RESUMO
BACKGROUND: The discovery of functionally relevant KRAS effectors in lung and pancreatic ductal adenocarcinoma (LUAD and PDAC) may yield novel molecular targets or mechanisms amenable to inhibition strategies. Phospholipids availability has been appreciated as a mechanism to modulate KRAS oncogenic potential. Thus, phospholipid transporters may play a functional role in KRAS-driven oncogenesis. Here, we identified and systematically studied the phospholipid transporter PITPNC1 and its controlled network in LUAD and PDAC. METHODS: Genetic modulation of KRAS expression as well as pharmacological inhibition of canonical effectors was completed. PITPNC1 genetic depletion was performed in in vitro and in vivo LUAD and PDAC models. PITPNC1-deficient cells were RNA sequenced, and Gene Ontology and enrichment analyses were applied to the output data. Protein-based biochemical and subcellular localization assays were run to investigate PITPNC1-regulated pathways. A drug repurposing approach was used to predict surrogate PITPNC1 inhibitors that were tested in combination with KRASG12C inhibitors in 2D, 3D, and in vivo models. RESULTS: PITPNC1 was increased in human LUAD and PDAC, and associated with poor patients' survival. PITPNC1 was regulated by KRAS through MEK1/2 and JNK1/2. Functional experiments showed PITPNC1 requirement for cell proliferation, cell cycle progression and tumour growth. Furthermore, PITPNC1 overexpression enhanced lung colonization and liver metastasis. PITPNC1 regulated a transcriptional signature which highly overlapped with that of KRAS, and controlled mTOR localization via enhanced MYC protein stability to prevent autophagy. JAK2 inhibitors were predicted as putative PITPNC1 inhibitors with antiproliferative effect and their combination with KRASG12C inhibitors elicited a substantial anti-tumour effect in LUAD and PDAC. CONCLUSIONS: Our data highlight the functional and clinical relevance of PITPNC1 in LUAD and PDAC. Moreover, PITPNC1 constitutes a new mechanism linking KRAS to MYC, and controls a druggable transcriptional network for combinatorial treatments.
Assuntos
Carcinoma Ductal Pancreático , Proteínas de Membrana Transportadoras , Neoplasias Pancreáticas , Humanos , Autofagia/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Pulmão/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Neoplasias PancreáticasRESUMO
Breast cancer (BC) is the most common malignancy in women worldwide. While the main systemic treatment option is anthracycline-containing chemotherapy, chemoresistance continues to be an obstacle to patient survival. Carnitine palmitoyltransferase 1C (CPT1C) has been described as a poor-prognosis marker for several tumour types, as it favours tumour growth and hinders cells from entering senescence. At the molecular level, CPT1C has been associated with lipid metabolism regulation and important lipidome changes. Since plasma membrane (PM) rigidity has been associated with reduced drug uptake, we explored whether CPT1C expression could be involved in PM remodelling and drug chemoresistance. Liquid chromatography-high resolution mass spectrometry (LC-HRMS) lipid analysis of PM-enriched fractions of MDA-MB-231 BC cells showed that CPT1C silencing increased PM phospholipid saturation, suggesting a rise in PM rigidity. Moreover, CPT1C silencing increased cell survival against doxorubicin (DOX) treatment in different BC cells due to reduced drug uptake. These findings, further complemented by ROC plotter analysis correlating lower CPT1C expression with a lower pathological complete response to anthracyclines in patients with more aggressive types of BC, suggest CPT1C as a novel predictive biomarker for BC chemotherapy.
