RESUMO
Antibodies targeting immune checkpoints were recently approved for metastatic melanoma. However, not all patients will respond to the treatment and some will experience grade III-IV immune-related adverse events. Therefore, early identification of non-responder patients would greatly aid clinical practice. Detection of circulating tumour DNA (ctDNA) is a non-invasive approach to monitor tumour response. Digital droplet PCR was used to quantify BRAF and NRAS mutations in the plasma of patients with metastatic melanoma treated with immunotherapy. In 16 patients, ctDNA variations mirrored tumour response (p = 0.034) and ctDNA augmentation during follow-up detected tumour progression with 100% specificity. In 13 patients, early ctDNA variation was associated with clinician decision at first evaluation (p = 0.0046), and early ctDNA increase with shorter progression-free survival (median 21 vs. 145 days; p = 0.001). Monitoring ctDNA variations early during immunotherapy may help clinicians rapidly to discriminate non-responder patients, allow early adaptation of therapeutic strategies, and reduce exposure to ineffective, expensive treatment.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais/genética , DNA Tumoral Circulante/genética , Imunoterapia/métodos , Melanoma/terapia , Neoplasias Cutâneas/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , DNA Tumoral Circulante/sangue , Progressão da Doença , Feminino , Humanos , Masculino , Melanoma/sangue , Melanoma/genética , Melanoma/imunologia , Pessoa de Meia-Idade , Intervalo Livre de Progressão , Estudo de Prova de Conceito , Estudos Retrospectivos , Neoplasias Cutâneas/sangue , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Fatores de TempoRESUMO
No specific biomarkers for prognostication or evaluation of tumour load in melanoma have been reported to our knowledge. MicroRNAs (miRNAs) are strongly implicated in oncogenesis and tumour progression, and their circulating forms have been studied as potential biomarkers in oncology. The aim of this prospective study was to identify a melanoma-specific profile of plasma miRNAs. A screening phase, using RNA microarray, was conducted on plasma from 14 patients with metastatic melanoma and 5 healthy subjects. Selected miRNAs were analysed by RTqPCR in 2 independent training and validation cohorts including, respectively, 29 and 31 patients and 16 and 43 control subjects. A profile of 2 miRNAs (miR-1246 and miR-185) significantly associated with metastatic melanoma with a sensitivity of 90.5% and a specificity of 89.1% was identified. This plasma miRNA profile may become an accurate non-invasive biomarker for melanoma.
Assuntos
Biomarcadores Tumorais/sangue , Perfilação da Expressão Gênica , Melanoma/sangue , MicroRNAs/sangue , Neoplasias Cutâneas/sangue , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Perfilação da Expressão Gênica/métodos , Humanos , Melanoma/genética , Melanoma/secundário , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologiaRESUMO
Drug-tolerance has emerged as one of the major non-genetic adaptive processes driving resistance to targeted therapy (TT) in non-small cell lung cancer (NSCLC). However, the kinetics and sequence of molecular events governing this adaptive response remain poorly understood. Here, we combine real-time monitoring of the cell-cycle dynamics and single-cell RNA sequencing in a broad panel of oncogenic addiction such as EGFR-, ALK-, BRAF- and KRAS-mutant NSCLC, treated with their corresponding TT. We identify a common path of drug adaptation, which invariably involves alveolar type 1 (AT1) differentiation and Rho-associated protein kinase (ROCK)-mediated cytoskeletal remodeling. We also isolate and characterize a rare population of early escapers, which represent the earliest resistance-initiating cells that emerge in the first hours of treatment from the AT1-like population. A phenotypic drug screen identify farnesyltransferase inhibitors (FTI) such as tipifarnib as the most effective drugs in preventing relapse to TT in vitro and in vivo in several models of oncogenic addiction, which is confirmed by genetic depletion of the farnesyltransferase. These findings pave the way for the development of treatments combining TT and FTI to effectively prevent tumor relapse in oncogene-addicted NSCLC patients.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Resistencia a Medicamentos Antineoplásicos , Farnesiltranstransferase , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Farnesiltranstransferase/antagonistas & inibidores , Farnesiltranstransferase/metabolismo , Farnesiltranstransferase/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral , Animais , Camundongos , Vício Oncogênico/genética , Terapia de Alvo Molecular , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Ensaios Antitumorais Modelo de Xenoenxerto , Oncogenes/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , QuinolonasRESUMO
RhoB is a small GTPase implicated in cytoskeletal organization, EGF receptor trafficking and cell transformation. It is an immediate-early gene, regulated at many levels of its biosynthetic pathway. Herein we show that the serine/threonine protein kinase CK1 phosphorylates RhoB in vitro but not RhoA or RhoC. With the use of specific CK1 inhibitors, IC261 and D4476, we show that the kinase phosphorylates also RhoB in HeLa cells. Mass spectrometry analysis demonstrates that RhoB is monophosphorylated by CK1, in its C-terminal end, on serine 185. The substitution of Ser185 by Ala dramatically inhibited the phosphorylation of RhoB in cultured cells. Lastly we show that the inhibition of CK1 activates RhoB and promotes RhoB dependent actin fiber formation and EGF-R level. Our data provide the first demonstration of RhoB phosphorylation and indicate that this post-translational maturation would be a novel critical mechanism to control the RhoB functions.
