host and how the host shapes the microbiota, in order to understand the biological pathways and mechanisms involved in their relationship and the consequences of their interactions in colorectal cancer. Thereby, it could be possible to find feasible measures and treatments to prevent or better diagnose colorectal cancer. In this review, we will try to summarize the role of the microbiota in colorectal cancer and its interactions with the host and the host genetics, coming to some conclusions that could be useful to find the gaps in our knowledge and propose future steps in this field.
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Lactate is a central metabolite in brain physiology but also contributes to tumor development. Glioblastoma (GB) is the most common and malignant primary brain tumor in adults, recognized by angiogenic and invasive growth, in addition to its altered metabolism. We show herein that lactate fuels GB anaplerosis by replenishing the tricarboxylic acid (TCA) cycle in absence of glucose. Lactate dehydrogenases (LDHA and LDHB), which we found spatially expressed in GB tissues, catalyze the interconversion of pyruvate and lactate. However, ablation of both LDH isoforms, but not only one, led to a reduction in tumor growth and an increase in mouse survival. Comparative transcriptomics and metabolomics revealed metabolic rewiring involving high oxidative phosphorylation (OXPHOS) in the LDHA/B KO group which sensitized tumors to cranial irradiation, thus improving mouse survival. When mice were treated with the antiepileptic drug stiripentol, which targets LDH activity, tumor growth decreased. Our findings unveil the complex metabolic network in which both LDHA and LDHB are integrated and show that the combined inhibition of LDHA and LDHB strongly sensitizes GB to therapy.
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Neoplasias Encefálicas , Glioblastoma , Lactato Desidrogenases , Animais , Camundongos , Ácido Láctico , Metabolômica , Glioblastoma/enzimologia , Glioblastoma/patologia , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/patologiaRESUMO
Activation of WNT/ß-catenin signaling has been associated with a non-T-cell-inflamed tumor microenvironment (TME) in several cancers. The aim of this work was to investigate the relationship between ß-catenin signaling and TME inflammation in head and neck squamous cell carcinomas (HNSCCs). Membrane and nuclear ß-catenin expression, PD-L1 expression, and CD8+ tumor-infiltrating lymphocyte (TIL) density were jointly evaluated by immunohistochemistry in a series of 372 HPV-negative HNSCCs. Membrane ß-catenin levels decreased in carcinomas compared to the normal epithelium. Positive nuclear ß-catenin was detected in 50 tumors (14.3%) and was significantly associated with a low CD8+ TIL density (168 cells/mm2 versus 293 cells/mm2 in nuclear-ß-catenin-negative cases; p = 0.01) and a tendency for a lower expression of PD-L1, resulting in association with a noninflamed TME (i.e., type II, immunological ignorance). Multivariate Cox analysis further demonstrated that low infiltration by CD8+ TILs (HR = 1.6, 95% CI = 1.19-2.14, p = 0.002) and nuclear ß-catenin expression (HR = 1.47, 95% CI = 1.01-2.16, p = 0.04) were both independently associated with a poorer disease-specific survival. In conclusion, tumor-intrinsic nuclear ß-catenin activation is associated with a non-inflamed TME phenotype and a poorer prognosis, thereby suggesting a possible implication as an immune exclusion mechanism for a subset of HNSCC patients.
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Antígeno B7-H1 , Neoplasias de Cabeça e Pescoço , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Linfócitos do Interstício Tumoral , Fenótipo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Microambiente Tumoral , beta Catenina/metabolismoRESUMO
Although the genetic contribution to colorectal cancer (CRC) has been studied in various populations, studies on the applicability of available genetic information in the Basque population are scarce. In total, 835 CRC cases and 940 controls from the Basque population were genotyped and genome-wide association studies were carried out. Mendelian Randomization analyses were used to discover the effect of modifiable risk factors and microbiota on CRC. In total, 25 polygenic risk score models were evaluated to assess their performance in CRC risk calculation. Moreover, 492 inflammatory bowel disease cases were used to assess whether that genetic information would not confuse both conditions. Five suggestive (p < 5 × 10−6) loci were associated with CRC risk, where genes previously associated with CRC were located (e.g., ABCA12, ATIC or ERBB4). Moreover, the analyses of CRC locations detected additional genes consistent with the biology of CRC. The possible contribution of cholesterol, BMI, Firmicutes and Cyanobacteria to CRC risk was detected by Mendelian Randomization. Finally, although polygenic risk score models showed variable performance, the best model performed correctly regardless of the location and did not misclassify inflammatory bowel disease cases. Our results are consistent with CRC biology and genetic risk models and could be applied to assess CRC risk in the Basque population.