Assuntos
Neoplasias da Mama , Carnitina O-Palmitoiltransferase , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Antraciclinas/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Membrana Celular/metabolismo , Regulação para BaixoRESUMO
BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a neoplasia of the biliary tract driven by genetic, epigenetic and transcriptional mechanisms. Herein, we investigated the role of the transcription factor FOSL1, as well as its downstream transcriptional effectors, in the development and progression of CCA. METHODS: FOSL1 was investigated in human CCA clinical samples. Genetic inhibition of FOSL1 in human and mouse CCA cell lines was performed in in vitro and in vivo models using constitutive and inducible short-hairpin RNAs. Conditional FOSL1 ablation was done using a genetically engineered mouse (GEM) model of CCA (mutant KRAS and Trp53 knockout). Follow-up RNA and chromatin immunoprecipitation (ChIP) sequencing analyses were carried out and downstream targets were validated using genetic and pharmacological inhibition. RESULTS: An inter-species analysis of FOSL1 in CCA was conducted. First, FOSL1 was found to be highly upregulated in human and mouse CCA, and associated with poor patient survival. Pharmacological inhibition of different signalling pathways in CCA cells converged on the regulation of FOSL1 expression. Functional experiments showed that FOSL1 is required for cell proliferation and cell cycle progression in vitro, and for tumour growth and tumour maintenance in both orthotopic and subcutaneous xenograft models. Likewise, FOSL1 genetic abrogation in a GEM model of CCA extended mouse survival by decreasing the oncogenic potential of transformed cholangiocytes. RNA and ChIP sequencing studies identified direct and indirect transcriptional effectors such as HMGCS1 and AURKA, whose genetic and pharmacological inhibition phenocopied FOSL1 loss. CONCLUSIONS: Our data illustrate the functional and clinical relevance of FOSL1 in CCA and unveil potential targets amenable to pharmacological inhibition that could enable the implementation of novel therapeutic strategies. LAY SUMMARY: Understanding the molecular mechanisms involved in cholangiocarcinoma (bile duct cancer) development and progression stands as a critical step for the development of novel therapies. Through an inter-species approach, this study provides evidence of the clinical and functional role of the transcription factor FOSL1 in cholangiocarcinoma. Moreover, we report that downstream effectors of FOSL1 are susceptible to pharmacological inhibition, thus providing new opportunities for therapeutic intervention.
Assuntos
Colangiocarcinoma/genética , Hidroximetilglutaril-CoA Sintase/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/efeitos adversos , Idoso , Colangiocarcinoma/diagnóstico , Colangiocarcinoma/etiologia , Feminino , Humanos , Hidroximetilglutaril-CoA Sintase/genética , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-fos/genética , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genéticaRESUMO
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.
Assuntos
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
BACKGROUND: Osteosarcoma is the most prevalent primary bone malignancy in children and young adults. These tumors are highly metastatic, leading to poor outcome. We previously demonstrated that Cysteine-rich protein 61 (CYR61/CCN1) expression level is correlated to osteosarcoma aggressiveness in preclinical model and in patient tumor samples. The aim of the present study was to investigate the CYR61-induced intracellular mechanisms leading to the acquisition of an invasive phenotype by osteosarcoma cells. METHODS: Modified murine and human osteosarcoma cell lines were evaluated for cell adhesion, aggregation (spheroid), motility (wound healing assay), phenotypic markers expression (RT-qPCR, western blot). Cell-derived xenograft FFPE samples and patients samples (TMA) were assessed by IHC. RESULTS: CYR61 levels controlled the expression of markers related to an Epithelial-mesenchymal transition (EMT)-like process, allowing tumor cells to migrate acquiring a competent morphology, and to be able to invade the surrounding stroma. This phenotypic shift indeed correlated with tumor grade and aggressiveness in patient samples and with the metastatic dissemination potential in cell-derived xenograft models. Unlike EGFR or PDGFR, IGF1Rß levels correlated with CYR61 and N-cadherin levels, and with the aggressiveness of osteosarcoma and overall survival. The expression levels of IGF1Rß/IGF1 axis were controlled by CYR61, and anti-IGF1 neutralizing antibody prevented the CYR61-induced phenotypic shift, aggregation, and motility abilities. CONCLUSIONS: Taken together, our study provides new evidence that CYR61 acts as a key inducing factor in the metastatic progression of osteosarcoma by playing a critical role in primary tumor dissemination, with a process associated with IGF1/IGFR stimulation. This suggests that CYR61 may represent a potential pivotal target for therapeutic management of metastases spreading in osteosarcoma, in correlation with IGF1/IGFR pathway.