Assuntos
Actinas/metabolismo , Caseína Quinase Ialfa/metabolismo , Receptores ErbB/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fibras de Estresse/metabolismo , Proteína rhoB de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Sequência de Aminoácidos/fisiologia , Substituição de Aminoácidos/fisiologia , Caseína Quinase Ialfa/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Dados de Sequência Molecular , Fosforilação , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Homologia de Sequência de Aminoácidos , Serina/metabolismo , Fibras de Estresse/ultraestrutura , Proteína rhoB de Ligação ao GTP/químicaRESUMO
OBJECTIVES: Pseudo-progression is a rare but worrying situation for both clinicians and patients during immunotherapy. Dedicated ir-RECIST criteria have been established to improve this situation. However, this can be sometimes considered inadequate and patients experiencing true progression may then receive inefficient treatments. Additional reliable tools to discriminate pseudo from true progression are thus needed. So far, no biomarker has been identified to distinguish pseudo from true progression. We hypothesize that biomarkers associated with the molecular characteristics of the tumor may be of interest. To avoid a tumor re-biopsy, circulating markers appear to be a less invasive and reproducible procedure. As ctDNA kinetics correlate with the response to treatment in KRAS-mutated adenocarcinoma, we anticipated that this analysis could be of interest. MATERIALS AND METHODS: We monitored the level of KRAS-mutated ctDNA by digital droplet PCR in serial plasma samples from two patients who had experienced pseudo-progression and compared the variations with those from of a patient that had true progression. RESULTS: ctDNA showed rapid and dramatic decreases in pseudo-progressive patients, whereas it was strongly increased in the progressive patient. CONCLUSIONS: ddPCR of ctDNA may thus be an additional tool to discriminate pseudo-progression from true progression for tumors that harbor an oncogenic addiction.
Assuntos
Adenocarcinoma/tratamento farmacológico , Anticorpos Monoclonais/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , DNA Tumoral Circulante/genética , Imunoterapia/métodos , Neoplasias Pulmonares/tratamento farmacológico , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Carcinoma Pulmonar de Células não Pequenas/patologia , Progressão da Doença , Humanos , Neoplasias Pulmonares/patologiaRESUMO
Liquid biopsies are a new non-invasive strategy to detect and monitor the biology of non-small-cell lung cancer (NSCLC) in the era of personalized medicine. KRAS is an oncogenic driver that is mutated in 30% of NSCLCs and is associated with a poor prognosis. 62 samples from 32 patients, treated for metastatic KRAS-mutated lung adenocarcinoma, had DNA extracted from plasma and circulating tumor cells (CTCs) prospectively tested for the presence of KRAS mutations using droplet digital PCR. A KRAS mutation was detected in 82% of patients. Sensitivity was 78% for circulating free DNA (cfDNA) and 34% for CTCs. The presence of a KRAS mutation in cfDNA was correlated with a poor response to chemotherapy or targeted therapy. When a KRAS-mutated-DNA was detected and then monitored in cfDNA, its variation during targeted or conventional therapy was correlated with response, according to RECIST criteria, in 87.5% of cases (n=14/16), whereas this correlation was observed in 37.5% of cases for CTCs (n=3/8). We report the usefulness of using digital droplet PCR on liquid biopsies to predict and monitor responses to treatment of KRAS-mutated lung adenocarcinoma. ctDNA was much more sensitive than CTCs in this context.
Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Carcinoma Pulmonar de Células não Pequenas/genética , DNA de Neoplasias/sangue , Neoplasias Pulmonares/genética , Mutação/genética , Células Neoplásicas Circulantes/metabolismo , Reação em Cadeia da Polimerase/métodos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Adenocarcinoma de Pulmão , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , DNA de Neoplasias/genética , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Masculino , Estudos Prospectivos , Resultado do TratamentoRESUMO
The response of BRAF-mutant melanoma patients to BRAF inhibitors is dramatically impaired by secondary resistances and rapid relapse. So far, the molecular mechanisms driving these resistances are not completely understood. Here, we show that, in BRAF-mutant melanoma cells, inhibition of BRAF or its target MEK induces RHOB expression by a mechanism that depends on the transcription factor c-Jun. In those cells, RHOB deficiency causes hypersensitivity to BRAF and MEK inhibitors-induced apoptosis. Supporting these results, loss of RHOB expression in metastatic melanoma tissues is associated with an increased progression-free survival of BRAF-mutant patients treated with vemurafenib. Following BRAF inhibition, RHOB activates AKT whose inhibition causes hypersensitivity of BRAF-mutant melanoma cells to BRAF inhibitors. In mice, AKT inhibition synergizes with vemurafenib to block tumor growth of BRAF-mutant metastatic melanoma. Our findings reveal that BRAF inhibition activates a c-Jun/RHOB/AKT pathway that promotes tumor cell survival and further support a role of this pathway in the resistance of melanoma to vemurafenib. Our data also highlight the importance of using RHOB tumor levels as a biomarker to predict vemurafenib patient's response and to select those that would benefit of the combination with AKT inhibitors.
Assuntos
Indóis/farmacologia , Melanoma/tratamento farmacológico , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Sulfonamidas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/genética , Camundongos , Camundongos Nus , Recidiva Local de Neoplasia/genética , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Vemurafenib , Proteína rhoB de Ligação ao GTP/metabolismoRESUMO
PURPOSE: A crucial event in lung adenocarcinoma progression is the switch from an aerogenous spread toward an infiltrating tumor. Loss of RhoB expression has been suggested to be critical for lung cancer invasion. Here, we tested RhoB expression as a prognostic biomarker in non-small cell lung cancer (NSCLC) with a special focus on lepidic pattern. EXPERIMENTAL DESIGN: We analyzed RhoB expression using both IHC and RT-qPCR in two series of operated patients (n = 100 and 48, respectively) and in a series of advanced lepidic adenocarcinoma (n = 31) from different hospitals. Next, we examined the role of RhoB in lung cancer progression in transgenic mice that express inducible EGFR(L858R) crossed with Rhob null mice. RESULTS: We identified that loss of RhoB expression was strongly associated with worse survival (P = 0.0001) and progression-free survival (P < 0.001) in the first series. We then confirmed these results after multivariate analyses of the second series. In the series of adenocarcinoma with lepidic features issued from a clinical trial (IFCT-0401), we showed that loss of RhoB expression was associated with higher aggressiveness of stage IV. Finally, we showed that EGFR(L858R)/Rhob(+/+) mice developed mainly diffuse lung tumors with a lepidic pattern, whereas EGFR(L858R)/Rhob(+/-) and EGFR(L858R)/Rhob(-/-) developed a greater number of tumors, and aggressive adenocarcinomas with invasive properties. CONCLUSIONS: We showed that RhoB is not only a strong prognostic factor in NSCLC but it is also critical for the acquisition of an aggressive phenotype of adenocarcinoma.
Assuntos
Adenocarcinoma/genética , Adenocarcinoma/patologia , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Proteína rhoB de Ligação ao GTP/genética , Adenocarcinoma/mortalidade , Adenocarcinoma de Pulmão , Adulto , Idoso , Animais , Biomarcadores Tumorais , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Progressão da Doença , Feminino , Expressão Gênica , Humanos , Neoplasias Pulmonares/mortalidade , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Fatores de Risco , Carga TumoralAssuntos
Adenocarcinoma/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , DNA Tumoral Circulante/genética , Neoplasias Pulmonares/patologia , Mutação , Células Neoplásicas Circulantes/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Adenocarcinoma/sangue , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , DNA Tumoral Circulante/sangue , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , PrognósticoRESUMO
Lung cancer is the leading cause of cancer-related death worldwide, mainly due to its highly metastatic properties. Previously, we reported an inverse correlation between RhoB expression and the progression of the lung cancer, occurring between preinvasive and invasive tumors. Herein, we mimicked the loss of RhoB observed throughout lung oncogenesis with RNA interference in nontumoral bronchial cell lines and analyzed the consequences on both cell transformation and invasion. Down-regulation of RhoB did not modify the cell growth properties but did promote migration and invasiveness. Furthermore, RhoB depletion was accompanied by modifications of actin and cell adhesion. The specific activation of the Akt1 isoform and Rac1 was found to be critical for this RhoB-mediated regulation of migration. Lastly, we showed that RhoB down-regulation consecutive to K-RasV12 cell transformation is critical for cell motility but not for cell proliferation. We propose that RhoB loss during lung cancer progression relates to the acquisition of invasiveness mediated by the phosphatidylinositol 3-kinase (PI3K)/AKT and Rac1 pathways rather than to tumor initiation.