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INTRODUCTION: SCLC is an extremely aggressive subtype of lung cancer without approved targeted therapies. Here we identified YES1 as a novel targetable oncogene driving SCLC maintenance and metastasis. METHODS: Association between YES1 levels and prognosis was evaluated in SCLC clinical samples. In vitro functional experiments for proliferation, apoptosis, cell cycle, and cytotoxicity were performed. Genetic and pharmacologic inhibition of YES1 was evaluated in vivo in cell- and patient-derived xenografts and metastasis. YES1 levels were evaluated in mouse and patient plasma-derived exosomes. RESULTS: Overexpression or gain/amplification of YES1 was identified in 31% and 26% of cases, respectively, across molecular subgroups, and was found as an independent predictor of poor prognosis. Genetic depletion of YES1 dramatically reduced cell proliferation, three-dimensional organoid formation, tumor growth, and distant metastasis, leading to extensive apoptosis and tumor regressions. Mechanistically, YES1-inhibited cells revealed alterations in the replisome and DNA repair processes, that conferred sensitivity to irradiation. Pharmacologic blockade with the novel YES1 inhibitor CH6953755 or dasatinib induced marked antitumor activity in organoid models and cell- and patient-derived xenografts. YES1 protein was detected in plasma exosomes from patients and mouse models, with levels matching those of tumors, suggesting that circulating YES1 could represent a biomarker for patient selection/monitoring. CONCLUSIONS: Our results provide evidence that YES1 is a new druggable oncogenic target and biomarker to advance the clinical management of a subpopulation of patients with SCLC.
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Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Camundongos , Animais , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Linhagem Celular Tumoral , Oncogenes , Proliferação de Células/genética , Apoptose , Carcinogênese/genética , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia , Proteínas Proto-Oncogênicas c-yes/genéticaRESUMO
YES1 is a nonreceptor tyrosine kinase that belongs to the SRC family of kinases (SFK) and controls multiple cancer signaling pathways. YES1 is amplified and overexpressed in many tumor types, where it promotes cell proliferation, survival, and invasiveness. Therefore, YES1 has been proposed as an emerging target in solid tumors. In addition, studies have shown that YES1 is a prognostic biomarker and a predictor of dasatinib activity. Several SFKs-targeting drugs have been developed, and some of them have reached clinical trials. However, these drugs have encountered challenges to their utilization in the clinical practice in unselected patients due to toxicity and lack of efficacy. In the case of YES1, novel specific inhibitors have been developed and tested in preclinical models, with impressive antitumor effects. In this review, we summarize the structure and activation of YES1 and describe its role in cancer as a target and prognostic and companion biomarker. We also address the efficacy of SFKs inhibitors that are currently in clinical trials, highlighting the main hindrances for their clinical use. Current available information strongly suggests that inhibiting YES1 in tumors with high expression of this protein is a promising strategy against cancer.
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Neoplasias , Quinases da Família src , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células , Dasatinibe/farmacologia , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteínas Proto-Oncogênicas c-yes/metabolismoRESUMO
Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.
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Neoplasias Encefálicas/patologia , Comunicação Celular , Movimento Celular , Técnicas de Cocultura/métodos , Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Neurônios/patologia , Animais , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Rastreamento de Células , Glioma/patologia , Humanos , Processamento de Imagem Assistida por Computador , Laminina/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ratos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologiaRESUMO
INTRODUCTION: The use of immune-checkpoint inhibitors has drastically improved the management of patients with non-small cell lung cancer (NSCLC), but innate and acquired resistances are hurdles needed to be solved. Immunomodulatory drugs that can reinvigorate the immune cytotoxic activity, in combination with antiprogrammed cell death 1 (PD-1) antibody, are a great promise to overcome resistance. We evaluated the impact of the SRC family kinases (SFKs) on NSCLC prognosis, and the immunomodulatory effect of the SFK inhibitor dasatinib, in combination with anti-PD-1, in clinically relevant mouse models of NSCLC. METHODS: A cohort of patients from University Clinic of Navarra (n=116) was used to study immune infiltrates by multiplex immunofluorescence (mIF) and YES1 protein expression in tumor samples. Publicly available resources (TCGA, Km Plotter, and CIBERSORT) were used to study patient's survival based on expression of SFKs and tumor infiltrates. Syngeneic NSCLC mouse models 393P and UNSCC680AJ were used for in vivo drug testing. RESULTS: Among the SFK members, YES1 expression showed the highest association with poor prognosis. Patients with high YES1 tumor levels also showed high infiltration of CD4+/FOXP3+ cells (regulatory T cells (Tregs)), suggesting an immunosuppressive phenotype. After testing for YES1 expression in a panel of murine cell lines, 393P and UNSCC680AJ were selected for in vivo studies. In the 393P model, dasatinib+anti-PD-1 treatment resulted in synergistic activity, with 87% tumor regressions and development of immunological memory that impeded tumor growth when mice were rechallenged. In vivo depletion experiments further showed that CD8+ and CD4+ cells are necessary for the therapeutic effect of the combination. The antitumor activity was accompanied by a very significant decrease in the number of Tregs, which was validated by mIF in tumor sections. In the UNSCC680AJ model, the antitumor effects of dasatinib+anti-PD-1 were milder but similar to the 393P model. In in vitro assays, we demonstrated that dasatinib blocks proliferation and transforming growth factor beta-driven conversion of effector CD4+ cells into Tregs through targeting of phospholymphocyte-specific protein tyrosine kinase and downstream effectors pSTAT5 and pSMAD3. CONCLUSIONS: YES1 protein expression is associated with increased numbers of Tregs in patients with NSCLC. Dasatinib synergizes with anti-PD-1 to impair tumor growth in NSCLC experimental models. This study provides the preclinical rationale for the combined use of dasatinib and PD-1/programmed death-ligand 1 blockade to improve outcomes of patients with NSCLC.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Dasatinibe/farmacologia , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-yes/antagonistas & inibidores , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Linfócitos do Interstício Tumoral/enzimologia , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos da Linhagem 129 , Fenótipo , Receptor de Morte Celular Programada 1/metabolismo , Proteínas Proto-Oncogênicas c-yes/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/enzimologia , Linfócitos T Reguladores/imunologia , Microambiente TumoralRESUMO
Celiac Disease (CeD) is an immune-mediated complex disease that is triggered by the ingestion of gluten and develops in genetically susceptible individuals. It has been known for a long time that the Human Leucocyte Antigen (HLA) molecules DQ2 and DQ8 are necessary, although not sufficient, for the disease development, and therefore other susceptibility genes and (epi)genetic events must participate in CeD pathogenesis. The advances in Genomics during the last 15 years have made CeD one of the immune-related disorders with the best-characterized genetic component. In the present work, we will first review the main Genome-Wide Association Studies (GWAS) carried out in the disorder, and emphasize post-GWAS discoveries, including diverse integrative strategies, SNP prioritization approaches, and insights into the Microbiome through the host Genomics. Second, we will explore CeD-related Epigenetics and Epigenomics, mostly focusing on the emerging knowledge of the celiac methylome, and the vast but yet under-explored non-coding RNA (ncRNA) landscape. We conclude that much has been done in the field although there are still completely unvisited areas in the post-Genomics of CeD. Chromatin conformation and accessibility, and Epitranscriptomics are promising domains that need to be unveiled to complete the big picture of the celiac Genome.
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Doença Celíaca/genética , Predisposição Genética para Doença , Genoma , Animais , Doença Celíaca/microbiologia , Metilação de DNA/genética , Genômica , Humanos , RNA não Traduzido/genética , RNA não Traduzido/metabolismoRESUMO
Glioblastoma (GBM) are the most common tumors of the central nervous system and among the deadliest cancers in adults. GBM overall survival has not improved over the last decade despite optimization of therapeutic standard-of-care. While immune checkpoint inhibitors (ICI) have revolutionized cancer care, they unfortunately have little therapeutic success in GBM. Here, we elaborate on normal brain and GBM-associated immune landscapes. We describe the role of microglia and tumor-associated macrophages (TAMs) in immune suppression and highlight the impact of energy metabolism in immune evasion. We also describe the challenges and opportunities of immunotherapies in GBM and discuss new avenues based on harnessing the anti-tumor activity of myeloid cells, vaccines, chimeric antigen receptors (CAR)-T and -NK cells, oncolytic viruses, nanocarriers, and combination therapies.