Assuntos
Neoplasias Ósseas/etiologia , Neoplasias Ósseas/metabolismo , Proteína Rica em Cisteína 61/genética , Transição Epitelial-Mesenquimal/genética , Osteossarcoma/etiologia , Osteossarcoma/metabolismo , Receptores de Somatomedina/metabolismo , Animais , Biomarcadores Tumorais , Neoplasias Ósseas/patologia , Caderinas/metabolismo , Adesão Celular/genética , Comunicação Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proteína Rica em Cisteína 61/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/secundário , Sistema de Sinalização das MAP Quinases , Camundongos , Osteossarcoma/patologia , Receptor IGF Tipo 1RESUMO
RATIONALE: C5aR1 (CD88), a receptor for complement anaphylatoxin C5a, is a potent immune mediator. Its impact on malignant growth and dissemination of non-small cell lung cancer cells is poorly understood. OBJECTIVES: To investigate the contribution of the C5a/C5aR1 axis to the malignant phenotype of non-small cell lung cancer cells, particularly in skeletal colonization, a preferential lung metastasis site. METHODS: Association between C5aR1 expression and clinical outcome was assessed in silico and validated by immunohistochemistry. Functional significance was evaluated by lentiviral gene silencing and ligand l-aptamer inhibition in in vivo models of lung cancer bone metastasis. In vitro functional assays for signaling, migration, invasion, metalloprotease activity, and osteoclastogenesis were also performed. MEASUREMENTS AND MAIN RESULTS: High levels of C5aR1 in human lung tumors were significantly associated with shorter recurrence-free survival, overall survival, and bone metastasis. Silencing of C5aR1 in lung cancer cells led to a substantial reduction in skeletal metastatic burden and osteolysis in in vivo models. Furthermore, metalloproteolytic, migratory, and invasive tumor cell activities were modulated in vitro by C5aR1 stimulation or gene silencing. l-Aptamer blockade or C5aR1 silencing significantly reduced the osseous metastatic activity of lung cancer cells in vivo. This effect was associated with decreased osteoclastogenic activity in vitro and was rescued by the exogenous addition of the chemokine CXCL16. CONCLUSIONS: Disruption of C5aR1 signaling in lung cancer cells abrogates their tumor-associated osteoclastogenic activity, impairing osseous colonization. This study unveils the role played by the C5a/C5aR1 axis in lung cancer dissemination and supports its potential use as a novel therapeutic target.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Quimiocina CXCL16/imunologia , Neoplasias Pulmonares/complicações , Neoplasias Pulmonares/imunologia , Metástase Neoplásica/imunologia , Receptor da Anafilatoxina C5a/imunologia , Transdução de Sinais/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Ósseas/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Survival rates for osteosarcoma, the most common primary bone cancer, have changed little over the past three decades and are particularly low for patients with metastatic disease. We conducted a multi-institutional genome-wide association study (GWAS) to identify germline genetic variants associated with overall survival in 632 patients with osteosarcoma, including 523 patients of European ancestry and 109 from Brazil. We conducted a time-to-event analysis and estimated hazard ratios (HR) and 95% confidence intervals (CI) using Cox proportional hazards models, with and without adjustment for metastatic disease. The results were combined across the European and Brazilian case sets using a random-effects meta-analysis. The strongest association after meta-analysis was for rs3765555 at 9p24.1, which was inversely associated with overall survival (HR = 1.76; 95% CI 1.41-2.18, p = 4.84 × 10-7 ). After imputation across this region, the combined analysis identified two SNPs that reached genome-wide significance. The strongest single association was with rs55933544 (HR = 1.9; 95% CI 1.5-2.4; p = 1.3 × 10-8 ), which localizes to the GLDC gene, adjacent to the IL33 gene and was consistent across both the European and Brazilian case sets. Using publicly available data, the risk allele was associated with lower expression of IL33 and low expression of IL33 was associated with poor survival in an independent set of patients with osteosarcoma. In conclusion, we have identified the GLDC/IL33 locus on chromosome 9p24.1 as associated with overall survival in patients with osteosarcoma. Further studies are needed to confirm this association and shed light on the biological underpinnings of this susceptibility locus.
Assuntos
Neoplasias Ósseas/genética , Neoplasias Ósseas/mortalidade , Interleucina-33/genética , Osteossarcoma/genética , Osteossarcoma/mortalidade , Adulto , Alelos , Brasil , Feminino , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla/métodos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Modelos de Riscos Proporcionais , Taxa de Sobrevida , População Branca/genéticaRESUMO
Osteosarcoma (OS) is the most prevalent osseous tumour in children and adolescents and, within this, lung metastases remain one of the factors associated with a dismal prognosis. At present, the genetic determinants driving pulmonary metastasis are poorly understood. We adopted a novel strategy using robust filtering analysis of transcriptomic profiling in tumour osteoblastic cell populations derived from human chemo-naive primary tumours displaying extreme phenotypes (indolent versus metastatic) to uncover predictors associated with metastasis and poor survival. We identified MGP, encoding matrix-Gla protein (MGP), a non-collagenous matrix protein previously associated with the inhibition of arterial calcification. Using different orthotopic models, we found that ectopic expression of Mgp in murine and human OS cells led to a marked increase in lung metastasis. This effect was independent of the carboxylation of glutamic acid residues required for its physiological role. Abrogation of Mgp prevented lung metastatic activity, an effect that was rescued by forced expression. Mgp levels dramatically altered endothelial adhesion, trans-endothelial migration in vitro and tumour cell extravasation ability in vivo. Furthermore, Mgp modulated metalloproteinase activities and TGFß-induced Smad2/3 phosphorylation. In the clinical setting, OS patients who developed lung metastases had high serum levels of MGP at diagnosis. Thus, MGP represents a novel adverse prognostic factor and a potential therapeutic target in OS. Microarray datasets may be found at: http://bioinfow.dep.usal.es/osteosarcoma/ Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Neoplasias Ósseas/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Neoplasias Pulmonares/secundário , Osteossarcoma/secundário , Animais , Neoplasias Ósseas/metabolismo , Movimento Celular/fisiologia , Humanos , Neoplasias Pulmonares/metabolismo , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Nus , Osteossarcoma/metabolismo , Fosforilação , Prognóstico , Proteínas Smad/metabolismo , Proteína de Matriz GlaRESUMO
The skeleton is a common site of metastases in lung cancer, an event associated with significant morbidities and poor outcomes. Current antiresorptive therapies provide limited benefit, and novel strategies of prevention and treatment are urgently needed. This review summarizes the latest advances and new perspectives on emerging experimental and clinical approaches to block this deleterious process. Progress propelled by preclinical models has led to a deeper understanding on the complex interplay of tumor cells in the osseous milieu, unveiling potential new targets for drug development. Improvements in early diagnosis through the use of sophisticated imaging techniques with bone serum biomarkers are also discussed in the context of identifying patients at risk and monitoring disease progression during the course of treatment.