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Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/terapia , Glioblastoma/imunologia , Glioblastoma/terapia , Imunoterapia/métodos , Animais , Humanos , Evasão Tumoral/imunologiaRESUMO
INTRODUCTION: The importance of immune tumor microenvironment in the prognosis of patients with head and neck squamous carcinomas (HNSCC) is increasingly recognized. We analyzed the prognostic relevance of PD-L1 and PD-1 expressions in relation to the infiltration by CD8+ and FOXP3+ tumor-infiltrating lymphocytes (TILs). METHODS: Samples from 372 surgically treated HPV-negative HNSCC patients were evaluated by immunohistochemistry for PD-L1 expression [both tumor proportion score (TPS) and combined proportion score (CPS)], PD-1 expression in immune cells, and density of infiltrating CD8+ and FOXP3+ TILs. PD-L1 expression and CD8+ TIL density were combined to establish the type of tumor microenvironment. RESULTS: 29.5% cases exhibited PD-L1 TPS positivity (≥ 1%), whereas PD-L1 CPS positivity (≥ 1%) was observed in 40% cases. 47.5% cases showed positive PD-1 expression (≥ 1%). PD-L1 and PD-1 positivity correlated with a high density of both CD8+ and FOXP3+ TILs. In univariate analysis, PD-L1 TPS positivity (P = 0.026), PD-L1 CPS positivity (P = 0.004), high density of CD8+ TIL (P = 0.001), and high density of FOXP3+ TIL (P = 0.004) were associated with a better disease-specific survival (DSS). However, in multivariate analysis, only high density of CD8+ TIL was associated with a better DSS (P = 0.002). The type of tumor microenvironment correlated with DSS (P = .008), with the better DSS observed in cases with type I (PD-L1 CPS positivity and high density of CD8+ TIL). CONCLUSIONS: High infiltration by CD8+ TIL is associated with better survival outcomes. Positive PD-L1 expression correlates with a high infiltration by TILs, explaining its association with better prognosis.
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Antígeno B7-H1/metabolismo , Neoplasias de Cabeça e Pescoço/imunologia , Linfócitos do Interstício Tumoral/imunologia , Papillomaviridae/imunologia , Infecções por Papillomavirus/complicações , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Microambiente Tumoral/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Feminino , Seguimentos , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Prognóstico , Estudos Retrospectivos , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Taxa de SobrevidaRESUMO
Aberrant SRC expression and activation is frequently detected in multiple cancers, and hence, targeting SRC has emerged as a promising therapeutic strategy. Different SRC inhibitors have demonstrated potent anti-tumor activity in preclinical models, although they largely lack clinical efficacy as monotherapy in late-stage solid tumors, including head and neck squamous cell carcinomas (HNSCC). Adequate selection and stratification of patients who may respond to and benefit from anti-SRC therapies is therefore needed to guide clinical trials and treatment efficacy. This study investigates the prognostic significance of active SRC expression in a homogeneous cohort of 122 human papillomavirus (HPV)-negative, surgically treated HNSCC patients. Immunohistochemical evaluation of the active form of SRC by means of anti-SRC Clone 28 monoclonal antibody was specifically performed and subsequently correlated with clinical data. The expression of p-SRC (Tyr419), total SRC, and downstream SRC effectors was also analyzed. Our results uncovered striking differences in the prognostic relevance of SRC expression in HNSCC patients depending on the tumor site. Active SRC expression was found to significantly associate with advanced disease stages, presence of lymph node metastasis, and tumor recurrences in patients with laryngeal tumors, but not in the pharyngeal subgroup. Multivariate Cox analysis further revealed active SRC expression as an independent predictor of cancer-specific mortality in patients with laryngeal carcinomas. Concordantly, expression of p-SRC (Tyr419) and the SRC substrates focal adhesion kinase (FAK) and the Arf GTPase-activating protein ASAP1 also showed specific associations with poor prognosis in the larynx. These findings could have important implications in ongoing Src family kinase (SFK)-based clinical trials, as these new criteria could help to improve patient selection and develop biomarker-stratified trials.
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Mitochondrial dysfunction plays critical roles in cancer development and related therapeutic response; however, exact molecular mechanisms remain unclear. Recently, alongside the discovery of mitochondrial-specific DNA methyltransferases, global and site-specific methylation of the mitochondrial genome has been described. Investigation of any functional consequences however remains unclear and debated due to insufficient evidence of the quantitative degree and frequency of mitochondrial DNA (mtDNA) methylation. This study uses WGBS to provide the first quantitative report of mtDNA methylation at single base pair resolution. The data show that mitochondrial genomes are extensively methylated predominantly at non-CpG sites. Importantly, these methylation patterns display notable differences between normal and cancer cells. Furthermore, knockdown of DNA methyltransferase enzymes resulted in a marked global reduction of mtDNA methylation levels, indicating these enzymes may be associated with the establishment and/or maintenance of mtDNA methylation. DNMT3B knockdown cells displayed a comparatively pronounced global reduction in mtDNA methylation with concomitant increases in gene expression, suggesting a potential functional link between methylation and gene expression. Together these results demonstrate reproducible, non-random methylation patterns of mtDNA and challenge the notion that mtDNA is lowly methylated. This study discusses key differences in methodology that suggest future investigations must allow for techniques that assess both CpG and non-CpG methylation.