Assuntos
Neoplasias Ósseas/secundário , Neoplasias Ósseas/terapia , Neoplasias Pulmonares/patologia , Carcinoma Pulmonar de Células não Pequenas/secundário , Ensaios Clínicos como Assunto , Humanos , Imunidade Celular , Imunidade Inata , Células Neoplásicas Circulantes , Neovascularização Patológica , Neoplasias da Coluna Vertebral/secundário , Neoplasias da Coluna Vertebral/terapiaRESUMO
New mouse models with specific drivers of genetic alterations are needed for preclinical studies. Herein, we created and characterized at the genetic level a new syngeneic model for lung cancer and metastasis in Balb-c mice. Tumor cell lines were obtained from a silica-mediated airway chronic inflammation that promotes tumorigenesis when combined with low doses of N-nitrosodimethylamine, a tobacco smoke carcinogen. Orthotopic transplantation of these cells induced lung adenocarcinomas, and their intracardiac injection led to prominent colonization of various organs (bone, lung, liver and brain). Driver gene alterations included a mutation in the codon 12 of KRAS (G-A transition), accompanied by a homozygous deletion of the WW domain-containing oxidoreductase (WWOX) gene. The mutant form of WWOX lacked exons 5-8 and displayed reduced protein expression level and activity. WWOX gene restoration decreased the in vitro and in vivo tumorigenicity, confirming the tumor suppressor function of this gene in this particular model. Interestingly, we found that cells displayed remarkable sphere formation ability with expression of specific lung cancer stem cell markers. Study of non-small-cell lung cancer patient cohorts demonstrated a deletion of WWOX in 30% of cases, with significant reduction in protein levels as compared to normal tissues. Overall, our new syngeneic mouse model provides a most valuable tool to study lung cancer metastasis in balb-c mice background and highlights the importance of WWOX deletion in lung carcinogenesis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/secundário , Modelos Animais de Doenças , Inflamação/patologia , Neoplasias Pulmonares/patologia , Recidiva Local de Neoplasia/patologia , Oxirredutases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética , Proteínas ras/genética , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Adenocarcinoma/secundário , Animais , Apoptose , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/secundário , Proliferação de Células , Hibridização Genômica Comparativa , Transição Epitelial-Mesenquimal , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Inflamação/genética , Inflamação/mortalidade , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação/genética , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/mortalidade , Estadiamento de Neoplasias , Oxirredutases/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/metabolismo , Oxidorredutase com Domínios WW , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/metabolismoRESUMO
RATIONALE: Efficient metastasis requires survival and adaptation of tumor cells to stringent conditions imposed by the extracellular milieu. Identification of critical survival signaling pathways in tumor cells might unveil novel targets relevant in disease progression. OBJECTIVES: To investigate the contribution of activated protein C (APC) and its receptor (endothelial protein C receptor [EPCR]) in animal models of lung cancer metastasis and in patients with lung adenocarcinoma. METHODS: Signaling pathway triggered by APC/EPCR and its relevance in apoptosis was studied in vitro. Functional significance was assessed by silencing and blocking antibodies in several in vivo models of lung cancer metastasis in athymic nude Foxn1(nu) mice. We examined EPCR levels using a microarray dataset of 107 patients. Immunohistochemical analysis was performed in an independent cohort of 295 patients with lung adenocarcinoma. MEASUREMENTS AND MAIN RESULTS: The effects of APC binding to EPCR rapidly triggered Akt and extracellular signal-regulated kinase signaling pathways, leading to attenuated in vitro apoptosis. In vivo, silencing of EPCR expression or blocking APC/EPCR interaction reduced infiltration in the target organ, resulting in impaired prometastatic activity. Moreover, overexpression of EPCR induced an increased metastatic activity to target organs. Analysis of clinical samples showed a robust association between high EPCR levels and poor prognosis, particularly in stage I patients. CONCLUSIONS: EPCR and its ligand APC promote cell survival that contributes to tumor cell endurance to stress favoring prometastatic activity of lung adenocarcinoma. EPCR/APC is a novel target of relevance in the clinical outcome of early-stage lung cancer.
Assuntos
Adenocarcinoma/secundário , Fatores de Coagulação Sanguínea/fisiologia , Neoplasias Pulmonares/patologia , Proteína C/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Apoptose/fisiologia , Sobrevivência Celular , Microambiente Celular/fisiologia , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Camundongos , Prognóstico , Análise Serial de Proteínas , Transdução de Sinais/fisiologiaRESUMO
ENPP1 (ecto-nucleotide pyrophosphatase/phosphodiesterase) participates in the hydrolysis of different purine nucleotides in an array of physiologic processes. However, ENPP1 is frequently overexpressed in local relapses and tumor metastases, which are associated with poor prognosis and survival in a range of solid tumors. ENPP1 promotes an immunosuppressive tumor microenvironment (TME) by tilting the balance of ATP/adenosine (Ado) in conjunction with other components (CD38, CD39/ENTPD1, and CD73/NT5E). Moreover, ENPP1 intersects with the stimulator of interferon genes (STING), impairing its robust immune response through the hydrolysis of the effector 2´,3´-cyclic GMP-AMP. Thus, ENPP1 blockade emerges as a unique target eliciting immune remodeling and leveraging the STING pathway. Several ENPP1 inhibitors have shown an immunostimulatory effect, and their combination with other therapeutic modalities, such as immune-checkpoint blockade, STING activation, DNA damage response (DDR) inhibitors, and radiotherapy (RT), represents a promising avenue to boost antitumor-immune responses and to improve current clinical outcomes in several tumors. This comprehensive review summarizes the current state of the art and opens new perspectives for novel treatment strategies.