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DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , DNA Mitocondrial/genética , Regulação da Expressão Gênica/genética , Animais , Ilhas de CpG/genética , Humanos , Mitocôndrias/genética , DNA Metiltransferase 3BRESUMO
The frequent dysregulation of SRC family kinases (SFK) in multiple cancers prompted various inhibitors to be actively tested in preclinical and clinical trials. Disappointingly, dasatinib and saracatinib failed to demonstrate monotherapeutic efficacy in patients with head and neck squamous cell carcinomas (HNSCC). Deeper functional and mechanistic knowledge of the actions of these drugs is therefore needed to improve clinical outcome and to develop more efficient combinational strategies. Even though the SFK inhibitors dasatinib and saracatinib robustly blocked cell migration and invasion in HNSCC cell lines, this study unveils undesirable stem cell-promoting functions that could explain the lack of clinical efficacy in HNSCC patients. These deleterious effects were targeted by the mithramycin analog EC-8042 that efficiently eliminated cancer stem cells (CSC)-enriched tumorsphere cultures as well as tumor bulk cells and demonstrated potent antitumor activity in vivo. Furthermore, combination treatment of dasatinib with EC-8042 provided favorable complementary anti-proliferative, anti-invasive, and anti-CSC functions without any noticeable adverse interactions of both agents. These findings strongly support combinational strategies with EC-8042 for clinical testing in HNSCC patients. These data may have implications on ongoing dasatinib-based trials.
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Rationale: The characterization of new genetic alterations is essential to assign effective personalized therapies in non-small cell lung cancer (NSCLC). Furthermore, finding stratification biomarkers is essential for successful personalized therapies. Molecular alterations of YES1, a member of the SRC (proto-oncogene tyrosine-protein kinase Src) family kinases (SFKs), can be found in a significant subset of patients with lung cancer.Objectives: To evaluate YES1 (v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1) genetic alteration as a therapeutic target and predictive biomarker of response to dasatinib in NSCLC.Methods: Functional significance was evaluated by in vivo models of NSCLC and metastasis and patient-derived xenografts. The efficacy of pharmacological and genetic (CRISPR [clustered regularly interspaced short palindromic repeats]/Cas9 [CRISPR-associated protein 9]) YES1 abrogation was also evaluated. In vitro functional assays for signaling, survival, and invasion were also performed. The association between YES1 alterations and prognosis was evaluated in clinical samples.Measurements and Main Results: We demonstrated that YES1 is essential for NSCLC carcinogenesis. Furthermore, YES1 overexpression induced metastatic spread in preclinical in vivo models. YES1 genetic depletion by CRISPR/Cas9 technology significantly reduced tumor growth and metastasis. YES1 effects were mainly driven by mTOR (mammalian target of rapamycin) signaling. Interestingly, cell lines and patient-derived xenograft models with YES1 gene amplifications presented a high sensitivity to dasatinib, an SFK inhibitor, pointing out YES1 status as a stratification biomarker for dasatinib response. Moreover, high YES1 protein expression was an independent predictor for poor prognosis in patients with lung cancer.Conclusions: YES1 is a promising therapeutic target in lung cancer. Our results provide support for the clinical evaluation of dasatinib treatment in a selected subset of patients using YES1 status as predictive biomarker for therapy.
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Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Proliferação de Células/genética , Dasatinibe/farmacologia , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas c-yes/genética , Células A549 , Animais , Antineoplásicos/uso terapêutico , Sistemas CRISPR-Cas , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dasatinibe/uso terapêutico , Amplificação de Genes , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Prognóstico , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-yes/antagonistas & inibidores , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
N6-methyladenosine (m6A) is the most common and abundant RNA modification. Recent studies have shown its importance in the regulation of several biological processes, including the immune response, and different approaches have been developed in order to map and quantify m6A marks. However, site specific detection of m6A methylation has been technically challenging, and existing protocols are long and tedious and often involve next-generation sequencing. Here, we describe a simple RT-QPCR based approach for the relative quantification of candidate m6A regions that takes advantage of the diminished capacity of BstI enzyme to retrotranscribe m6A residues. Using this technique, we have been able to confirm the recently described m6A methylation in the 3'UTR of SOCS1 and SOCS3 transcripts. Moreover, using the method presented here, we have also observed alterations in the relative levels of m6A in specific motifs of SOCS genes in celiac disease patients and in pancreatic ß-cells exposed to inflammatory stimuli.