Assuntos
Adenosina , Recidiva Local de Neoplasia , Humanos , Diester Fosfórico Hidrolases/genética , Microambiente TumoralRESUMO
PURPOSE: The identification of pancreatic ductal adenocarcinoma (PDAC) dysregulated genes may unveil novel molecular targets entering inhibitory strategies. Laminins are emerging as potential targets in PDAC given their role as diagnostic and prognostic markers. Here, we investigated the cellular, functional, and clinical relevance of LAMC2 and its regulated network, with the ultimate goal of identifying potential therapies. EXPERIMENTAL DESIGN: LAMC2 expression was analyzed in PDAC tissues, a panel of human and mouse cell lines, and a genetically engineered mouse model. Genetic perturbation in 2D, 3D, and in vivo allograft and xenograft models was done. Expression profiling of a LAMC2 network was performed by RNA-sequencing, and publicly available gene expression datasets from experimental and clinical studies examined to query its human relevance. Dual inhibition of pharmacologically targetable LAMC2-regulated effectors was investigated. RESULTS: LAMC2 was consistently upregulated in human and mouse experimental models as well as in human PDAC specimens, and associated with tumor grade and survival. LAMC2 inhibition impaired cell cycle, induced apoptosis, and sensitized PDAC to MEK1/2 inhibitors (MEK1/2i). A LAMC2-regulated network was featured in PDAC, including both classical and quasi-mesenchymal subtypes, and contained downstream effectors transcriptionally shared by the KRAS signaling pathway. LAMC2 regulated a functional FOSL1-AXL axis via AKT phosphorylation. Furthermore, genetic LAMC2 or pharmacological AXL inhibition elicited a synergistic antiproliferative effect in combination with MEK1/2is that was consistent across 2D and 3D human and mouse PDAC models, including primary patient-derived organoids. CONCLUSIONS: LAMC2 is a molecular target in PDAC that regulates a transcriptional network that unveils a dual drug combination for cancer treatment.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Humanos , Camundongos , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Laminina/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fosforilação , Transdução de Sinais , Neoplasias PancreáticasRESUMO
Circulating tumor cells are the key link between a primary tumor and distant metastases, but once in the bloodstream, loss of adhesion induces cell death. To identify the mechanisms relevant for melanoma circulating tumor cell survival, we performed RNA sequencing and discovered that detached melanoma cells and isolated melanoma circulating tumor cells rewire lipid metabolism by upregulating fatty acid (FA) transport and FA beta-oxidationârelated genes. In patients with melanoma, high expression of FA transporters and FA beta-oxidation enzymes significantly correlates with reduced progression-free and overall survival. Among the highest expressed regulators in melanoma circulating tumor cells were the carnitine transferases carnitine O-octanoyltransferase and carnitine acetyltransferase, which control the shuttle of peroxisome-derived medium-chain FAs toward mitochondria to fuel mitochondrial FA beta-oxidation. Knockdown of carnitine O-octanoyltransferase or carnitine acetyltransferase and short-term treatment with peroxisomal or mitochondrial FA beta-oxidation inhibitors thioridazine or ranolazine suppressed melanoma metastasis in mice. Carnitine O-octanoyltransferase and carnitine acetyltransferase depletion could be rescued by medium-chain FA supplementation, indicating that the peroxisomal supply of FAs is crucial for the survival of nonadherent melanoma cells. Our study identifies targeting the FA-based cross-talk between peroxisomes and mitochondria as a potential therapeutic opportunity to challenge melanoma progression. Moreover, the discovery of the antimetastatic activity of the Food and Drug Administrationâapproved drug ranolazine carries translational potential.
Assuntos
Melanoma , Células Neoplásicas Circulantes , Camundongos , Animais , Carnitina O-Acetiltransferase/genética , Carnitina O-Acetiltransferase/metabolismo , Carnitina Aciltransferases/genética , Carnitina Aciltransferases/metabolismo , Ranolazina , Oxirredução , Ácidos Graxos/metabolismo , Melanoma/tratamento farmacológico , Carnitina/metabolismoRESUMO
Drug combinations are key to circumvent resistance mechanisms compromising response to single anti-cancer targeted therapies. The implementation of combinatorial approaches involving MEK1/2 or KRASG12C inhibitors in the context of KRAS-mutated lung cancers focuses fundamentally on targeting KRAS proximal activators or effectors. However, the antitumor effect is highly determined by compensatory mechanisms arising in defined cell types or tumor subgroups. A potential strategy to find drug combinations targeting a larger fraction of KRAS-mutated lung cancers may capitalize on the common, distal gene expression output elicited by oncogenic KRAS. By integrating a signature-driven drug repurposing approach with a pairwise pharmacological screen, here we show synergistic drug combinations consisting of multi-tyrosine kinase PKC inhibitors together with MEK1/2 or KRASG12C inhibitors. Such combinations elicit a cytotoxic response in both in vitro and in vivo models, which in part involves inhibition of the PKC inhibitor target AURKB. Proteome profiling links dysregulation of MYC expression to the effect of both PKC inhibitor-based drug combinations. Furthermore, MYC overexpression appears as a resistance mechanism to MEK1/2 and KRASG12C inhibitors. Our study provides a rational framework for selecting drugs entering combinatorial strategies and unveils MEK1/2- and KRASG12C-based therapies for lung cancer.
Assuntos
Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Reposicionamento de Medicamentos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Combinação de Medicamentos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Mutação , Linhagem Celular TumoralRESUMO
INTRODUCTION: Several studies have linked increased risk of osteosarcoma with tall stature, high birthweight, and early puberty, although evidence is inconsistent. We used genetic risk scores (GRS) based on established genetic loci for these traits and evaluated associations between genetically inferred birthweight, height, and puberty timing with osteosarcoma. METHODS: Using genotype data from two genome-wide association studies, totaling 1039 cases and 2923 controls of European ancestry, association analyses were conducted using logistic regression for each study and meta-analyzed to estimate pooled odds ratios (ORs) and 95% confidence intervals (CIs). Subgroup analyses were conducted by case diagnosis age, metastasis status, tumor location, tumor histology, and presence of a known pathogenic variant in a cancer susceptibility gene. RESULTS: Genetically inferred higher birthweight was associated with an increased risk of osteosarcoma (OR =1.59, 95% CI 1.07-2.38, P = 0.02). This association was strongest in cases without metastatic disease (OR =2.46, 95% CI 1.44-4.19, P = 9.5 ×10-04). Although there was no overall association between osteosarcoma and genetically inferred taller stature (OR=1.06, 95% CI 0.96-1.17, P = 0.28), the GRS for taller stature was associated with an increased risk of osteosarcoma in 154 cases with a known pathogenic cancer susceptibility gene variant (OR=1.29, 95% CI 1.03-1.63, P = 0.03). There were no significant associations between the GRS for puberty timing and osteosarcoma. CONCLUSION: A genetic propensity to higher birthweight was associated with increased osteosarcoma risk, suggesting that shared genetic factors or biological pathways that affect birthweight may contribute to osteosarcoma pathogenesis.
RESUMO
Bone tumors strongly influence normal tissues and stimulate bone cells for the production of cytokines supporting proliferation and abnormal survival in cancer cells. We previously reported that the proteoglycan syndecan-2 controls the activity of various cytokines and growth factors and also modulates apoptosis and response to cytotoxic agents in osteosarcoma cell lines. Here, we show that syndecan-2 has a stronger tumor suppressor activity in vivo. We identify calpain-6 as a target gene downregulated by syndecan-2 in cells and in vivo. We demonstrate that calpain-6 expression in osteosarcoma cells depends on endothelin-1, a mediator of the tumor progression in bone. Syndecan-2 overexpression alters ERK1/2, PI3K/AKT and NFκB pathways that are calpain-6-promoting signals downstream of endothelin-1. Immunohistochemical analysis shows that calpain-6 is expressed in human bone tumors and metastases. A high expression of calpain-6 was specially found in recurrent osteosarcoma. Moreover, calpain-6 levels in primary tumors were inversely related to the response to chemotherapy. Consistently, calpain-6 was increased by doxorubicin and was found to be expressed at higher levels in doxorubicin-resistant U2OS osteosarcoma-derived cells as compared to responsive cells. Inhibition of calpain-6 with shRNA resulted in decreased proliferation, increased spontaneous apoptosis and increased sensitivity to doxorubicin and also methotrexate in responsive and resistant osteosarcoma cells. Taken together, our data show that syndecan-2 exerts its pro-apoptotic function through modulation of the endothelin-1/NFκB signaling and through downregulation of calpain-6, a protective factor that contributes to abnormal cell survival. Thus, this study identifies calpain-6 as a new possible therapeutic target in chemoresistant osteosarcoma.
Assuntos
Neoplasias Ósseas/patologia , Calpaína/fisiologia , Resistencia a Medicamentos Antineoplásicos , Endotelina-1/fisiologia , Osteossarcoma/patologia , Transdução de Sinais/fisiologia , Apoptose , Neoplasias Ósseas/tratamento farmacológico , Linhagem Celular Tumoral , Humanos , NF-kappa B/fisiologia , Osteossarcoma/tratamento farmacológico , Sindecana-2/fisiologiaRESUMO
The analysis of circulating tumor cells (CTCs) in blood is a powerful noninvasive alternative to conventional tumor biopsy. Inertial-based separation is a promising high-throughput, marker-free sorting strategy for the enrichment and isolation of CTCs. Here, we present and validate a double spiral microfluidic device that efficiently isolates CTCs with a fine-tunable cut-off value of 9 µm and a separation range of 2 µm. We designed the device based on computer simulations that introduce a novel, customized inertial force term, and provide practical fabrication guidelines. We validated the device using calibration beads, which allowed us to refine the simulations and redesign the device. Then we validated the redesigned device using blood samples and a murine model of metastatic breast cancer. Finally, as a proof of principle, we tested the device using peripheral blood from a patient with hepatocellular carcinoma, isolating more than 17 CTCs/ml, with purity/removal values of 96.03% and 99.99% of white blood cell and red blood cells, respectively. These results confirm highly efficient CTC isolation with a stringent cut-off value and better separation results than the state of the art.
RESUMO
Myeloid-derived suppressor cells (MDSCs) play a major role in cancer progression. In this study, we investigated the mechanisms by which complement C5a increases the capacity of polymorphonuclear MDSCs (PMN-MDSCs) to promote tumor growth and metastatic spread. Stimulation of PMN-MDSCs with C5a favored the invasion of cancer cells via a process dependent on the formation of neutrophil extracellular traps (NETs). NETosis was dependent on the production of high mobility group box 1 (HMGB1) by cancer cells. Moreover, C5a induced the surface expression of the HMGB1 receptors TLR4 and RAGE in PMN-MDSCs. In a mouse lung metastasis model, inhibition of C5a, C5a receptor-1 (C5aR1) or NETosis reduced the number of circulating-tumor cells (CTCs) and the metastatic burden. In support of the translational relevance of these findings, C5a was able to stimulate migration and NETosis in PMN-MDSCs obtained from lung cancer patients. Furthermore, myeloperoxidase (MPO)-DNA complexes, as markers of NETosis, were elevated in lung cancer patients and significantly correlated with C5a levels. In conclusion, C5a induces the formation of NETs from PMN-MDSCs in the presence of cancer cells, which may facilitate cancer cell dissemination and metastasis.